Substitutions for glutamate 101 in subunit II of cytochrome c oxidase from Rhodobacter sphaeroides result in blocking the proton-conducting K-channel

Two functional input pathways for protons have been characterized in the heme-copper oxidases: the D-channel and the K-channel. These two proton-conducting channels have different functional roles and have been defined both by X-ray crystallography and by the characterization of site-directed mutants. Whereas the entrance of the D-channel is well-defined as D132^I (subunit I; Rhodobacter sphaeroides numbering), the entrance of the K-channel has not been clearly defined. Previous mutagenesis studies of the cytochrome bo₃ quinol oxidase from Escherichia coli implicated an almost fully conserved glutamic acid residue within subunit II as a likely candidate for the entrance of the K-channel. The current work examines the properties of mutants of this conserved glutamate in the oxidase from R. sphaeroides (E101^III,A,C,Q,D,N,H) and residues in the immediate vicinity of E101^II. It is shown that virtually any substitution for E101^II, including E101^IID, strongly reduces oxidase turnover (to 8-29%). Furthermore, the low steady-state activity correlates with an inhibition of the rate of reduction of heme a₃ prior to the reaction with O₂. These are phenotypes expected of K-channel mutants. It is concluded that the predominant entry point for protons going into the K-channel of cytochrome oxidase is the surface-exposed glutamic acid E101^II in subunit II.