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Amadei M, Polticelli F, Musci G, Bonaccorsi di Patti MC. The Ferroxidase-Permease System for Transport of Iron Across Membranes: From Yeast to Humans. Int J Mol Sci 2025; 26:875. [PMID: 39940646 PMCID: PMC11817551 DOI: 10.3390/ijms26030875] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/23/2024] [Revised: 01/16/2025] [Accepted: 01/19/2025] [Indexed: 02/16/2025] Open
Abstract
Transport of iron across the cell membrane is a tightly controlled process carried out by specific proteins in all living cells. In yeast and in mammals, a system formed by an enzyme with ferroxidase activity coupled to a membrane transporter supports iron uptake or iron efflux, respectively. Ferroxidase belongs to the family of blue multicopper oxidases, enzymes able to couple the one-electron oxidation of substrate(s) to full reduction of molecular oxygen to water. On the other hand, the permeases are widely different and are specific to Fe3+ and Fe2+ in yeast and multicellular organisms, respectively. This review will describe the yeast and human ferroxidase-permease systems, highlighting similarities and differences in structure, function and regulation of the respective protein components.
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Affiliation(s)
- Matteo Amadei
- Department of Biochemical Sciences ‘A. Rossi Fanelli’, Sapienza University of Rome, 00185 Rome, Italy;
| | | | - Giovanni Musci
- Department of Biosciences and Territory, University of Molise, 86090 Pesche, Italy;
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2
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Novikova IV, Soldatova AV, Moser TH, Thibert SM, Romano CA, Zhou M, Tebo BM, Evans JE, Spiro TG. Cryo-EM Structure of the Mnx Protein Complex Reveals a Tunnel Framework for the Mechanism of Manganese Biomineralization. J Am Chem Soc 2024; 146:22950-22958. [PMID: 39056168 DOI: 10.1021/jacs.3c06537] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 07/28/2024]
Abstract
The global manganese cycle relies on microbes to oxidize soluble Mn(II) to insoluble Mn(IV) oxides. Some microbes require peroxide or superoxide as oxidants, but others can use O2 directly, via multicopper oxidase (MCO) enzymes. One of these, MnxG from Bacillus sp. strain PL-12, was isolated in tight association with small accessory proteins, MnxE and MnxF. The protein complex, called Mnx, has eluded crystallization efforts, but we now report the 3D structure of a point mutant using cryo-EM single particle analysis, cross-linking mass spectrometry, and AlphaFold Multimer prediction. The β-sheet-rich complex features MnxG enzyme, capped by a heterohexameric ring of alternating MnxE and MnxF subunits, and a tunnel that runs through MnxG and its MnxE3F3 cap. The tunnel dimensions and charges can accommodate the mechanistically inferred binuclear manganese intermediates. Comparison with the Fe(II)-oxidizing MCO, ceruloplasmin, identifies likely coordinating groups for the Mn(II) substrate, at the entrance to the tunnel. Thus, the 3D structure provides a rationale for the established manganese oxidase mechanism, and a platform for further experiments to elucidate mechanistic details of manganese biomineralization.
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Affiliation(s)
- Irina V Novikova
- Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, 3335 Innovation Blvd, Richland, Washington 99354, United States
| | - Alexandra V Soldatova
- Department of Chemistry, University of Washington, Box 351700, Seattle, Washington 98195, United States
| | - Trevor H Moser
- Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, 3335 Innovation Blvd, Richland, Washington 99354, United States
| | - Stephanie M Thibert
- Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, 3335 Innovation Blvd, Richland, Washington 99354, United States
| | - Christine A Romano
- Division of Environmental and Biomolecular Systems, Institute of Environmental Health, Oregon Health & Science University, Portland, Oregon 97239, United States
| | - Mowei Zhou
- Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, 3335 Innovation Blvd, Richland, Washington 99354, United States
| | - Bradley M Tebo
- Department of Chemistry, University of Washington, Box 351700, Seattle, Washington 98195, United States
- Division of Environmental and Biomolecular Systems, Institute of Environmental Health, Oregon Health & Science University, Portland, Oregon 97239, United States
| | - James E Evans
- Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, 3335 Innovation Blvd, Richland, Washington 99354, United States
| | - Thomas G Spiro
- Department of Chemistry, University of Washington, Box 351700, Seattle, Washington 98195, United States
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3
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Singha A, Sekretareva A, Tao L, Lim H, Ha Y, Braun A, Jones SM, Hedman B, Hodgson KO, Britt RD, Kosman DJ, Solomon EI. Tuning the Type 1 Reduction Potential of Multicopper Oxidases: Uncoupling the Effects of Electrostatics and H-Bonding to Histidine Ligands. J Am Chem Soc 2023. [PMID: 37294874 PMCID: PMC10392966 DOI: 10.1021/jacs.3c03241] [Citation(s) in RCA: 11] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/11/2023]
Abstract
In multicopper oxidases (MCOs), the type 1 (T1) Cu accepts electrons from the substrate and transfers these to the trinuclear Cu cluster (TNC) where O2 is reduced to H2O. The T1 potential in MCOs varies from 340 to 780 mV, a range not explained by the existing literature. This study focused on the ∼350 mV difference in potential of the T1 center in Fet3p and Trametes versicolor laccase (TvL) that have the same 2His1Cys ligand set. A range of spectroscopies performed on the oxidized and reduced T1 sites in these MCOs shows that they have equivalent geometric and electronic structures. However, the two His ligands of the T1 Cu in Fet3p are H-bonded to carboxylate residues, while in TvL they are H-bonded to noncharged groups. Electron spin echo envelope modulation spectroscopy shows that there are significant differences in the second-sphere H-bonding interactions in the two T1 centers. Redox titrations on type 2-depleted derivatives of Fet3p and its D409A and E185A variants reveal that the two carboxylates (D409 and E185) lower the T1 potential by 110 and 255-285 mV, respectively. Density functional theory calculations uncouple the effects of the charge of the carboxylates and their difference in H-bonding interactions with the His ligands on the T1 potential, indicating 90-150 mV for anionic charge and ∼100 mV for a strong H-bond. Finally, this study provides an explanation for the generally low potentials of metallooxidases relative to the wide range of potentials of the organic oxidases in terms of different oxidized states of their TNCs involved in catalytic turnover.
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Affiliation(s)
- Asmita Singha
- Department of Chemistry, Stanford University, Stanford, California 94305, United States
| | - Alina Sekretareva
- Department of Chemistry, Stanford University, Stanford, California 94305, United States
| | - Lizhi Tao
- Department of Chemistry, University of California at Davis, Davis, California 95616, United States
| | - Hyeongtaek Lim
- Department of Chemistry, Stanford University, Stanford, California 94305, United States
| | - Yang Ha
- Department of Chemistry, Stanford University, Stanford, California 94305, United States
| | - Augustin Braun
- Department of Chemistry, Stanford University, Stanford, California 94305, United States
| | - Stephen M Jones
- Department of Chemistry, Stanford University, Stanford, California 94305, United States
| | - Britt Hedman
- Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Stanford University, Menlo Park, California 94025, United States
| | - Keith O Hodgson
- Department of Chemistry, Stanford University, Stanford, California 94305, United States
- Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Stanford University, Menlo Park, California 94025, United States
| | - R David Britt
- Department of Chemistry, University of California at Davis, Davis, California 95616, United States
| | - Daniel J Kosman
- Department of Biochemistry, The University at Buffalo, Buffalo, New York 14214, United States
| | - Edward I Solomon
- Department of Chemistry, Stanford University, Stanford, California 94305, United States
- Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Stanford University, Menlo Park, California 94025, United States
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4
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Helman SL, Zhou J, Fuqua BK, Lu Y, Collins JF, Chen H, Vulpe CD, Anderson GJ, Frazer DM. The biology of mammalian multi-copper ferroxidases. Biometals 2023; 36:263-281. [PMID: 35167013 PMCID: PMC9376197 DOI: 10.1007/s10534-022-00370-z] [Citation(s) in RCA: 33] [Impact Index Per Article: 16.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/28/2021] [Accepted: 02/04/2022] [Indexed: 12/24/2022]
Abstract
The mammalian multicopper ferroxidases (MCFs) ceruloplasmin (CP), hephaestin (HEPH) and zyklopen (ZP) comprise a family of conserved enzymes that are essential for body iron homeostasis. Each of these enzymes contains six biosynthetically incorporated copper atoms which act as intermediate electron acceptors, and the oxidation of iron is associated with the four electron reduction of dioxygen to generate two water molecules. CP occurs in both a secreted and GPI-linked (membrane-bound) form, while HEPH and ZP each contain a single C-terminal transmembrane domain. These enzymes function to ensure the efficient oxidation of iron so that it can be effectively released from tissues via the iron export protein ferroportin and subsequently bound to the iron carrier protein transferrin in the blood. CP is particularly important in facilitating iron release from the liver and central nervous system, HEPH is the major MCF in the small intestine and is critical for dietary iron absorption, and ZP is important for normal hair development. CP and HEPH (and possibly ZP) function in multiple tissues. These proteins also play other (non-iron-related) physiological roles, but many of these are ill-defined. In addition to disrupting iron homeostasis, MCF dysfunction perturbs neurological and immune function, alters cancer susceptibility, and causes hair loss, but, despite their importance, how MCFs co-ordinately maintain body iron homeostasis and perform other functions remains incompletely understood.
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Affiliation(s)
- Sheridan L Helman
- Molecular Nutrition Laboratory, QIMR Berghofer Medical Research Institute, Brisbane, Australia
| | - Jie Zhou
- Department of Physiological Sciences, University of Florida, Gainsville, FL, USA
| | - Brie K Fuqua
- David Geffen School of Medicine at UCLA, Los Angeles, CA, USA
| | - Yan Lu
- Iron Metabolism Laboratory, QIMR Berghofer Medical Research Institute, 300 Herston Road, Brisbane, QLD, 4006, Australia
- Mucosal Immunology Group, QIMR Berghofer Medical Research Institute, Brisbane, Australia
| | - James F Collins
- Food Science and Human Nutrition Department, University of Florida, Gainsville, FL, USA
| | - Huijun Chen
- Jiangsu Key Laboratory of Molecular Medicine, Medical School of Nanjing University, Nanjing, China
| | - Christopher D Vulpe
- Department of Physiological Sciences, University of Florida, Gainsville, FL, USA
| | - Gregory J Anderson
- Iron Metabolism Laboratory, QIMR Berghofer Medical Research Institute, 300 Herston Road, Brisbane, QLD, 4006, Australia.
