Binding of O-2 and its reduction are both retarded by replacement of valine 279 by isoleucine in cytochrome c oxidase from Paracoccus denitrificans

The crystal structure of the heme-copper oxidases suggested a putative chan nel of oxygen entry into the heme-copper site of O-2 reduction. Changing a conserved valine near this center in cytochrome bo(3) of Escherichia coli t o isoleucine caused a significant increase in the apparent K-M for oxygen w ith little or no change in V-max suggesting that oxygen diffusion had been partially blocked [Riistama, S., Puustinen, A., Garcia-Horsman, A., Iwata, S., Michel, H., and Wikstrom, M. (1996) Biochim. Biophys. Acta 1275, 1-4]. To study this phenotype further using rapid kinetic methods, the correspond ing change (V279I) has been made in cytochrome aa(3) from Paracoccus dentri ficans. in this mutant, the apparent KM for oxygen is 8 times higher than i n the wild-type enzyme, whereas V-max is decreased only to approximately ha lf of the wild-type value. Flow-flash kinetic measurements show that the in itial binding of oxygen to the heme of the binuclear site is indeed much sl ower in the mutant than in the wild-type enzyme. However, the subsequent ph ases of the reaction with O-2 are also slow although the pure heme-to-heme electron transfer process is essentially unperturbed. It is suggested that the mutation sterically hinders O-2 entry into the binuclear site and that it may also perturb the structure of local water molecules involved in prot on transfer to this site.