Electron transfer kinetics of caa3 oxidase from Bacillus stearothermophilus: A hypothesis for thermophilicity

The O2 reaction and the reverse electron transfer of the thermophilic caa3 terminal oxidase of Bacillus stearothermophilus have been studied by laser flash-photolysis. The results show that both reactions, although studied at a temperature of 20°C, far from the optimal temperature of > 60°C for caa3, follow a kinetic behavior essentially identical to that observed with the electrostatic complex between mammalian cyt c and cyt c oxidase. In the O2 reaction cyt a and cyt a3 are very quickly oxidized; cyt a is then re-reduced via Cu(A), whereas cyt c oxidation is apparently rate- limited by the oxidation of Cu(A). Upon photodissociation of the mixed valence-CO caa3, reverse electron transfer from the binuclear center to cyt a3+ (?1 = 3 ?s) and Cu(A)/2+ (?2 = 64 ?s) is observed, while cyt c is not reduced by any detectable level. These results seem to rule out accounting for enzymatic thermophilicity by altered kinetics of intramolecular electron transfer involving the cyt center in the reduced configuration, which is very fast. On the basis of these results and previous data, we propose that thermophilicity involves an increased activation barrier for the reduction of cyt a3-Cu(B) in the configuration typical of the oxidized site.