- School of Chemistry and Molecular Bioscience, University of Queensland, Brisbane, Australia.
| | - David M Frazer
- Molecular Nutrition Laboratory, QIMR Berghofer Medical Research Institute, Brisbane, Australia
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Sokolov AV, Vasilyev VB, Samygina VR. X-Ray Analysis of the Monoclinic Crystal Form of Human Ceruloplasmin. CRYSTALLOGR REP+ 2022. [DOI: 10.1134/s1063774522060232] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
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Abstract
Zn2+ ions are essential in many physiological processes, including enzyme catalysis, protein structural stabilization, and the regulation of many proteins. The affinities of proteins for Zn2+ ions span several orders of magnitude, with catalytic Zn2+ ions generally held more tightly than structural or regulatory ones. Metal carrier proteins, most of which are not specific for Zn2+, bind these ions with a broad range of affinities that overlap those of catalytic, structural, and regulatory Zn2+ ions and are thought to be responsible for distributing the metal through most cells, tissues, and fluid compartments. While little is known about how many proteins obtain or release these ions, there is now considerable experimental evidence suggesting that metal carrier proteins may be responsible for transferring metals to and from some Zn2+-dependent proteins, thus serving as a major regulatory factor for them. In this review, the biological roles of Zn2+ and structures of Zn2+ binding sites are examined, and experimental evidence demonstrating the direct participation of metal carrier proteins in enzyme regulation is discussed. Mechanisms of metal ion transfer are also offered, and the potential physiological significance of this phenomenon is explored.
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Sakajiri T, Nakatsuji M, Teraoka Y, Furuta K, Ikuta K, Shibusa K, Sugano E, Tomita H, Inui T, Yamamura T. Zinc mediates the interaction between ceruloplasmin and apo-transferrin for the efficient transfer of Fe(III) ions. Metallomics 2021; 13:6427378. [PMID: 34791391 DOI: 10.1093/mtomcs/mfab065] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/04/2020] [Accepted: 11/02/2021] [Indexed: 11/14/2022]
Abstract
Fe(II) exported from cells is oxidized to Fe(III), possibly by a multi-copper ferroxidase (MCF) such as ceruloplasmin (CP), to efficiently bind with the plasma iron transport protein transferrin (TF). As unbound Fe(III) is highly insoluble and reactive, its release into the blood during the transfer from MCF to TF must be prevented. A likely mechanism for preventing the release of unbound Fe(III) is via direct interaction between MCF and TF; however, the occurrence of this phenomenon remains controversial. This study aimed to reveal the interaction between these proteins, possibly mediated by zinc. Using spectrophotometric, isothermal titration calorimetric, and surface plasmon resonance methods, we found that Zn(II)-bound CP bound to iron-free TF (apo-TF) with a Kd of 4.2 μM and a stoichiometry CP:TF of ∼2:1. Computational modeling of the complex between CP and apo-TF predicted that each of the three Zn(II) ions that bind to CP further binds to acidic amino acid residues of apo-TF to play a role as a cross-linker connecting both proteins. Domain 4 of one CP molecule and domain 6 of the other CP molecule fit tightly into the clefts in the N- and C-lobes of apo-TF, respectively. Upon the binding of two Fe(III) ions to apo-TF, the resulting diferric TF [Fe(III)2TF] dissociated from CP by conformational changes in TF. In human blood plasma, zinc deficiency reduced the production of Fe(III)2TF and concomitantly increased the production of non-TF-bound iron. Our findings suggest that zinc may be involved in the transfer of iron between CP and TF.
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Affiliation(s)
- Tetsuya Sakajiri
- Laboratory of Biological Macromolecules, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japan.,Faculty of Nutritional Sciences, the University of Morioka, 808 Sunakomi, Takizawa, Iwate 020-0694, Japan.,Qualtec Co. Ltd., 4-230 Sambo-cho, Sakai, Osaka 590-0906, Japan.,Department of Nutrition, Kyushu Nutrition Welfare University, 5-1-1 Shimoitozu, Kitakyushu Kokurakita-ku, Fukuoka 803-0846, Japan
| | - Masatoshi Nakatsuji
- Laboratory of Biological Macromolecules, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japan
| | - Yoshiaki Teraoka
- Laboratory of Biological Macromolecules, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japan
| | - Kosuke Furuta
- Laboratory of Biological Macromolecules, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japan
| | - Katsuya Ikuta
- Division of Gastroenterology and Hematology/Oncology, Department of Medicine, Asahikawa Medical University, 2-1-1-1 Midorigaoka-Higashi, Asahikawa, Hokkaido 078-8510, Japan.,Japanese Red Cross Hokkaido Blood Center, 2-1 Nijuyonken, Nishi-ku, Sapporo, Hokkaido 063-0802, Japan
| | - Kotoe Shibusa
- Division of Gastroenterology and Hematology/Oncology, Department of Medicine, Asahikawa Medical University, 2-1-1-1 Midorigaoka-Higashi, Asahikawa, Hokkaido 078-8510, Japan.,Hokkaido System Science Co., Ltd., 2-1 Shinkawa Nishi, Kita-ku, Sapporo, Hokkaido 001-0932, Japan
| | - Eriko Sugano
- Department of Chemistry and Biological Sciences, Faculty of Science and Engineering, Iwate University, 4-3-5 Ueda, Morioka, Iwate 020-8551, Japan
| | - Hiroshi Tomita
- Department of Chemistry and Biological Sciences, Faculty of Science and Engineering, Iwate University, 4-3-5 Ueda, Morioka, Iwate 020-8551, Japan
| | - Takashi Inui
- Laboratory of Biological Macromolecules, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japan
| | - Takaki Yamamura
- Laboratory of Biological Macromolecules, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japan.,Faculty of Nutritional Sciences, the University of Morioka, 808 Sunakomi, Takizawa, Iwate 020-0694, Japan
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8
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Chan JC, Zhang B, Martinez M, Kuruba B, Brozik J, Kang C, Zhang X. Structural studies of Myceliophthora Thermophila Laccase in the presence of deep eutectic solvents. Enzyme Microb Technol 2021; 150:109890. [PMID: 34489043 DOI: 10.1016/j.enzmictec.2021.109890] [Citation(s) in RCA: 14] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/15/2021] [Revised: 07/08/2021] [Accepted: 07/28/2021] [Indexed: 11/24/2022]
Abstract
In this work, we elucidated the interactions between Myceliophthora thermophila laccase and deep eutectic solvent (DES) by crystallographic and kinetics analyses. Four types of DESs with different hydrogen bond acceptor (HBA) and hydrogen bond donor (HBD), including lactic acid: betaine, glycerol: choline chloride, lactic acid: choline chloride and glycerol: betaine was used. The results revealed that different DES have different effects on laccase activity. Lactic acid-betaine (2:1) DES has shown to enhance laccase activity up to 300 % at a concentration ranged from 2% to 8% v/v, while glycerol: choline chloride and lactic acid: choline chloride DES choline chloride-based DES have found to possess inhibitory effects on laccase under the same concentration range. Detailed kinetic study showed that glycerol: choline chloride DES is a S-parabolic-I-parabolic mixed non-competitive inhibitor, where conformational changes can occur. The crystal structures of laccase with lactic acid: choline chloride DES (LCDES) were obtained at 1.6 Å. Crystallographic analysis suggested that the addition of LCDES causes changes in the laccase active site, but the increase in water molecules observed in the resulting crystal prevented laccase from experiencing drastic structural change. Fluorescence and circular dichroism spectroscopies were also applied to determine the effects of DES on the structural conformation of laccase. The results have confirmed that the presence of DES can trigger changes in the local environments of the amino acids in the active site of laccase which contributes to the changes in its activity and stability.
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Affiliation(s)
- Jou Chin Chan
- Voiland School of Chemical Engineering and Bioengineering - Washington State University, 2710 Crimson Way, Richland, WA, 99354, USA
| | - Bixia Zhang
- Department of Chemistry, Washington State University, Pullman, WA, 99164, USA
| | - Michael Martinez
- Department of Chemistry, Washington State University, Pullman, WA, 99164, USA
| | - Balaganesh Kuruba
- Voiland School of Chemical Engineering and Bioengineering - Washington State University, 2710 Crimson Way, Richland, WA, 99354, USA
| | - James Brozik
- Department of Chemistry, Washington State University, Pullman, WA, 99164, USA
| | - ChulHee Kang
- Department of Chemistry, Washington State University, Pullman, WA, 99164, USA.
| | - Xiao Zhang
- Voiland School of Chemical Engineering and Bioengineering - Washington State University, 2710 Crimson Way, Richland, WA, 99354, USA; Pacific Northwest National Laboratory - 902 Battelle Boulevard, P.O. Box 999, MSIN P8-60, Richland, WA, 99352, USA.
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9
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Yang D, Wang T, Liu J, Wang H, Kang YJ. Reverse regulation of hepatic ceruloplasmin production in rat model of myocardial ischemia. J Trace Elem Med Biol 2021; 64:126686. [PMID: 33249375 DOI: 10.1016/j.jtemb.2020.126686] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/26/2020] [Revised: 10/09/2020] [Accepted: 11/12/2020] [Indexed: 02/05/2023]
Abstract
BACKGROUND Ceruloplasmin (Cp) is a major copper-binding protein produced in the liver and delivers copper to extrahepatic organs. Patients with myocardial infarction are often featured by an elevation of serum copper concentrations due to copper efflux from ischemic hearts. The present study was undertaken to test the hypothesis that serum copper elevation leads to up-regulation of hepatic Cp in myocardial infarction. METHODS Adult male Sprague-Dawley rats were subjected to left anterior descending (LAD) coronary artery ligation to induce myocardial infarction. Serum copper and Cp levels, as well as changes in hepatic Cp and copper-transporting P-type ATPase (Atp7b), were determined from blood and liver samples collected on day 1, 4, or 7 after the operation. RESULTS Serum copper concentrations were significantly increased on day 4 after LAD ligation, accompanied by an increase in serum Cp levels and activities. Concomitantly, the protein levels of Cp and copper exporter, Atp7b, were also significantly increased in the liver. Furthermore, inhibiting the increase of serum copper by a copper chelator, triethylenetetramine (TETA), effectively abolished the elevated Cp activity after LAD ligation. CONCLUSION These results indicate that serum Cp elevation in response to myocardial ischemia most likely resulted from the increased hepatic Cp production, which in turn was more responsive to serum copper elevation than inflammatory response following myocardial ischemia.
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Affiliation(s)
- Dan Yang
- Regenerative Medicine Research Center, Sichuan University West China Hospital, Chengdu, Sichuan, 610041, China
| | - Tao Wang
- Regenerative Medicine Research Center, Sichuan University West China Hospital, Chengdu, Sichuan, 610041, China
| | - Jiaming Liu
- Regenerative Medicine Research Center, Sichuan University West China Hospital, Chengdu, Sichuan, 610041, China
| | - Haitao Wang
- Regenerative Medicine Research Center, Sichuan University West China Hospital, Chengdu, Sichuan, 610041, China
| | - Y James Kang
- Regenerative Medicine Research Center, Sichuan University West China Hospital, Chengdu, Sichuan, 610041, China.
