Electron transfer kinetics of caa(3) oxidase from Bacillus stearothermophilus: A hypothesis for thermophilicity

The O-2 reaction and the reverse electron transfer of the thermophilic caa, terminal oxidase of Bacillus stearothermophilus have been studied by laser flash-photolysis. The results show that both reactions, although studied a t a temperature of 20 degrees C, far from the optimal temperature of > 60 d egrees C for caa(3), follow a kinetic behavior essentially identical to tha t observed with the electrostatic complex between mammalian cyt c and cyt c oxidase. In the O-2 reaction cyt a and cyt a(3) are very quickly oxidized; cyt a is then re-reduced via Cu,, whereas cyt c oxidation is apparently ra te-limited by the oxidation of Cu-A, Upon photodissociation of the mixed va lence-CO caa(3), reverse electron transfer from the binuclear center to cyt a(3+) (tau(1) = 3 mu s) and Cu-A(2+) (tau(2) = 64 mu 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 cent er in the reduced configuration, which is very fast. On the basis of these results and previous data, we propose that thermophilicity involves an incr eased activation barrier for the reduction of cyt a(3)-Cu-B in the configur ation typical of the oxidized site.