Involvement of a labile axial histidine in coupling electron and proton transfer in Methylophilus methylotrophus cytochrome c″

Abstract

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 pKa values within the pH range studied: pK0a1= 6.4, pKTa1= 5.4 and pKTa2= 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 pKTa was assigned to a haem propionic acid substituent and the other to the axial histidine ligand which becomes detached upon reduction, which has a pKOa 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 haemiron coordination. Possible implications for the physiological role of the protein are discussed. Copyright © 1992, Wiley Blackwell. All rights reserved