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10
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Jones SM, Heppner DE, Vu K, Kosman DJ, Solomon EI. Rapid Decay of the Native Intermediate in the Metallooxidase Fet3p Enables Controlled Fe II Oxidation for Efficient Metabolism. J Am Chem Soc 2020; 142:10087-10101. [PMID: 32379440 DOI: 10.1021/jacs.0c02384] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
The multicopper oxidases (MCOs) couple four 1e- oxidations of substrate to the 4e- reduction of O2 to H2O. These divide into two groups: those that oxidize organic substrates with high turnover frequencies (TOFs) up to 560 s-1 and those that oxidize metal ions with low TOFs, ∼1 s-1 or less. The catalytic mechanism of the organic oxidases has been elucidated, and the high TOF is achieved through rapid intramolecular electron transfer (IET) to the native intermediate (NI), which only slowly decays to the resting form. Here, we uncover the factors that govern the low TOF in Fet3p, a prototypical metallooxidase, in the context of the MCO mechanism. We determine that the NI decays rapidly under optimal turnover conditions, and the mechanism thereby becomes rate-limited by slow IET to the resting enzyme. Development of a catalytic model leads to the important conclusions that proton delivery to the NI controls the mechanism and enables the slow turnover in Fet3p that is functionally significant in Fe metabolism enabling efficient ferroxidase activity while avoiding ROS generation.
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Affiliation(s)
- Stephen M Jones
- Department of Chemistry, Stanford University, 333 Campus Drive Stanford, California 94305, United States
| | - David E Heppner
- Department of Chemistry, Stanford University, 333 Campus Drive Stanford, California 94305, United States
| | - Kenny Vu
- Department of Biochemistry, The University at Buffalo, 140 Farber Hall, 3435 Main Street, Buffalo, New York 14214, United States
| | - Daniel J Kosman
- Department of Biochemistry, The University at Buffalo, 140 Farber Hall, 3435 Main Street, Buffalo, New York 14214, United States
| | - Edward I Solomon
- Department of Chemistry, Stanford University, 333 Campus Drive Stanford, California 94305, United States
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11
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ROMP polymer supported manganese porphyrins: Influence of C C bonds along polymer chains on catalytic behavior in oxidation of low concentration Fe2+. REACT FUNCT POLYM 2020. [DOI: 10.1016/j.reactfunctpolym.2020.104530] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/03/2023]
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12
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Sharma P, Reichert M, Lu Y, Markello TC, Adams DR, Steinbach PJ, Fuqua BK, Parisi X, Kaler SG, Vulpe CD, Anderson GJ, Gahl WA, Malicdan MCV. Biallelic HEPHL1 variants impair ferroxidase activity and cause an abnormal hair phenotype. PLoS Genet 2019; 15:e1008143. [PMID: 31125343 PMCID: PMC6534290 DOI: 10.1371/journal.pgen.1008143] [Citation(s) in RCA: 18] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/07/2018] [Accepted: 04/16/2019] [Indexed: 11/18/2022] Open
Abstract
Maintenance of the correct redox status of iron is functionally important for critical biological processes. Multicopper ferroxidases play an important role in oxidizing ferrous iron, released from the cells, into ferric iron, which is subsequently distributed by transferrin. Two well-characterized ferroxidases, ceruloplasmin (CP) and hephaestin (HEPH) facilitate this reaction in different tissues. Recently, a novel ferroxidase, Hephaestin like 1 (HEPHL1), also known as zyklopen, was identified. Here we report a child with compound heterozygous mutations in HEPHL1 (NM_001098672) who presented with abnormal hair (pili torti and trichorrhexis nodosa) and cognitive dysfunction. The maternal missense mutation affected mRNA splicing, leading to skipping of exon 5 and causing an in-frame deletion of 85 amino acids (c.809_1063del; p.Leu271_ala355del). The paternal mutation (c.3176T>C; p.Met1059Thr) changed a highly conserved methionine that is part of a typical type I copper binding site in HEPHL1. We demonstrated that HEPHL1 has ferroxidase activity and that the patient's two mutations exhibited loss of this ferroxidase activity. Consistent with these findings, the patient's fibroblasts accumulated intracellular iron and exhibited reduced activity of the copper-dependent enzyme, lysyl oxidase. These results suggest that the patient's biallelic variants are loss-of-function mutations. Hence, we generated a Hephl1 knockout mouse model that was viable and had curly whiskers, consistent with the hair phenotype in our patient. These results enhance our understanding of the function of HEPHL1 and implicate altered ferroxidase activity in hair growth and hair disorders.
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Affiliation(s)
- Prashant Sharma
- NIH Undiagnosed Diseases Program, Common Fund, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland, United States of America
- Office of the Clinical Director, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland, United States of America
| | - Marie Reichert
- NIH Undiagnosed Diseases Program, Common Fund, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland, United States of America
- Office of the Clinical Director, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland, United States of America
| | - Yan Lu
- Iron Metabolism Laboratory, QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia
| | - Thomas C. Markello
- NIH Undiagnosed Diseases Program, Common Fund, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland, United States of America
- Office of the Clinical Director, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland, United States of America
- Medical Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland Bethesda, Maryland, United States of America
| | - David R. Adams
- NIH Undiagnosed Diseases Program, Common Fund, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland, United States of America
- Office of the Clinical Director, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland, United States of America
| | - Peter J. Steinbach
- Center for Molecular Modeling, Center for Information Technology, National Institutes of Health, Bethesda, Maryland, United States of America
| | - Brie K. Fuqua
- Department of Medicine, University of California, Los Angeles, United States of America
| | - Xenia Parisi
- NIH Undiagnosed Diseases Program, Common Fund, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland, United States of America
- Office of the Clinical Director, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland, United States of America
| | - Stephen G. Kaler
- Section on Translational Neuroscience, Molecular Medicine Branch, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, United States of America
| | - Christopher D. Vulpe
- Center for Environmental and Human Toxicology, Department of Physiological Sciences, University of Florida, Gainesville, Florida, United States of America
| | - Gregory J. Anderson
- Iron Metabolism Laboratory, QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia
| | - William A. Gahl
- NIH Undiagnosed Diseases Program, Common Fund, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland, United States of America
- Office of the Clinical Director, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland, United States of America
- Medical Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland Bethesda, Maryland, United States of America
| | - May Christine V. Malicdan
- NIH Undiagnosed Diseases Program, Common Fund, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland, United States of America
- Office of the Clinical Director, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland, United States of America
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13
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Looking for a partner: ceruloplasmin in protein-protein interactions. Biometals 2019; 32:195-210. [PMID: 30895493 DOI: 10.1007/s10534-019-00189-1] [Citation(s) in RCA: 35] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/27/2018] [Accepted: 03/18/2019] [Indexed: 10/27/2022]
Abstract
Ceruloplasmin (CP) is a mammalian blood plasma ferroxidase. More than 95% of the copper found in plasma is carried by this protein, which is a member of the multicopper oxidase family. Proteins from this group are able to oxidize substrates through the transfer of four electrons to oxygen. The essential role of CP in iron metabolism in humans is particularly evident in the case of loss-of-function mutations in the CP gene resulting in a neurodegenerative syndrome known as aceruloplasminaemia. However, the functions of CP are not limited to the oxidation of ferrous iron to ferric iron, which allows loading of the ferric iron into transferrin and prevents the deleterious reactions of Fenton chemistry. In recent years, a number of novel CP functions have been reported, and many of these functions depend on the ability of CP to form stable complexes with a number of proteins.
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14
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Grillet L, Lan P, Li W, Mokkapati G, Schmidt W. IRON MAN is a ubiquitous family of peptides that control iron transport in plants. NATURE PLANTS 2018; 4:953-963. [PMID: 30323182 DOI: 10.1038/s41477-018-0266-y] [Citation(s) in RCA: 157] [Impact Index Per Article: 22.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/04/2015] [Accepted: 08/28/2018] [Indexed: 05/09/2023]
Abstract
Iron (Fe) is an essential mineral nutrient that severely affects the growth, yield and nutritional quality of plants if not supplied in sufficient quantities. Here, we report that a short C-terminal amino-acid sequence consensus motif (IRON MAN; IMA) conserved across numerous, highly diverse peptides in angiosperms is essential for Fe uptake in plants. Overexpression of the IMA sequence in Arabidopsis induced Fe uptake genes in roots, causing accumulation of Fe and manganese in all plant parts including seeds. Silencing of all eight IMA genes harboured in the Arabidopsis genome abolished Fe uptake and caused severe chlorosis; increasing the Fe supply or expressing IMA1 restored the wild-type phenotype. IMA1 is predominantly expressed in the phloem, preferentially in leaves, and reciprocal grafting showed that IMA1 peptides in shoots positively regulate Fe uptake in roots. IMA homologues are highly responsive to the Fe status and functional when heterologously expressed across species. IMA constitutes a novel family of peptides that are critical for the acquisition and cellular homeostasis of Fe across land plants.
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Affiliation(s)
- Louis Grillet
- Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan
| | - Ping Lan
- Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan
- State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, Nanjing, China
| | - Wenfeng Li
- Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan
- Collaborative Innovation Center of Sustainable Forestry in Southern China of Jiangsu Province, Nanjing Forestry University, Nanjing, China
| | - Girish Mokkapati
- Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan
- Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, Taiwan
- Molecular Biology and Agricultural Sciences Program, Taiwan International Graduate program, Academia Sinica and National ChungHsing University, Taipei, Taiwan
| | - Wolfgang Schmidt
- Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan.
- Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, Taiwan.
- Genome and Systems Biology Degree Program, College of Life Science, National Taiwan University, Taipei, Taiwan.
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15
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Das S, Sahoo PK. Ceruloplasmin, a moonlighting protein in fish. FISH & SHELLFISH IMMUNOLOGY 2018; 82:460-468. [PMID: 30144565 DOI: 10.1016/j.fsi.2018.08.043] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/19/2018] [Revised: 08/15/2018] [Accepted: 08/21/2018] [Indexed: 06/08/2023]
Abstract
Ceruloplasmin is an ancient multicopper oxidase evolved to insure a safe handling of oxygen in some metabolic pathways of vertebrates. The current knowledge of its structure provides a glimpse of its plasticity, revealing a multitude of binding sites that point to an elaborate mechanism of multifunctional activity. Ceruloplasmin is highly conserved throughout the vertebrate evolution. Cupredoxin, a multi-cupper blue protein is believed to be the evolutionary precursor of ceruloplasmin with three trinuclear and three mononuclear copper binding sites. There are 20 copper-binding residues in ceruloplasmin gene out of which 16 residues are conserved in fish. This ceruloplasmin gene is being characterized in zebrafish (Danio rerio), rohu (Labeo rohita), Indian medaka (Oryzias melastigama), catfish (Ictalurus punctatus), icefish (Chionodraco rastrospinosus), goldfish (Carassius auratus) and yellow perch (Perca flaviscens). The complete coding sequence of fish ceruloplasmin gene is around 3.2 kb which codes for 1000 to 1100 amino acid residues. The size of ceruloplasmin gene sequence in fish ranges around 13 kb containing 20 exons and 19 introns. Liver is the major site of synthesis in fish. Increased expression of this gene during bacterial infection in channel catfish and rohu suggested its potential involvement in bacterial disease response in fish. It has been found to serve as an indirect marker for selection against Aeromonas hydrophila resistance in rohu carp. Ceruloplasmin expression is also evident during parasitic infection in few fish species. The role of this gene is well studied during inflammatory response to hormonal, drug and heavy metal mediated toxicity in fish. Overall, ceruloplasmin represents an example of a 'moonlighting' protein that overcomes the one gene-one structure-one function concept to follow the changes of the organism in its physiological and pathological conditions.
