INVOLVEMENT OF A LABILE AXIAL HISTIDINE IN COUPLING ELECTRON AND PROTON-TRANSFER IN METHYLOPHILUS-METHYLOTROPHUS CYTOCHROME-C''

Methylophilus methylotrophus cytochrome c" is an unusual monohaem protein (15 kDa) undergoing a redox-linked spin-state transition [Santos, H. & Turner, D. L. (1988) Biochim. Biophys. Acta 954, 277 - 286]. The midpoint redox potential of cytochrome c" was measured over the pH range 4 - 10. The pH dependence of the midpoint redox potential was interpreted in terms of a model that considers the redox-state dependence of the ionization of two distinct and non-interacting protonated groups in the protein. This analysis led to the following pK(a) values within the pH range studied: pK(a1)0 = 6.4, pK(a1)T = 5.4 and PK(a2)T = 8.1. Proton-NMR spectroscopy was used to assist the characterization of the two ionizing groups responsible for the observed redox-Bohr effect: the group ionizing with a lower pK(a)T was assigned to a haem propionic acid substituent and the other to the axial histidine ligand which becomes detached upon reduction, which has a pK(a)0 too low to be measured. It is shown that M. methylotrophus cytochrome c" is able to couple electron and proton transfer in the physiological pH range through a mechanism involving reversible change in the haem-iron coordination. Possible implications for the physiological role of the protein are discussed.