Structural and functional mechanisms of Desulfovibrio vulgaris cytochrome c3 revealed by site specific mutagenesis

The structural basis for the redox properties of the tetrahaem Desulfovibrio vulgaris cytochrome c3, has been investigated by site specific mutagenesis. Thermodynamic and structural characterisation of mutated cytochromes revealed the functional role of Lys45 and Thr24 in the wild type protein. Mutation of Lys45, located in the vicinity of the propionates of haem I, by the uncharged residues Thr and Gin, allowed to conclude that Lys45 controls the pKa of such propionate groups and confirms the involvement of this residue in the redox-Bohr effect ~. More recently, the replacement of Thr24, located in the vicinity ofhaem III, by Val showed how crucial Thr24 is for the stabilisation of the oxidised form of the protein. This single mutation increases the microscopic redox potential ofhaem III by 106 mV. This mutation also destabilizes the concerted two-electron step between the intermediate oxidation stages, 1 and 3, which can occur in the wild type protein. Therefore, Thr24 is responsible for the balance of the global network of cooperativities tuned to control thermodynamically the directionality of the stepwise electron transfer 2