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Affiliation(s)
- Sweta Das
- Peninsular and Marine Fish Genetic Resources Centre, ICAR-National Bureau of Fish Genetic Resources, CMFRI Campus, Kochi 682 018, India
| | - Pramoda Kumar Sahoo
- ICAR-Central Institute of Freshwater Aquaculture, Kausalyaganga, Bhubaneswar 751 002, India.
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16
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Oxidation of cysteine by ceruloplasmin leads to formation of hydrogen peroxide, which can be utilized by myeloperoxidase. Biochem Biophys Res Commun 2018; 503:2146-2151. [DOI: 10.1016/j.bbrc.2018.08.003] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/30/2018] [Accepted: 08/01/2018] [Indexed: 01/08/2023]
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17
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Tao L, Stich TA, Soldatova AV, Tebo BM, Spiro TG, Casey WH, Britt RD. Mn(III) species formed by the multi-copper oxidase MnxG investigated by electron paramagnetic resonance spectroscopy. J Biol Inorg Chem 2018; 23:1093-1104. [PMID: 29968177 DOI: 10.1007/s00775-018-1587-z] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2018] [Accepted: 06/22/2018] [Indexed: 01/24/2023]
Abstract
The multi-copper oxidase (MCO) MnxG from marine Bacillus bacteria plays an essential role in geochemical cycling of manganese by oxidizing Mn2+(aq) to form manganese oxide minerals at rates that are three to five orders of magnitude faster than abiotic rates. The MCO MnxG protein is isolated as part of a multi-protein complex, denoted as Mnx, which includes one MnxG unit and a hexamer of MnxE3F3 subunit. During the oxidation of Mn2+(aq) catalyzed by the Mnx protein complex, an enzyme-bound Mn(III) species was trapped recently in the presence of pyrophosphate (PP) and analyzed using parallel-mode electron paramagnetic resonance (EPR) spectroscopy. Herein, we provide a full analysis of this enzyme-bound Mn(III) intermediate via temperature dependence studies and spectral simulations. This Mnx-bound Mn(III) species is characterized by a hyperfine-coupling value of A(55Mn) = 4.2 mT (corresponding to 120 MHz) and a negative zero-field splitting (ZFS) value of D = - 2.0 cm-1. These magnetic properties suggest that the Mnx-bound Mn(III) species could be either six-coordinate with a 5B1g ground state or square-pyramidal five-coordinate with a 5B1 ground state. In addition, as a control, Mn(III)PP is also analyzed by parallel-mode EPR spectroscopy. It exhibits distinctly different magnetic properties with a hyperfine-coupling value of A(55Mn) = 4.8 mT (corresponding to 140 MHz) and a negative ZFS value of D = - 2.5 cm-1. The different ZFS values suggest differences in ligand environment of Mnx-bound Mn(III) and aqueous Mn(III)PP species. These studies provide further insights into the mechanism of biological Mn2+(aq) oxidation.
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Affiliation(s)
- Lizhi Tao
- Department of Chemistry, University of California, One Shields Avenue, Davis, CA, 95616, USA
| | - Troy A Stich
- Department of Chemistry, University of California, One Shields Avenue, Davis, CA, 95616, USA
| | - Alexandra V Soldatova
- Department of Chemistry, University of Washington, Box 351700, Seattle, WA, 98195, USA
| | - Bradley M Tebo
- Division of Environmental and Biomolecular Systems, Oregon Health and Science University, Portland, OR, 97239, USA
| | - Thomas G Spiro
- Department of Chemistry, University of Washington, Box 351700, Seattle, WA, 98195, USA
| | - William H Casey
- Department of Chemistry, University of California, One Shields Avenue, Davis, CA, 95616, USA
- Department of Geology, University of California, One Shields Avenue, Davis, CA, 95616, USA
| | - R David Britt
- Department of Chemistry, University of California, One Shields Avenue, Davis, CA, 95616, USA.
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18
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Samygina VR, Sokolov AV, Bourenkov G, Schneider TR, Anashkin VA, Kozlov SO, Kolmakov NN, Vasilyev VB. Rat ceruloplasmin: a new labile copper binding site and zinc/copper mosaic. Metallomics 2017; 9:1828-1838. [PMID: 29177316 DOI: 10.1039/c7mt00157f] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
Abstract
Ceruloplasmin (Cp) is a copper-containing multifunctional oxidase of plasma, an antioxidant, an acute-phase protein and a free radical scavenger. The structural organization of Cp causes its sensitivity to proteolysis and ROS (reactive oxygen species), which can alter some of the important Cp functions. Elucidation of the orthorhombic crystal structure of rat Cp at 2.3 Å resolution revealed the basis for stronger resistance of rat Cp to proteolysis and a new labile copper binding site. The presence of this site appears as a very rare and distinctive feature of rat Cp as was shown by sequence alignment of ceruloplasmin, hephaestin and zyklopen in the Deuterostomia taxonomic group. The trigonal crystal form of rat Cp at 3.2 Å demonstrates unexpected partial substitution of copper by zinc.
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Affiliation(s)
- V R Samygina
- Shubnikov Institute of Crystallography of FSRC "Crystallography and Photonics" RAS, Leninsky pr.59, Moscow 117333, Russia. and NRC Kurchatov Institute, Kurchatov pl. 1, Moscow 123098, Russia
| | - A V Sokolov
- Institute of Experimental Medicine, ul. Academica Pavlova, 12, Saint-Petersburg 197376, Russia and Saint-Petersburg State Universisty, Universitetskaya nab. 7-9, Saint-Petersburg 199034, Russia and Centre of Preclinical Translational Research, Almazov National Medical Research Centre, ul. Dolgoozernaya, 43, Saint-Petersburg 197371, Russia
| | - G Bourenkov
- EMBL, Notkestrasse 85, 22607 Hamburg, Germany
| | | | - V A Anashkin
- Shubnikov Institute of Crystallography of FSRC "Crystallography and Photonics" RAS, Leninsky pr.59, Moscow 117333, Russia. and Belozersky Institute of Physico-Chemical Biology and Department of Chemistry, Lomonosov Moscow State University, Moscow 119899, Russia
| | - S O Kozlov
- Institute of Experimental Medicine, ul. Academica Pavlova, 12, Saint-Petersburg 197376, Russia
| | - N N Kolmakov
- Institute of Experimental Medicine, ul. Academica Pavlova, 12, Saint-Petersburg 197376, Russia
| | - V B Vasilyev
- Institute of Experimental Medicine, ul. Academica Pavlova, 12, Saint-Petersburg 197376, Russia and Saint-Petersburg State Universisty, Universitetskaya nab. 7-9, Saint-Petersburg 199034, Russia
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19
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Deshpande CN, Xin V, Lu Y, Savage T, Anderson GJ, Jormakka M. Large scale expression and purification of secreted mouse hephaestin. PLoS One 2017; 12:e0184366. [PMID: 28880952 PMCID: PMC5589216 DOI: 10.1371/journal.pone.0184366] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/22/2017] [Accepted: 08/22/2017] [Indexed: 01/04/2023] Open
Abstract
Hephaestin is a large membrane-anchored multicopper ferroxidase involved in mammalian iron metabolism. Newly absorbed dietary iron is exported across the enterocyte basolateral membrane by the ferrous iron transporter ferroportin, but hephaestin increases the efficiency of this process by oxidizing the transported iron to its ferric form and promoting its release from ferroportin. Deletion or mutation of the hephaestin gene leads to systemic anemia with iron accumulation in the intestinal epithelium. The crystal structure of human ceruloplasmin, another multicopper ferroxidase with 50% sequence identity to hephaestin, has provided a framework for comparative analysis and modelling. However, detailed structural information for hephaestin is still absent, leaving questions relating to metal coordination and binding sites unanswered. To obtain structural information for hephaestin, a reliable protocol for large-scale purification is required. Here, we present an expression and purification protocol of soluble mouse hephaestin, yielding milligram amounts of enzymatically active, purified protein using the baculovirus/insect cell system.
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Affiliation(s)
- Chandrika N. Deshpande
- Structural Biology Program, Centenary Institute, Sydney, New South Wales, Australia
- Sydney Medical School, University of Sydney, Sydney, New South Wales, Australia
| | - Vicky Xin
- Structural Biology Program, Centenary Institute, Sydney, New South Wales, Australia
- Sydney Medical School, University of Sydney, Sydney, New South Wales, Australia
| | - Yan Lu
- Iron Metabolism Laboratory, QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia
| | - Tom Savage
- School of Geosciences, University of Sydney, Sydney, New South Wales, Australia
| | - Gregory J. Anderson
- Iron Metabolism Laboratory, QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia
- School of Chemistry and Molecular Bioscience, University of Queensland, Brisbane, Queensland, Australia
| | - Mika Jormakka
- Structural Biology Program, Centenary Institute, Sydney, New South Wales, Australia
- Sydney Medical School, University of Sydney, Sydney, New South Wales, Australia
- * E-mail:
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20
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Soldatova AV, Tao L, Romano CA, Stich TA, Casey WH, Britt RD, Tebo BM, Spiro TG. Mn(II) Oxidation by the Multicopper Oxidase Complex Mnx: A Binuclear Activation Mechanism. J Am Chem Soc 2017; 139:11369-11380. [PMID: 28712284 DOI: 10.1021/jacs.7b02771] [Citation(s) in RCA: 25] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
The bacterial protein complex Mnx contains a multicopper oxidase (MCO) MnxG that, unusually, catalyzes the two-electron oxidation of Mn(II) to MnO2 biomineral, via a Mn(III) intermediate. Although Mn(III)/Mn(II) and Mn(IV)/Mn(III) reduction potentials are expected to be high, we find a low reduction potential, 0.38 V (vs Normal Hydrogen Electrode, pH 7.8), for the MnxG type 1 Cu2+, the electron acceptor. Indeed the type 1 Cu2+ is not reduced by Mn(II) in the absence of molecular oxygen, indicating that substrate oxidation requires an activation step. We have investigated the enzyme mechanism via electronic absorption spectroscopy, using chemometric analysis to separate enzyme-catalyzed MnO2 formation from MnO2 nanoparticle aging. The nanoparticle aging time course is characteristic of nucleation and particle growth; rates for these processes followed expected dependencies on Mn(II) concentration and temperature, but exhibited different pH optima. The enzymatic time course is sigmoidal, signaling an activation step, prior to turnover. The Mn(II) concentration and pH dependence of a preceding lag phase indicates weak Mn(II) binding. The activation step is enabled by a pKa > 8.6 deprotonation, which is assigned to Mn(II)-bound H2O; it induces a conformation change (consistent with a high activation energy, 106 kJ/mol) that increases Mn(II) affinity. Mnx activation is proposed to decrease the Mn(III/II) reduction potential below that of type 1 Cu(II/I) by formation of a hydroxide-bridged binuclear complex, Mn(II)(μ-OH)Mn(II), at the substrate site. Turnover is found to depend cooperatively on two Mn(II) and is enabled by a pKa 7.6 double deprotonation. It is proposed that turnover produces a Mn(III)(μ-OH)2Mn(III) intermediate that proceeds to the enzyme product, likely Mn(IV)(μ-O)2Mn(IV) or an oligomer, which subsequently nucleates MnO2 nanoparticles. We conclude that Mnx exploits manganese polynuclear chemistry in order to facilitate an otherwise difficult oxidation reaction, as well as biomineralization. The mechanism of the Mn(III/IV) conversion step is elucidated in an accompanying paper .
