As the final electron acceptor in the respiratory chain of eukaryotic and many prokaryotic organisms, cytochrome c oxidase (EC 1.9.3.1) catalyzes the reduction of oxygen to water and generates a proton gradient. To test for proton pathways through the oxidase, site-directed mutagenesis was applied to subunit I of the Rhodobacter sphaeroides enzyme. Mutants were characterized in three highly conserved regions of the peptide, comprising possible proton loading, unloading, and transfer sites: an interior loop between helices II and III (Aspl32Asn/Ala), an exterior loop between helices IX and X (His4l]Ala, Asp412Asn, Thr413Asn, Tyr414Phe), and the predicted transmembrane helix VIII (Thr352Ala, Pro358Ala, Thr359Ala, Lys362Met). Most of the mutants had lower activity than wild type, but only mutants at residue 132 lost proton pumping while retaining electron transfer activity. Although electron transfer was substantially inhibited, no major structural alteration appears to have occurred in D132 mutants, since resonance Raman and visible absorbance spectra were normal. However, lower CO binding (70-85% of wild type) suggests some minor change to the binuclear center. In addition, the activity of the reconstituted Aspl32 mutants was inhibited rather than stimulated by ionophores or uncoupler. The inhibition was not observed with the purified enzyme and a direct pH effect was ruled out, suggesting an altered response to the electrical or pH gradient. The results support an important role for the conserved II-I-I loop in the proton pumping process and are consistent with the possibility of involvement of residues in helix VIII and the IX-X loop.Cytochrome c oxidase (EC 1.9.3.1), a key enzyme in aerobic energy metabolism, reduces oxygen to water, yielding substantial energy that drives the formation of a proton gradient; however, the mechanism of coupling between oxygen reduction and proton translocation remains obscure.Recognition of the strong homology between mitochondrial and bacterial enzymes (1, 2) has stimulated the application of molecular genetic tools to the analysis of the oxidase mechanism. The genes for cytochrome c oxidase from Rhodobacter sphaeroides have been cloned, sequenced, deleted, and reintroduced into the bacterium, and sequence comparisons reveal a high degree of homology with the three mitochondrially encoded subunits of mammalian oxidase (3-7). Extensive sitedirected mutagenesis of the largest subunit, COX I, has permitted the assignment of the ligands for the three redox active metal centers, heme a, heme a3, and CUB (7-10), suggesting that all three metal centers are located in COX I toward the outer side of the membrane, while substrate and pumped protons come from the inside (11). Thus some kind of proton channel or relay system is required to convey protons to the site of oxygen reduction, the heme a3-CuB center, and beyond. It is reasonable to look for residues involved in proton pumpingThe publication costs of this article were defrayed in part by page charge payment. This article m...

Cytochrome aa3 of Rhodobacter sphaeroides and cytochrome bo of E. coli are useful models of the more complex cytochrome c oxidase of eukaryotes, as demonstrated by the genetic, spectroscopic, and functional studies reviewed here. A summary of site-directed mutants of conserved residues in these two enzymes is presented and discussed in terms of a current model of the structure of the metal centers and evidence for regions of the protein likely to be involved in proton transfer. The model of ligation of the heme a3 (or o)-CuB center, in which both hemes are bound to helix X of subunit I, has important implications for the pathways and control of electron transfer.

No abstract

either an alanine (DI32A) or an asparagine (D132N) strongly diminishes oxygen reduction activity and abolishes proton pumping. D132 is a substantial distance from the oxygen-reactive site and mutation of the residue does not perturb the spectral properties of the metal centers [4]. Similar effects are seen when the corresponding residue in Escherichia coli cytochrome bo3 is mutated [5,6]).Another effect of the D132N/A mutations is that the reconstituted enzymes show anomalous responses to uncouplers and ionophores [4]. Valinomycin and CCCP, both singly and in combination, inhibit instead of stimulating turnover in reconstituted enzyme. The ionophores do not inhibit the purified enzyme directly and the oxygen reduction activity shows a similar decrease with increasing pH as wild-type, indicating that the inhibition is likely caused by changes in membrane potential (A~) and/or pH gradient (ApH) [4]. An understanding of these responses might clarify the pumping process itself. The present paper will show that (i) a normal membrane potential is formed by COV containing mutant enzyme and (ii) the defect of the D132A/N mutations can partially be overcome by addition of free fatty acids. The fatty acids also rectify the anomolous responses to uncouplers and ionophores, but repair of proton pumping has not been demonstrated. Materials and methods

No abstract