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...
The sequential layer-by-layer formation of peptide-supported bimolecular lipid membranes at solid supports is described. In the first step, thiol-derivatized peptide sequences of 5 and 7 amino acids are assembled on a Au substrate. After activation of their COOH-terminus phospholipid molecules (DMPE) are covalently attached via an amid bond to form a tethered monolayer on the Au electrode. The different preparation steps are analyzed by Fourier transform IR, X-ray reflectometry, and surface plasmon spectroscopy. The latter technique is then also used to on-line monitor at the solid/solution interface the formation of a bilayer by fusion of vesicles prepared from a fluid lipid mixture with and without reconstituted proteins. The obtained thicknesses and capacitance values are compatible with the tethered bilayer model and point to an incorporation of ATPase into these membrane matrices.
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