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Affiliation(s)
- Alexandra V Soldatova
- Department of Chemistry, University of Washington , Box 351700, Seattle, Washington 98195, United States
| | | | - Christine A Romano
- Division of Environmental and Biomolecular Systems, Institute of Environmental Health, Oregon Health & Science University , Portland, Oregon 97239, United States
| | | | | | | | - Bradley M Tebo
- Division of Environmental and Biomolecular Systems, Institute of Environmental Health, Oregon Health & Science University , Portland, Oregon 97239, United States
| | - Thomas G Spiro
- Department of Chemistry, University of Washington , Box 351700, Seattle, Washington 98195, United States
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21
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Tao L, Stich TA, Liou SH, Soldatova AV, Delgadillo DA, Romano CA, Spiro TG, Goodin DB, Tebo BM, Casey WH, Britt RD. Copper Binding Sites in the Manganese-Oxidizing Mnx Protein Complex Investigated by Electron Paramagnetic Resonance Spectroscopy. J Am Chem Soc 2017; 139:8868-8877. [DOI: 10.1021/jacs.7b02277] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/19/2022]
Affiliation(s)
| | | | | | - Alexandra V. Soldatova
- Department
of Chemistry, University of Washington, Box 351700, Seattle, Washington 98195, United States
| | - David A. Delgadillo
- Department of Chemistry & Chemical Biology, University of California, 5200 North Lake Road, Merced, California 95343, United States
| | - Christine A. Romano
- Division of Environmental and Biomolecular Systems, Institute of Environmental Health, Oregon Health & Science University, Portland, Oregon 97239, United States
| | - Thomas G. Spiro
- Department
of Chemistry, University of Washington, Box 351700, Seattle, Washington 98195, United States
| | | | - Bradley M. Tebo
- Division of Environmental and Biomolecular Systems, Institute of Environmental Health, Oregon Health & Science University, Portland, Oregon 97239, United States
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22
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Liu W, Worms IAM, Herlin-Boime N, Truffier-Boutry D, Michaud-Soret I, Mintz E, Vidaud C, Rollin-Genetet F. Interaction of silver nanoparticles with metallothionein and ceruloplasmin: impact on metal substitution by Ag(i), corona formation and enzymatic activity. NANOSCALE 2017; 9:6581-6594. [PMID: 28474724 DOI: 10.1039/c7nr01075c] [Citation(s) in RCA: 25] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/23/2023]
Abstract
The release of Ag(i) from silver nanoparticles (AgNPs) unintentionally spread in the environment is suspected to impair some key biological functions. In comparison with AgNO3, in-depth investigations were carried out into the interactions between citrate-coated AgNPs (20 nm) and two metalloproteins, intracellular metallothionein 1 (MT1) and plasmatic ceruloplasmin (Cp), both involved in metal homeostasis. These were chosen for their physiological relevance and the diversity of their various native metals bound because of thiol groups and/or their structural differences. Transmission electron microscopy (TEM), and dynamic light scattering (DLS), UV-vis and circular dichroism (CD) spectroscopies were used to study the effects of such intricate interactions on AgNP dissolution and proteins in terms of metal exchanges and structural modifications. The isolation of the different populations formed together with on-line quantifications of their metal content were performed by asymmetrical flow field-flow fractionation (AF4) linked to inductively coupled plasma mass spectrometry (ICP-MS). For the 2 proteins, Ag(i) dissolved from the AgNPs, substituted for the native metal, to different extents and with different types of dynamics for the corona formed: the MT1 rapidly surrounded the AgNPs with the transient reticulate corona thus promoting their dissolution associated with the metal substitution, whereas the Cp established a more stable layer around the AgNPs, with a limited substitution of Cu and a decrease in its ferroxidase activity. The accessibility and lability of the metal binding sites inside these proteins and their relative affinities for Ag(i) are discussed, taking into account the structural characteristics of the proteins.
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Affiliation(s)
- Wei Liu
- CEA, DRF-BIAM, Site de Marcoule, F-30207 Bagnols-sur-Cèze, France.
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23
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Cortes L, Roberts BR, Wedd AG, Xiao Z. Molecular Aspects of a Robust Assay for Ferroxidase Function of Ceruloplasmin. Inorg Chem 2017; 56:5275-5284. [PMID: 28414228 DOI: 10.1021/acs.inorgchem.7b00372] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
Ceruloplasmin (Cp) is one of the most complex multicopper oxidase enzymes and plays an essential role in the metabolism of iron in mammals. Ferrous ion supplied by the ferroportin exporter is converted by Cp to ferric ion that is accepted by plasma metallo-chaperone transferrin. Study of the enzyme at the atomic and molecular level has been hampered by the lack of a suitable ferrous substrate. We have developed the classic chromophoric complex FeIIHx(Tar)2 (H2Tar, 4-(2-thiazolylazo)resorcinol; x = 0-2; overall charge omitted) as a robust substrate for evaluation of the ferroxidase function of Cp and related enzymes. The catalysis can be followed conveniently in real time by monitoring the solution absorbance at 720 nm, a fingerprint of FeIIHx(Tar)2. The complex is oxidized to its ferric form FeIIIHx(Tar)2 via the overall reaction sequence FeIIHx(Tar)2 → FeII-Cp → FeIII-Cp → FeIIIHx(Tar)2: i.e., Fe(II) is transferred formally from FeIIHx(Tar)2 to the substrate docking/oxidation (SDO) site(s) in Cp, followed by oxidation to product Fe(III) that is trapped again by the ligand. Each Tar ligand in the above bis-complex coordinates the metal center in a meridional tridentate mode involving a pH-sensitive -OH group (pKa > 12), and this imposes rapid Fe(II) and Fe(III) transfer kinetics to facilitate the catalytic process. The formation constants of both the ferrous and ferric complexes at pH 7.0 were determined (log β2' = 13.6 and 21.6, respectively), as well as an average dissociation constant of the SDO site(s) in Cp (log KD' = -7.2).
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Affiliation(s)
- Laura Cortes
- School of Chemistry and Bio21 Molecular Science and Biotechnology Institute, University of Melbourne , Parkville, Victoria 3010, Australia.,The Florey Institute of Neuroscience and Mental Health, The University of Melbourne , Parkville, Victoria 3010, Australia
| | - Blaine R Roberts
- The Florey Institute of Neuroscience and Mental Health, The University of Melbourne , Parkville, Victoria 3010, Australia
| | - Anthony G Wedd
- School of Chemistry and Bio21 Molecular Science and Biotechnology Institute, University of Melbourne , Parkville, Victoria 3010, Australia
| | - Zhiguang Xiao
- School of Chemistry and Bio21 Molecular Science and Biotechnology Institute, University of Melbourne , Parkville, Victoria 3010, Australia.,The Florey Institute of Neuroscience and Mental Health, The University of Melbourne , Parkville, Victoria 3010, Australia
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24
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Ion channelopathies and migraine pathogenesis. Mol Genet Genomics 2017; 292:729-739. [DOI: 10.1007/s00438-017-1317-1] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/12/2015] [Accepted: 03/30/2017] [Indexed: 10/19/2022]
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25
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Di Bella LM, Alampi R, Biundo F, Toscano G, Felice MR. Copper chelation and interleukin-6 proinflammatory cytokine effects on expression of different proteins involved in iron metabolism in HepG2 cell line. BMC BIOCHEMISTRY 2017; 18:1. [PMID: 28118841 PMCID: PMC5259844 DOI: 10.1186/s12858-017-0076-2] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 08/25/2016] [Accepted: 01/09/2017] [Indexed: 12/21/2022]
Abstract
Background In vertebrates, there is an intimate relationship between copper and iron homeostasis. Copper deficiency, which leads to a defect in ceruloplasmin enzymatic activity, has a strong effect on iron homeostasis resulting in cellular iron retention. Much is known about the mechanisms underlying cellular iron retention under “normal” conditions, however, less is known about the effect of copper deficiency during inflammation. Results We show that copper deficiency and the inflammatory cytokine interleukin-6 have different effects on the expression of proteins involved in iron and copper metabolism such as the soluble and glycosylphosphtidylinositol anchored forms of ceruloplasmin, hepcidin, ferroportin1, transferrin receptor1, divalent metal transporter1 and H-ferritin subunit. We demonstrate, using the human HepG2 cell line, that in addition to ceruloplasmin isoforms, copper deficiency affects other proteins, some posttranslationally and some at the transcriptional level. The addition of interleukin-6, moreover, has different effects on expression of ferroportin1 and ceruloplasmin, in which ferroportin1 is decreased while ceruloplasmin is increased. These effects are stronger when a copper chelating agent and IL-6 are used simultaneously. Conclusions These results suggest that copper chelation has effects not only on ceruloplasmin but also on other proteins involved in iron metabolism, sometimes at the mRNA level and, in inflammatory conditions, the functions of ferroportin and ceruloplasmin may be independent.
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Affiliation(s)
- Luca Marco Di Bella
- Department of Chemical, Biological, Pharmaceutical, and Environmental Sciences, University of Messina, Viale F. Stagno D'Alcontres, 31, 98166, Messina, Italy.,Inter University National Group of Marine Sciences (CoNISMa), Piazzale Flaminio, 9, 00196, Rome, Italy
| | - Roberto Alampi
- Department of Chemical, Biological, Pharmaceutical, and Environmental Sciences, University of Messina, Viale F. Stagno D'Alcontres, 31, 98166, Messina, Italy
| | - Flavia Biundo
- Department of Chemical, Biological, Pharmaceutical, and Environmental Sciences, University of Messina, Viale F. Stagno D'Alcontres, 31, 98166, Messina, Italy
| | - Giovanni Toscano
- Department of Chemical, Biological, Pharmaceutical, and Environmental Sciences, University of Messina, Viale F. Stagno D'Alcontres, 31, 98166, Messina, Italy
| | - Maria Rosa Felice
- Department of Chemical, Biological, Pharmaceutical, and Environmental Sciences, University of Messina, Viale F. Stagno D'Alcontres, 31, 98166, Messina, Italy.
