Fatty Acids Stimulate & Restore Respiratory Control in a Proton Channel Mutant of Cytochrome Oxidase

John Fetter, Martyn Sharpe, Jie Qian, Denise Mills, Shelagh Ferguson-Miller & Peter Nicholls FEBS Lett. 393 155-160 (1996).

SUMMARY:(1) Removal of a COOH at residue 132 of subunit I of Rhodobacter sphaeroides cytochrome c oxidase significantly inhibits electron transfer and makes proton pumping undetectable (Fetter et al. (1995) Proc Natl. Acad. Sci USA 92 1604-1608). When reconstituted into phospholipid vesicles (COV) wild-type oxidase shows respiratory control that is partially released by either valinomycin or by nigericin and fully released the two ionophores combined. Under the same conditions thew D132A mutant COV show anomalous ionophore responses, including inhibition by valinomycin or CCCP. Nevertheless, oxidase activity results in development of a similar membrane potential in COV containing either wild-type or D132A oxidase, and the ionophore response of the membrane potential is the same for both enzymes.
(2)Long chain fatty acids e.g. arachidonic acid (20:4) but not fatty alcohols stimulate steady-state electron transfer 3-7-fold, with either detergent solubilized (purified) D312A oxidase mor the reconstituted form. The effect is specific for this mutant and is not seen with either wild-type or other mutants of similar activity. Arachidonate-treated D132A COV show normal ionophore responses to valinomycin and nigericin mand full release of respiration in presence of both ionophores or of CCCP. Thus, arachidonate and some other fatty acids abolish the ionophore anomalies seen when THE D132A enzyme is reconstituted in their absence.
(3) Fatty acid oxidation does nor restore proton pumping,likely because fatty acids also induce proton permeability and some degree of uncoupling. A model of D132A function is presented and possible roles for the fatty acids in "chemical rescue" of the mutant are discussed.