Characterisation of ionisations that influence the redox potential of mitochondrial cytochrome c and photosynthetic bacterial cytochromes c2

Abstract

Several cytochromes c 2 from the Rhodospirillaceae show a pH dependence of redox potential in the physiological pH range which can be described by equations involving an ionisation in the oxidised form (p K o) and one in the reduced form (p K r). These cytochromes fall into one of two groups according to the degree of separation of p K o and p K r. In group A, represented here by the Rhodomicrobium vannielii cytochrome c 2, the separation is approx. one pH unit and the ionisation is that of a haem propionic acid. Members of this group are unique among both cytochromes c 2 and mitochondrial cytochromes c in lacking the conserved residue Arg-38. We propose that the role of Arg-38 is to lower the p K of the nearby propionic acid, so that it lies out of the physiological pH range. Substitution of this residue by an uncharged amino acid leads to a raised p K for the propionic acid. In group B, represented here by Rhodopseudomonas viridis cytochrome c 2, the separation between p K o and p K r is approx. 0.4 pH unit and the ionisable group is a histidine at position 39. This was established by NMR spectroscopy and confirmed by chemical modification. Only a few other members of the cytochrome c 2/mitochondrial cytochrome c family have a histidine at this position and of these, both Crithidia cytochrome c-557 and yeast cytochrome c were found to have a pH-dependent redox potential similar to that of Rps. viridis cytochrome c 2. Using Coulomb's law, it was found that the energy required to separate p K o and p K r could be accounted for by simple electrostatic interactions between the haem iron and the ionisable group.