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Tao L, Stich TA, Butterfield CN, Romano CA, Spiro TG, Tebo BM, Casey WH, Britt RD. Mn(II) Binding and Subsequent Oxidation by the Multicopper Oxidase MnxG Investigated by Electron Paramagnetic Resonance Spectroscopy. J Am Chem Soc 2015; 137:10563-75. [DOI: 10.1021/jacs.5b04331] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Affiliation(s)
| | | | - Cristina N. Butterfield
- Division of Environmental and Biomolecular Systems, Institute of Environmental Health, Oregon Health & Science University, Portland, Oregon 97239, United States
| | - Christine A. Romano
- Division of Environmental and Biomolecular Systems, Institute of Environmental Health, Oregon Health & Science University, Portland, Oregon 97239, United States
| | - Thomas G. Spiro
- Department
of Chemistry, University of Washington, Box 351700, Seattle, Washington 98195, United States
| | - Bradley M. Tebo
- Division of Environmental and Biomolecular Systems, Institute of Environmental Health, Oregon Health & Science University, Portland, Oregon 97239, United States
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Kostevich VA, Sokolov AV, Grudinina NA, Zakharova ET, Samygina VR, Vasilyev VB. Interaction of macrophage migration inhibitory factor with ceruloplasmin: role of labile copper ions. Biometals 2015; 28:817-26. [DOI: 10.1007/s10534-015-9868-2] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/09/2015] [Accepted: 06/11/2015] [Indexed: 02/06/2023]
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Eid C, Hémadi M, Ha-Duong NT, El Hage Chahine JM. Iron uptake and transfer from ceruloplasmin to transferrin. Biochim Biophys Acta Gen Subj 2014; 1840:1771-81. [DOI: 10.1016/j.bbagen.2014.01.011] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/17/2013] [Revised: 12/19/2013] [Accepted: 01/03/2014] [Indexed: 01/03/2023]
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Musci G, Polticelli F, Bonaccorsi di Patti MC. Ceruloplasmin-ferroportin system of iron traffic in vertebrates. World J Biol Chem 2014; 5:204-215. [PMID: 24921009 PMCID: PMC4050113 DOI: 10.4331/wjbc.v5.i2.204] [Citation(s) in RCA: 46] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/13/2014] [Accepted: 02/19/2014] [Indexed: 02/05/2023] Open
Abstract
Safe trafficking of iron across the cell membrane is a delicate process that requires specific protein carriers. While many proteins involved in iron uptake by cells are known, only one cellular iron export protein has been identified in mammals: ferroportin (SLC40A1). Ceruloplasmin is a multicopper enzyme endowed with ferroxidase activity that is found as a soluble isoform in plasma or as a membrane-associated isoform in specific cell types. According to the currently accepted view, ferrous iron transported out of the cell by ferroportin would be safely oxidized by ceruloplasmin to facilitate loading on transferrin. Therefore, the ceruloplasmin-ferroportin system represents the main pathway for cellular iron egress and it is responsible for physiological regulation of cellular iron levels. The most recent findings regarding the structural and functional features of ceruloplasmin and ferroportin and their relationship will be described in this review.
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Multicopper oxidases: intramolecular electron transfer and O2 reduction. J Biol Inorg Chem 2014; 19:541-54. [DOI: 10.1007/s00775-013-1080-7] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/16/2013] [Accepted: 12/18/2013] [Indexed: 12/29/2022]
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Kataoka K, Sakurai T. Role of Hydrogen Bond Connecting Ligands for Substrate and Type I Copper in Copper(I) Oxidase CueO. CHEM LETT 2013. [DOI: 10.1246/cl.130422] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022]
Affiliation(s)
- Kunishige Kataoka
- Graduate School of Natural Science and Technology, Kanazawa University
| | - Takeshi Sakurai
- Graduate School of Natural Science and Technology, Kanazawa University
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Butterfield CN, Soldatova AV, Lee SW, Spiro TG, Tebo BM. Mn(II,III) oxidation and MnO2 mineralization by an expressed bacterial multicopper oxidase. Proc Natl Acad Sci U S A 2013; 110:11731-5. [PMID: 23818588 PMCID: PMC3718108 DOI: 10.1073/pnas.1303677110] [Citation(s) in RCA: 103] [Impact Index Per Article: 8.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
Reactive Mn(IV) oxide minerals are ubiquitous in the environment and control the bioavailability and distribution of many toxic and essential elements and organic compounds. Their formation is thought to be dependent on microbial enzymes, because spontaneous Mn(II) to Mn(IV) oxidation is slow. Several species of marine Bacillus spores oxidize Mn(II) on their exosporium, the outermost layer of the spore, encrusting them with Mn(IV) oxides. Molecular studies have identified the mnx (Mn oxidation) genes, including mnxG, encoding a putative multicopper oxidase (MCO), as responsible for this two-electron oxidation, a surprising finding because MCOs only catalyze single-electron transfer reactions. Characterization of the enzymatic mechanism has been hindered by the lack of purified protein. By purifying active protein from the mnxDEFG expression construct, we found that the resulting enzyme is a blue (absorption maximum 590 nm) complex containing MnxE, MnxF, and MnxG proteins. Further, by analyzing the Mn(II)- and (III)-oxidizing activity in the presence of a Mn(III) chelator, pyrophosphate, we found that the complex facilitates both electron transfers from Mn(II) to Mn(III) and from Mn(III) to Mn(IV). X-ray absorption spectroscopy of the Mn mineral product confirmed its similarity to Mn(IV) oxides generated by whole spores. Our results demonstrate that Mn oxidation from soluble Mn(II) to Mn(IV) oxides is a two-step reaction catalyzed by an MCO-containing complex. With the purification of active Mn oxidase, we will be able to uncover its mechanism, broadening our understanding of Mn mineral formation and the bioinorganic capabilities of MCOs.
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Affiliation(s)
- Cristina N. Butterfield
- Division of Environmental and Biomolecular Systems, Institute of Environmental Health, Oregon Health and Science University, Beaverton, OR 97006; and
| | | | - Sung-Woo Lee
- Division of Environmental and Biomolecular Systems, Institute of Environmental Health, Oregon Health and Science University, Beaverton, OR 97006; and
| | - Thomas G. Spiro
- Department of Chemistry, University of Washington, Seattle, WA 98195
| | - Bradley M. Tebo
- Division of Environmental and Biomolecular Systems, Institute of Environmental Health, Oregon Health and Science University, Beaverton, OR 97006; and
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Multi-copper oxidases and human iron metabolism. Nutrients 2013; 5:2289-313. [PMID: 23807651 PMCID: PMC3738974 DOI: 10.3390/nu5072289] [Citation(s) in RCA: 136] [Impact Index Per Article: 11.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/11/2013] [Revised: 05/29/2013] [Accepted: 06/06/2013] [Indexed: 01/13/2023] Open
Abstract
Multi-copper oxidases (MCOs) are a small group of enzymes that oxidize their substrate with the concomitant reduction of dioxygen to two water molecules. Generally, multi-copper oxidases are promiscuous with regards to their reducing substrates and are capable of performing various functions in different species. To date, three multi-copper oxidases have been detected in humans—ceruloplasmin, hephaestin and zyklopen. Each of these enzymes has a high specificity towards iron with the resulting ferroxidase activity being associated with ferroportin, the only known iron exporter protein in humans. Ferroportin exports iron as Fe2+, but transferrin, the major iron transporter protein of blood, can bind only Fe3+ effectively. Iron oxidation in enterocytes is mediated mainly by hephaestin thus allowing dietary iron to enter the bloodstream. Zyklopen is involved in iron efflux from placental trophoblasts during iron transfer from mother to fetus. Release of iron from the liver relies on ferroportin and the ferroxidase activity of ceruloplasmin which is found in blood in a soluble form. Ceruloplasmin, hephaestin and zyklopen show distinctive expression patterns and have unique mechanisms for regulating their expression. These features of human multi-copper ferroxidases can serve as a basis for the precise control of iron efflux in different tissues. In this manuscript, we review the biochemical and biological properties of the three human MCOs and discuss their potential roles in human iron homeostasis.
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Soldatova AV, Butterfield C, Oyerinde OF, Tebo BM, Spiro TG. Multicopper oxidase involvement in both Mn(II) and Mn(III) oxidation during bacterial formation of MnO(2). J Biol Inorg Chem 2012; 17:1151-8. [PMID: 22892957 PMCID: PMC3743667 DOI: 10.1007/s00775-012-0928-6] [Citation(s) in RCA: 46] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/25/2012] [Accepted: 07/18/2012] [Indexed: 12/19/2022]
Abstract
Global cycling of environmental manganese requires catalysis by bacteria and fungi for MnO(2) formation, since abiotic Mn(II) oxidation is slow under ambient conditions. Genetic evidence from several bacteria indicates that multicopper oxidases (MCOs) are required for MnO(2) formation. However, MCOs catalyze one-electron oxidations, whereas the conversion of Mn(II) to MnO(2) is a two-electron process. Trapping experiments with pyrophosphate (PP), a Mn(III) chelator, have demonstrated that Mn(III) is an intermediate in Mn(II) oxidation when mediated by exosporium from the Mn-oxidizing bacterium Bacillus SG-1. The reaction of Mn(II) depends on O(2) and is inhibited by azide, consistent with MCO catalysis. We show that the subsequent conversion of Mn(III) to MnO(2) also depends on O(2) and is inhibited by azide. Thus, both oxidation steps appear to be MCO-mediated, likely by the same enzyme, which is indicated by genetic evidence to be the MnxG gene product. We propose a model of how the manganese oxidase active site may be organized to couple successive electron transfers to the formation of polynuclear Mn(IV) complexes as precursors to MnO(2) formation.
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Affiliation(s)
- Alexandra V. Soldatova
- Department of Chemistry, University of Washington, Box 351700, Seattle, Washington 98195 USA
| | - Cristina Butterfield
- Division of Environmental and Biomolecular Systems, Oregon Health & Science University, 20000 NW Walker Road, Beaverton, Oregon 97006 USA
| | - Oyeyemi F. Oyerinde
- Department of Chemistry, University of Washington, Box 351700, Seattle, Washington 98195 USA
| | - Bradley M. Tebo
- Division of Environmental and Biomolecular Systems, Oregon Health & Science University, 20000 NW Walker Road, Beaverton, Oregon 97006 USA
| | - Thomas G. Spiro
- Department of Chemistry, University of Washington, Box 351700, Seattle, Washington 98195 USA
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Functional role of the putative iron ligands in the ferroxidase activity of recombinant human hephaestin. J Biol Inorg Chem 2012; 17:1187-95. [PMID: 22961397 DOI: 10.1007/s00775-012-0932-x] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/25/2012] [Accepted: 08/16/2012] [Indexed: 01/10/2023]
Abstract
Hephaestin is a multicopper ferroxidase expressed mainly in the mammalian small intestine. The ferroxidase activity of hephaestin is thought to play an important role during iron export from intestinal enterocytes and the subsequent iron loading of the blood protein transferrin, which delivers iron to the tissues. Structurally, the ectodomain of hephaestin is predicted to resemble ceruloplasmin, the soluble ferroxidase of blood. In this study, the human hephaestin ectodomain was expressed in baby hamster kidney cells and purified to electrophoretic homogeneity. Ion exchange chromatography of purified recombinant human hephaestin (rhHp) resulted in the isolation of hephaestin fractions with distinct catalytic and spectroscopic properties. The fraction of rhHp with the highest enzymatic activity also showed an enhanced molar absorptivity at 600 nm, characteristic of type 1 copper sites. Kinetic analysis revealed that rhHp possesses both high-affinity and low-affinity binding sites for ferrous iron. To investigate the role of particular residues in iron specificity of hephaestin, mutations of putative iron ligands were introduced into rhHp using site-directed mutagenesis. Kinetic analysis of ferroxidation rates of wild-type rhHp and mutants demonstrated the important roles of hephaestin residues E960 and H965 in the observed ferroxidase activity.
