Mutations in the putative H-channel in the cytochrome c oxidase from Rhodobacter sphaeroides show that this channel is not important for proton conduction but reveal modulation of the properties of heme a

AS the final electron acceptor in the respiratory chain of eukaryotic and m any prokaryotic organisms, cytochrome c oxidase catalyzes the reduction of oxygen to water, concomitantly generating a proton gradient, X-ray structur es of two cytodlrome c oxidases have been reported, and in each structure t hree possible pathways for proton translocation are indicated: the D-, K-, and H-channels. The putative H-channel is most clearly delineated in the bo vine heart oxidase and has been proposed to be functionally important for t he translocation of pumped prc,tons in the mammalian oxidase [Yoshikawa et al. (1998) Science 280, 1723-1729]. In the present work, the functional imp ortance of residues lining the putative H-channel in the oxidase from Rhodo bacter sphaeroides are examined by site-directed mutagenesis. Mutants were generated in eight different sites and the enzymes have been purified and c haracterized. The results suggest that the I-I-channel is not functionally important in the prokaryotic oxidase, in agreement with the conclusion from previous work with the oxidase from Paracoccus denitrificans [Pfitzner et al. (1998) J, Biomembr. Bioenerg. 30, 89-93]. Each of the mutants in R. sph aeroides, with an exception at only one position, is enzymatically active a nd pumps protons in reconstituted proteoliposomes. This includes H456A, whe re in the P, dentrificans oxidase a leucine residue substituted for the cor responding residue resulted in inactive enzyme. The only mutations that res ult in completely inactive enzyme in the set examined in the R, sphaeroides oxidase are in R52, a residue that, along with Q471, appears to be hydroge n-bonded to the formyl group of heme a in the X-ray structures. To characte rize the interactions between this residue and the heme group, resonance Ra man spectra of the R52 mutants were obtained. The frequency of the heme a f ormyl stretching mode in the R52A mutant is characteristic of that seen in non-hydrogen-bonded model heme a complexes, Thus the data confirm the prese nce of hydrogen bonding between the heme a formyl group and the R52 side ch ain, as suggested from crystallographic data. In the R52K mutant, this hydr ogen bonding is maintained by the lysine residue, and this mutant enzyme re tains near wild-type activity. The heme n formyl frequency is also affected by mutation of Q471, confirming the X-ray models that show this residue al so has hydrogen-bonding interactions with the formyl group. Unlike R52, how ever, Q471 does not appear to be critical for the enzyme function.