Flow-flash study of the reaction between cytochrome bo and oxygen

The reaction between reduced cytochrome bo from Escherichia coli and oxygen has been studied using flash photolysis of the CO complex of the reduced protein after rapid mixing with oxygen. Absorbance changes were monitored in the alpha and Soret spectral regions. Two kinetic phases taking place at catalytically competent rates could be detected. The apparent rate constant obtained for both the first and second phase showed a hyperbolic dependence on the oxygen concentration. For the first phase, we obtained limiting first- and second-order rate constants at saturating and low oxygen concentrations of 4.5 x 10(4) s-1 and 1.6 x 10(8) M-1 s-1, respectively. The corresponding values for the second phase were 5 x 10(3) s-1 and 1.7 x 10(7) M-1 s-1. The first phase accounted for 30% of the total absorbance change in the Soret band (430 nm) and 15% of the total absorbance change in the alpha band (555 nm). These reactions are followed by a very slow phase with a lifetime of about 1 s. We have also studied the interaction between the fully oxidized enzyme and hydrogen peroxide, and we have found that peroxide binding induces an absorbance increase in the alpha band and a red shift of the Soret band. A consideration of the magnitude of the absorbance changes taking place during the first phase suggests that this reaction includes at least partial oxidation of the low-spin cytochrome b.