AXIAL COORDINATION AND REDUCTION POTENTIALS OF THE 16 HEMES IN HIGH-MOLECULAR-MASS CYTOCHROME-C FROM DESULFOVIBRIO-VULGARIS (HILDENBOROUGH)

A spectroelectrochemical study is described of the sixteen hemes in th e high-molecular-mass, monomeric cytochrome c (Hmc) from the periplasm ic space of Desulfovibrio vulgaris, strain Hildenborough, One of the h emes has special properties. In the oxidized state at pH 7 it is predo minantly high-spin, S = 5/2, with a g(perpendicular to) value of less than 6 indicative of quantum-mechanical mixing with a low-lying (800 c m(-1)) S = 3/2 state; the balance is probably a low-spin derivative. T he high-spin heme has an E(m,7.5) value of + 61 mV. The fifteen other hemes are low-spin bis-histidine coordinated with E(m,7.5) values of a pproximately -0.20 V. Two of these hemes exhibit very anisotropic EPR spectra with a g(1) value of 3.65 characteristic for strained bis-hist idine coordination. A previous proposal, namely that methionine is coo rdinated to one of the hemes [Pollock, W. B. R., Loufti, M. Bruschi, M . Rapp-Giles, B. J., Wall, J. and Voordouw, G. (1991) J. Bacteriol. 17 3, 220] is disproved using spectroscopic evidence. Contrasting electro chemical data sets from two previous studies [Tan, J. & Cowan, J. A. ( 1990) Biochemistry 29, 4886; Bruschi, M., Bertrand, P., More, C., Lero y, G., Bonicel, J., Haladjian, J., Chottard, G., Pollock, W. B. R. and Voordouw, G. (1992) Biochemistry 31, 3281] are not consistent with ou r EPR titration results and are not reproducible. Hmc can be reduced b y D. vulgaris Fe-hydrogenase in the presence of molecular hydrogen.