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 cytoc hrome P450 enzymes. Two of the three histidines of human ferredoxin ar e strictly conserved in the sequences of all known vertebrate ferredox ins, and one of these (His(56)) is adjacent to Cys(55), which serves a s one of the ligands to the iron-sulfur cluster. All but 16 of its res idues show sequence identity with those of bovine ferredoxin. It has b een proposed for bovine ferredoxin that His(56) hydrogen bonds with a labile sulfur and that the reduction of the iron-sulfur center is acco mpanied by the uptake of a proton by this histidine [Lambeth, J. D., S eybert, D. W., Lancaster, J. R., Jr., Salemo, J. C., & Kamin, H. (1982 ) Mol. Cell. Biochem. 45, 13-31]. In this paper, we report procedures for labeling human ferredoxin uniformly with N-15 using (NH4Cl)-N-15 a nd selectively with C-13 by the incorporation of [U-C-13]histidine. Mo st of the imidazole H-1, C-13, and N-15 resonances of the three histid ines have been assigned by heteronuclear two-dimensional single- and m ultiple-bond correlation spectroscopy. Site-directed mutagenesis was u sed in assigning the NMR signals from His(56). The pK(a) values of His (10) (6.5) and His(62) (5.8) in oxidized human ferredoxin were found t o be similar to those reported previously for the corresponding residu es of bovine ferredoxin [Greenfield, N. J., Wu, X., & Jordan, F. (1989 ) Biochim. Biophys. Acta 995, 246-254; Miura, S., Tamita, S., & Ichika wa, Y. (1991) J. Biol. Chem. 266, 19212-19216]. The pK(a) value for Hi s(56) was found to be abnormal; the residue does not titrate between p H 6.0 and 8.6 in either the oxidized or the reduced state, and analysi s of the N-15 chemical shift indicates that its pK(a) value is <5. pH dependence (pH(mid) similar to 7.2) was observed for the reduction pot ential of the iron-sulfur cluster in this class of ferredoxins [Cooper , D. Y., Schleyer, H., Levin, S. S., & Rosenthal, O. (1973) Ann. N. Y. Acad. Sci. 212, 227-247]; the results show that such a pH dependence, if present in human ferredoxin, would not involve histidine residues, as proposed for bovine ferredoxin. The lack of a strong temperature d ependence for the H-1 NMR chemical shifts of any of the histidines rul es out a direct interaction between any of these residues and the iron -sulfur cluster.