Intermediate forms of cytochrome oxidase observed in transient kinetic experiments and those visited in the catalytic cycle.

The cytochrome oxidase family of heme-copper oxidases has been the subject of intense kinetic and mechanistic enquiry. Much of this work has focussed on transient kinetic studies of the partial reactions of the enzyme with the goal being to build a kinetic model describing the catalytic cycle that the enzyme undergoes to direct the oxidation of substrate, reduction of oxygen and vectorial proton transfer. A key aspect of such a model is to define the structures of each of the intermediate forms the enzyme takes up as it traverses the catalytic cycle. One complication that has been prevalent with mitochondrial cytochrome c oxidase is the existence of structural variants of the enzyme, as isolated, that may not be participants in catalysis. Studies of structurally simpler procaryotic members of the family may offer new insight on the intermediates of catalysis. In this paper transient-state and steady-state kinetic studies of cytochrome aa(3)-600 from Bacillus subtilis are integrated into a model of the catalytic cycle. This model specifies that the P intermediate accumulates in the steady-state and it is proposed that the step following its formation is limited by proton uptake.