Binding of O2 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 channel 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 to isoleucine caused a significant increase in the apparent K-M for oxygen with 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 corresponding change (V279I) has been made in cytochrome aa(3) from Paracoccus dentrificans. in this mutant, the apparent KM for oxygen is 8 times higher than in the wild-type enzyme, whereas V-max is decreased only to approximately half of the wild-type value. Flow-flash kinetic measurements show that the initial binding of oxygen to the heme of the binuclear site is indeed much slower in the mutant than in the wild-type enzyme. However, the subsequent phases 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 proton transfer to this site.