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Artyukhov VG, Basharina OV, Bragin MV, Sukhanov DY, Vashanov GA. Spectral and functional properties of ceruloplasmin under UV irradiation. Biophysics (Nagoya-shi) 2012. [DOI: 10.1134/s0006350912030037] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
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Ghiladi RA, Rheingold AL, Siegler MA, Karlin KD. Synthesis and Characterization of New Trinuclear Copper Complexes. Inorganica Chim Acta 2012; 389:131-137. [PMID: 22773847 DOI: 10.1016/j.ica.2012.02.020] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/28/2022]
Abstract
This report describes our approach towards modelling the copper cluster active sites of nitrous oxide reductase and the multicopper oxidases/oxygenases. We have synthesized two mesitylene-based trinucleating ligands, MesPY1 and MesPY2, which employ bis(2-picolyl)amine (PY1) and bis(2-pyridylethyl)amine (PY2) tridentate copper chelates, respectively. Addition of cuprous salts to these ligands leads to the isolation of tricopper(I) complexes [(Mes-PY1)Cu(I) (3)(CH(3)CN)(3)](ClO(4))(3)·0.25Et(2)O (1) and [(Mes-PY2)Cu(I) (3)](PF(6))(3) (3) Each of the three copper centers in 1 is most likely four-coordinate, with ligated acetonitrile as the fourth ligand; by contrast, the copper centers in 3 are three-coordinate, as determined by X-ray crystallography The synthesis of [(Mes-PY1)Cu(II) (3)(CH(3)CN)(2)(CH(3)OH)(2)](ClO(4))(6)·(CH(3)OH) (2) was accomplished by addition of three equivalents of the copper(II) salt, Cu(ClO(4))(2)·6H(2)O, to the ligand. The structure of 2 shows that two of the copper centers are tetracoordinate (with MeCN solvent ligation), but have additional weak axial (fifth ligand) interactions with the perchlorate anions; the third copper is unique in that it is coordinated by two MeOH solvent molecules, making it overall five-coordinate. For complexes 2 and 3, one copper ion center is located on the opposite side of the mesitylene plane as the other two. These observations, although in the solid state, must be taken into account for future studies where intramolecular tricopper(I)/O(2) (or other small molecules of interest) interactions in solution are desirable.
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Affiliation(s)
- Reza A Ghiladi
- Department of Chemistry, The Johns Hopkins University, Baltimore, Maryland 21218, USA
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Silva CS, Durão P, Fillat A, Lindley PF, Martins LO, Bento I. Crystal structure of the multicopper oxidase from the pathogenic bacterium Campylobacter jejuniCGUG11284: characterization of a metallo-oxidase. Metallomics 2012; 4:37-47. [DOI: 10.1039/c1mt00156f] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
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El Hage Chahine JM, Hémadi M, Ha-Duong NT. Uptake and release of metal ions by transferrin and interaction with receptor 1. Biochim Biophys Acta Gen Subj 2011; 1820:334-47. [PMID: 21872645 DOI: 10.1016/j.bbagen.2011.07.008] [Citation(s) in RCA: 61] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/29/2011] [Revised: 07/12/2011] [Accepted: 07/13/2011] [Indexed: 10/17/2022]
Abstract
BACKGROUND For a metal to follow the iron acquisition pathway, four conditions are required: 1-complex formation with transferrin; 2-interaction with receptor 1; 3-metal release in the endosome; and 4-metal transport to cytosol. SCOPE OF THE REVIEW This review deals with the mechanisms of aluminum(III), cobalt(III), uranium(VI), gallium(III) and bismuth(III) uptake by transferrin and interaction with receptor 1. MAJOR CONCLUSIONS The interaction of the metal-loaded transferrin with receptor 1 takes place in one or two steps: a very fast first step (μs to ms) between the C-lobe and the helical domain of the receptor, and a second slow step (2-6h) between the N-lobe and the protease-like domain. In transferrin loaded with metals other than iron, the dissociation constants for the interaction of the C-lobe with TFR are in a comparable range of magnitudes 10 to 0.5μM, whereas those of the interaction of the N-lobe are several orders of magnitudes lower or not detected. Endocytosis occurs in minutes, which implies a possible internalization of the metal-loaded transferrin with only the C-lobe interacting with the receptor. GENERAL SIGNIFICANCE A competition with iron is possible and implies that metal internalization is more related to kinetics than thermodynamics. As for metal release in the endosome, it is faster than the recycling time of transferrin, which implies its possible liberation in the cell. This article is part of a Special Issue entitled Transferrins: Molecular mechanisms of iron transport and disorders.
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Affiliation(s)
- Jean-Michel El Hage Chahine
- Université Paris Diderot Sorbonne Paris Cité–CNRS, Interfaces, Traitements, Organisation Dynamique des Systèmes–UMR 7086, Bâtiment Lavoisier, 15 rue Jean-Antoine de Baïf,75205 Paris Cedex 13, France.
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Kallio JP, Gasparetti C, Andberg M, Boer H, Koivula A, Kruus K, Rouvinen J, Hakulinen N. Crystal structure of an ascomycete fungal laccase from Thielavia arenaria - common structural features of asco-laccases. FEBS J 2011; 278:2283-95. [PMID: 21535408 DOI: 10.1111/j.1742-4658.2011.08146.x] [Citation(s) in RCA: 64] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Affiliation(s)
- Juha P Kallio
- Department of Chemistry, University of Eastern Finland, Joensuu, Finland
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Abstract
Multi-copper oxidases are a large family of enzymes prevalent in all three domains of life. They couple the one-electron oxidation of substrate to the four-electron reduction of dioxygen to water and feature at least four Cu atoms, traditionally divided into three sites: T1, T2, and (binuclear) T3. The T1 site catalyzes substrate oxidation while a trinuclear cluster (comprising combined T2 and T3 centres) catalyzes the reduction of dioxygen. Substrate oxidation at the T1 Cu site occurs via an outer-sphere mechanism and consequently substrate specificities are determined primarily by the nature of a substrate docking/oxidation (SDO) site associated with the T1 Cu centre. Many of these enzymes ‘moonlight’, i.e. display broad specificities towards many different substrates and may have multiple cellular functions. A sub-set are robust catalysts for the oxidation of low-valent transition metal ions such as FeII, CuI, and MnII and are termed ‘metallo-oxidases’. They play essential roles in nutrient metal uptake and homeostasis, with the ferroxidase ceruloplasmin being a prominent member. Their SDO sites are tailored to facilitate specific binding and facile oxidation of these low-valent metal ions and this is the focus of this review.
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Ha-Duong NT, Eid C, Hémadi M, El Hage Chahine JM. In Vitro Interaction between Ceruloplasmin and Human Serum Transferrin. Biochemistry 2010; 49:10261-3. [DOI: 10.1021/bi1014503] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Affiliation(s)
- Nguyêt-Thanh Ha-Duong
- Laboratoire ITODYS (Interfaces, Traitements et Organisation des Systèmes), Université Paris-Diderot, CNRS UMR 7086, Bâtiment Lavoisier, 15 rue Jean-Antoine de Baïf, 75205 Paris Cedex 13, France
| | - Chantal Eid
- Laboratoire ITODYS (Interfaces, Traitements et Organisation des Systèmes), Université Paris-Diderot, CNRS UMR 7086, Bâtiment Lavoisier, 15 rue Jean-Antoine de Baïf, 75205 Paris Cedex 13, France
| | - Miryana Hémadi
- Laboratoire ITODYS (Interfaces, Traitements et Organisation des Systèmes), Université Paris-Diderot, CNRS UMR 7086, Bâtiment Lavoisier, 15 rue Jean-Antoine de Baïf, 75205 Paris Cedex 13, France
| | - Jean-Michel El Hage Chahine
- Laboratoire ITODYS (Interfaces, Traitements et Organisation des Systèmes), Université Paris-Diderot, CNRS UMR 7086, Bâtiment Lavoisier, 15 rue Jean-Antoine de Baïf, 75205 Paris Cedex 13, France
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Analysis of the high-affinity iron uptake system at the Chlamydomonas reinhardtii plasma membrane. EUKARYOTIC CELL 2010; 9:815-26. [PMID: 20348389 DOI: 10.1128/ec.00310-09] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
Multicopper ferroxidases play a vital role in iron metabolism in bacteria, fungi, algae, and mammals. Saccharomyces cerevisiae utilizes a channeling mechanism to couple the ferroxidase activity of Fet3p to Fe(3+) transport into the cell by Ftr1p. In contrast, the mechanisms by which mammals couple the ferroxidase reaction to iron trafficking is unclear. The human ferroxidases ceruloplasmin and hephaestin are twice the size of Fet3p and interact with proteins that are not expressed in fungi. Chlamydomonas FOX1 is a homolog of the human ferroxidases but likely supports iron uptake in a manner similar to that of yeast, since Chlamydomonas reinhardtii expresses a ferric iron permease homolog, FTR1. The results presented support this hypothesis. We show that FOX1 is trafficked to the plasma membrane and is oriented with its multicopper oxidase/ferroxidase domain in the exocytoplasmic space. Our analysis of FTR1 indicates its topology is similar to that of S. cerevisiae Ftr1p, with a potential exocytoplasmic iron channeling motif and two potential iron permeation motifs in membrane-spanning regions. We demonstrate that high-affinity iron uptake is dependent on FOX1 and the copper status of the cell. Kinetic inhibition of high-affinity iron uptake by a ferric iron chelator does not reflect the strength of the chelator, supporting a ferric iron channeling mechanism for high-affinity iron uptake in Chlamydomonas. Last, recombinant FOX1 (rFOX1) has been isolated in a partially holo form that exhibits the UV-visible absorbance spectrum of a multicopper oxidase and the catalytic activity of a ferroxidase.
