Multinuclear magnetic resonance and mutagenesis studies of the histidine residues of human mitochondrial ferredoxin.
Human mitochondrial ferredoxin is a [2Fe-2S] protein that functions to transfer electrons from NADPH-dependent ferredoxin reductase to cytochrome P450 enzymes. Two of the three histidines of human ferredoxin are strictly conserved in the sequences of all known vertebrate ferredoxins, and one of these (His56) is adjacent to Cys55, which serves as one of the ligands to the iron-sulfur cluster. All but 16 of its residues show sequence identity with those of bovine ferredoxin. It has been proposed for bovine ferredoxin that His56 hydrogen bonds with a labile sulfur and that the reduction of the iron-sulfur center is accompanied by the uptake of a proton by this histidine [Lambeth, J. D., Seybert, D. W., Lancaster, J. R., Jr., Salerno, J. C., & Kamin, H. (1982) Mol. Cell. Biochem. 45, 13-31]. In this paper, we report procedures for labeling human ferredoxin uniformly with 15N using 15NH4Cl and selectively with 13C by the incorporation of [U-13C]histidine. Most of the imidazole 1H, 13C, and 15N resonances of the three histidines have been assigned by heteronuclear two-dimensional single- and multiple-bond correlation spectroscopy. Site-directed mutagenesis was used in assigning the NMR signals from His56. The pKa values of His10 (6.5) and His62 (5.8) in oxidized human ferredoxin were found to be similar to those reported previously for the corresponding residues of bovine ferredoxin [Greenfield, N. J., Wu, X., & Jordan, F. (1989) Biochim. Biophys. Acta 995, 246-254; Miura, S., Tamita, S., & Ichikawa, Y. (1991) J. Biol. Chem. 266, 19212-19216].(ABSTRACT TRUNCATED AT 250 WORDS)
Xia
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Graduate Biophysics Program, College of Agricultural and Life Sciences, University of Wisconsin, Madison 53706.
Cheng
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Skjeldal
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Coghlan
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Vickery
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Markley
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D000595:Amino Acid Sequence; D002247:Carbon Isotopes; D004926:Escherichia coli / Q000235:genetics; D005288:Ferredoxins / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry; D006801:Humans; D006859:Hydrogen; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D008928:Mitochondria / Q000737:chemistry*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009587:Nitrogen Isotopes; D010084:Oxidation-Reduction; D017386:Sequence Homology, Amino Acid; D013816:Thermodynamics
D002247:Carbon Isotopes; D005288:Ferredoxins; D009587:Nitrogen Isotopes; D006639:Histidine; D006859:Hydrogen