Oxygen Kinetics of Frozen Cytochrome Oxidase

Cytochrome c oxidase (EC 1.9.3.1) is the site of 02 utilization in the inner mitochondrial membrane of all higher organisms. It contains two haems (a and a3) and two copper atoms. Reduced haem a3 is able to bind 02 and catalyse its reduction to water. Flash photolysis of CO-inhibited cytochrome oxidase in the presence of 02 at low temperatures (-95 to -1250C) leads to the formation of oxycytochrome oxidase in the initial phase of the reaction (Chance et al., 1975c). The equilibrium of 02 and the enzyme can be demonstrated (KD= 320,M at -940C), and a linear increase in the pseudo-firstorder rate constant for 02 binding is observed with increasing 02 concentration, indicating a bimolecular reaction at the active site of the enzyme, and suggesting the presence of an 02 pocket for a number of ligand molecules, which retains fluidity well below the macroscopic freezing point of the sample. The 02 concentration inside the pocket is proportional to that in the bulk of the solution (Chance, 1977). The kinetics of the reaction of CO with cytochrome oxidase in the frozen state are pseudo-firstorder (Sharrock & Yonetani, 1976, 1977). The C-O stretch band at 1963.5 cm-l for carbonmonoxycytochrome oxidase has a very narrow bandwidth (Yoshikawa et al., 1977). Both of these observations suggest the existence of a pocket for CO, and that the bound CO is isolated from the polar solvent and is in a hydrophobic environment. The present study further examines the effect of 02 on the kinetics of formation of oxycytochrome oxidase in order to estimate the capacity of this pocket in membrane-bound and isolated cytochrome oxidase.