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Djoko KY, Chong LX, Wedd AG, Xiao Z. Reaction Mechanisms of the Multicopper Oxidase CueO from Escherichia coli Support Its Functional Role as a Cuprous Oxidase. J Am Chem Soc 2010; 132:2005-15. [DOI: 10.1021/ja9091903] [Citation(s) in RCA: 84] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Affiliation(s)
- Karrera Y. Djoko
- School of Chemistry and Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Victoria 3010, Australia
| | - Lee Xin Chong
- School of Chemistry and Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Victoria 3010, Australia
| | - Anthony G. Wedd
- School of Chemistry and Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Victoria 3010, Australia
| | - Zhiguang Xiao
- School of Chemistry and Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Victoria 3010, Australia
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Abstract
Microorganisms control the redox cycling of manganese in the natural environment. Although the homogeneous oxidation of Mn(II) to form manganese oxide minerals is slow, solid MnO(2) is the stable form of manganese in the oxygenated portion of the biosphere. Diverse bacteria and fungi have evolved the ability to catalyze this process, producing the manganese oxides found in soils and sediments. Other bacteria have evolved to utilize MnO(2) as a terminal electron acceptor in respiration. This Account summarizes the properties of Mn oxides produced by bacteria (bacteriogenic MnO(2)) and our current thinking about the biochemical mechanisms of bacterial Mn(II) oxidation. According to X-ray absorption spectroscopy and X-ray scattering studies, the MnO(2) produced by bacteria consists of stacked hexagonal sheets of MnO(6) octahedra, but these particles are extremely small and have numerous structural defects, particularly cation vacancies. The defects provide coordination sites for binding exogenous metal ions, which can be adsorbed to a high loading. As a result, bacterial production of MnO(2) influences the bioavailability of these metals in the natural environment. Because of its high surface area and oxidizing power, bacteriogenic MnO(2) efficiently degrades biologically recalcitrant organic molecules to lower-molecular-mass compounds, spurring interest in using these properties in the bioremediation of xenobiotic organic compounds. Finally, bacteriogenic MnO(2) is reduced to soluble Mn(II) rapidly in the presence of exogenous ligands or sunlight. It can therefore help to regulate the bioavailability of Mn(II), which is known to protect organisms from superoxide radicals and is required to assemble the water-splitting complex in photosynthetic organisms. Bioinorganic chemists and microbiologists have long been interested in the biochemical mechanism of Mn(IV) oxide production. The reaction requires a two-electron oxidation of Mn(II), but genetic and biochemical evidence for several bacteria implicate multicopper oxidases (MCOs), which are only known to engage one-electron transfers from substrate to O(2). In experiments with the exosporium of a Mn(II)-oxidizing Bacillus species, we could trap the one-electron oxidation product, Mn(III), as a pyrophosphate complex in an oxygen-dependent reaction inhibited by azide, consistent with MCO catalysis. The Mn(III) pyrophosphate complex can further act as a substrate, reacting in the presence of the exosporium to produce Mn(IV) oxide. Although this process appears to be unprecedented in biology, it is reminiscent of the oxidation of Fe(II) to form Fe(2)O(3) in the ferritin iron storage protein. However, it includes a critical additional step of Mn(III) oxidation or disproportionation. We shall continue to investigate this biochemically unique process with purified enzymes.
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Affiliation(s)
- Thomas G. Spiro
- Department of Chemistry, University of Washington, Seattle, Washington 98195
| | - John R. Bargar
- Stanford Synchrotron Radiation Lightsource, 2575 Sand Hill Road, Menlo Park, California 94025
| | - Garrison Sposito
- Department of Environmental Science, Policy and Management, University of California, Berkeley, California 94720-3114
| | - Bradley M. Tebo
- Division of Environmental and Biomolecular Systems, Oregon Health & Science University, 20000 NW Walker Road, Beaverton, Oregon 97006
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Kosman DJ. Multicopper oxidases: a workshop on copper coordination chemistry, electron transfer, and metallophysiology. J Biol Inorg Chem 2009; 15:15-28. [PMID: 19816718 DOI: 10.1007/s00775-009-0590-9] [Citation(s) in RCA: 128] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/29/2009] [Accepted: 09/15/2009] [Indexed: 01/01/2023]
Abstract
Multicopper oxidases (MCOs) are unique among copper proteins in that they contain at least one each of the three types of biologic copper sites, type 1, type 2, and the binuclear type 3. MCOs are descended from the family of small blue copper proteins (cupredoxins) that likely arose as a complement to the heme-iron-based cytochromes involved in electron transport; this event corresponded to the aerobiosis of the biosphere that resulted in the conversion of Fe(II) to Fe(III) as the predominant redox state of this essential metal and the solubilization of copper from Cu(2)S to Cu(H(2)O)( n ) (2+). MCOs are encoded in genomes in all three kingdoms and play essential roles in the physiology of essentially all aerobes. With four redox-active copper centers, MCOs share with terminal copper-heme oxidases the ability to catalyze the four-electron reduction of O(2) to two molecules of water. The electron transfers associated with this reaction are both outer and inner sphere in nature and their mechanisms have been fairly well established. A subset of MCO proteins exhibit specificity for Fe(2+), Cu(+), and/or Mn(2+) as reducing substrates and have been designated as metallooxidases. These enzymes, in particular the ferroxidases found in all fungi and metazoans, play critical roles in the metal metabolism of the expressing organism.
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Affiliation(s)
- Daniel J Kosman
- Department of Biochemistry, The University at Buffalo, NY 14214, USA.
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Kallio JP, Auer S, Jänis J, Andberg M, Kruus K, Rouvinen J, Koivula A, Hakulinen N. Structure-function studies of a Melanocarpus albomyces laccase suggest a pathway for oxidation of phenolic compounds. J Mol Biol 2009; 392:895-909. [PMID: 19563811 DOI: 10.1016/j.jmb.2009.06.053] [Citation(s) in RCA: 82] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/09/2009] [Revised: 06/11/2009] [Accepted: 06/14/2009] [Indexed: 10/20/2022]
Abstract
Melanocarpus albomyces laccase crystals were soaked with 2,6-dimethoxyphenol, a common laccase substrate. Three complex structures from different soaking times were solved. Crystal structures revealed the binding of the original substrate and adducts formed by enzymatic oxidation of the substrate. The dimeric oxidation products were identified by mass spectrometry. In the crystals, a 2,6-dimethoxy-p-benzoquinone and a C-O dimer were observed, whereas a C-C dimer was the main product identified by mass spectrometry. Crystal structures demonstrated that the substrate and/or its oxidation products were bound in the pocket formed by residues Ala191, Pro192, Glu235, Leu363, Phe371, Trp373, Phe427, Leu429, Trp507 and His508. Substrate and adducts were hydrogen-bonded to His508, one of the ligands of type 1 copper. Therefore, this surface-exposed histidine most likely has a role in electron transfer by laccases. Based on our mutagenesis studies, the carboxylic acid residue Glu235 at the bottom of the binding site pocket is also crucial in the oxidation of phenolics. Glu235 may be responsible for the abstraction of a proton from the OH group of the substrate and His508 may extract an electron. In addition, crystal structures revealed a secondary binding site formed through weak dimerization in M. albomyces laccase molecules. This binding site most likely exists only in crystals, when the Phe427 residues are packed against each other.
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Affiliation(s)
- J P Kallio
- Department of Chemistry, University of Joensuu, P.O. Box 111, FIN-80101 Joensuu, Finland
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Lawton TJ, Sayavedra-Soto LA, Arp DJ, Rosenzweig AC. Crystal structure of a two-domain multicopper oxidase: implications for the evolution of multicopper blue proteins. J Biol Chem 2009; 284:10174-80. [PMID: 19224923 PMCID: PMC2665071 DOI: 10.1074/jbc.m900179200] [Citation(s) in RCA: 57] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/09/2009] [Revised: 02/06/2009] [Indexed: 11/06/2022] Open
Abstract
The two-domain multicopper oxidases are proposed to be key intermediates in the evolution of three-domain multicopper oxidases. A number of two-domain multicopper oxidases have been identified from genome sequences and are classified as type A, type B, or type C on the basis of the predicted location of the type 1 copper center. The crystal structure of blue copper oxidase, a type C two-domain multicopper oxidase from Nitrosomonas europaea, has been determined to 1.9 A resolution. Blue copper oxidase is a trimer, of which each subunit comprises two cupredoxin domains. Each subunit houses a type 1 copper site in domain 1 and a type 2/type 3 trinuclear copper cluster at the subunit-subunit interface. The coordination geometry at the trinuclear copper site is consistent with reduction of the copper ions. Although the overall architecture of blue copper oxidase is similar to nitrite reductases, detailed structural alignments show that the fold and domain orientation more closely resemble the three-domain multicopper oxidases. These observations have important implications for the evolution of nitrite reductases and multicopper oxidases.
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Affiliation(s)
- Thomas J Lawton
- Departments of Biochemistry, Molecular Biology, and Cell Biology and of Chemistry, Northwestern University, Evanston, Illinois 60208, USA
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Terzulli AJ, Kosman DJ. The Fox1 ferroxidase of Chlamydomonas reinhardtii: a new multicopper oxidase structural paradigm. J Biol Inorg Chem 2009; 14:315-25. [PMID: 19023602 PMCID: PMC2675754 DOI: 10.1007/s00775-008-0450-z] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/19/2008] [Accepted: 11/06/2008] [Indexed: 12/19/2022]
Abstract
Multicopper oxidases (MCO) contain at least four copper atoms arrayed in three distinct ligand fields supported by two canonical structural features: (1) multiples of the cupredoxin fold and (2) four unique sequence elements that include the ten histidine and one cysteine ligands to the four copper atoms. Ferroxidases are a subfamily of MCO proteins that contain residues supporting a specific reactivity towards ferrous iron; these MCOs play a vital role in iron metabolism in bacteria, algae, fungi, and mammals. In contrast to the fungal ferroxidases, e.g., Fet3p from Saccharomyces cerevisiae, the mammalian ceruloplasmin (Cp) is twice as large (six vs. three cupredoxin domains) and contains three type 1, or "blue," copper sites. Chlamydomonas reinhardtii expresses a putative ferroxidase, Fox1, which has sequence similarity to human Cp (hCp). Eschewing the standard sequence-based modeling paradigm, we have constructed a function-based model of the Fox1 protein which replicates hCp's six copper-site ligand arrays with an overall root mean square deviation of 1.4 A. Analysis of this model has led also to assignment of motifs in Fox1 that are unique to ferroxidases, the strongest evidence to date that the well-characterized fungal high-affinity iron uptake system is essential to iron homeostasis in green algae. The model of Fox1 also establishes a subfamily of MCO proteins with a noncanonical copper-ligand organization. These diverse structures suggest alternative mechanisms for intramolecular electron transfer and require a new trajectory for the evolution of the MCO superfamily.
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Affiliation(s)
- Alaina J Terzulli
- Department of Biochemistry, School of Medicine and Biomedical Sciences, State University of New York at Buffalo, Buffalo, NY 14214, USA
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