POSSIBLE PROTON RELAY PATHWAYS IN CYTOCHROME-C-OXIDASE

As the final electron acceptor in the respiratory chain of eukaryotic and many prokaryotic organisms, cytochrome c oxidase (EC 1.9.3.1) cata lyzes the reduction of oxygen to water and generates a proton gradient . To test for proton pathways through the oxidase, site-directed mutag enesis was applied to subunit I of the Rhodobacter sphaeroides enzyme. Mutants were characterized in three highly conserved regions of the p eptide, comprising possible proton loading, unloading, and transfer si tes: an interior loop between helices II and III (Asp132Asn/Ala), an e xterior loop between helices IX and X (His411Ala, Asp412Asn, Thr413Asn , Tyr414Phe), and the predicted transmembrane helix VIII (Thr352Ala, P ro358Ala, Thr359Ala, Lys362Met). Most of the mutants had lower activit y 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 t o have occurred in D132 mutants, since resonance Raman and visible abs orbance spectra were normal. However, fewer CO binding (70-85% of wild type) suggests some minor change to the binuclear center. In addition , the activity of the reconstituted Asp132 mutants was inhibited rathe r than stimulated by ionophores or uncoupler. The inhibition was not o bserved 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-III loop in the proton pumping process and are consistent with the possibility of inv olvement of residues in helix VIII and the IX-X loop.