ABSTRACT
A spectroelectrochemical study is described of the sixteen hemes in the high-molecular-mass, monomeric cytochrome c (Hmc) from the periplasmic space of Desulfovibrio vulgaris, strain Hildenborough. One of the hemes has special properties. In the oxidized state at pH 7 it is predominantly high-spin, S = 5/2, with a g perpendicular value of less than 6 indicative of quantum-mechanical mixing with a low-lying (800 cm-1) S = 3/2 state; the balance is probably a low-spin derivative. The high-spin heme has an Em.7.5 value of +61 mV. The fifteen other hemes are low-spin bis-histidine coordinated with Em.7.5 values of approximately -0.20 V. Two of these hemes exhibit very anisotropic EPR spectra with a g1 value of 3.65 characteristic for strained bis-histidine coordination. A previous proposal, namely that methionine is coordinated to one of the hemes [Pollock, W.B.R., Loufti, M. Bruschi, M. Rapp-Giles, B.J., Wall, J. & Voordouw, G. (1991) J. Bacteriol. 173, 220] is disproved using spectroscopic evidence. Contrasting electrochemical data sets from two previous studies [Tan, J. & Cowan, J.A. (1990) Biochemistry 29, 4886; Bruschi, M., Bertrand, P., More, C., Leroy, G., Bonicel, J., Haladjian, J., Chottard, G., Pollock, W.B.R. & Voordouw, G. (1992) Biochemistry 31, 3281] are not consistent with our EPR titration results and are not reproducible. Hmc can be reduced by D. vulgaris Fe-hydrogenase in the presence of molecular hydrogen.
