Fatty acids stimulate activity and restore respiratory control in a proton channel mutant of cytochrome c oxidase.
(1) Removal of a carboxyl 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 valimomycin or nigericin and fully released by the two ionophores combined. Under the same conditions, the D132A mutant COV show anomalous ionophore responses, including inhibition by valinomycin or by CCCP. Nevertheless, oxidase activity results in development of a similar membrane potential in COV containing either wild-type or D132A oxidase, and the ionophore responses of the membrane potential are similar for both enzymes. (2) Long chain fatty acids such as arachidonic acid, but not fatty alcohols, stimulate steady-state electron transfer activity 3-7-fold, with either detergent-solubilized (purified) D132A oxidase or the reconstituted form. The effect is specific for this mutant and is not seen with wild-type or other mutants of similar overall activity. Arachidonate-treated D132A COV show normal ionophore responses to valinomycin and nigericin and 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 addition does not 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.
Fetter
J
J
Dept. Biochemistry, Michigan State University, East Lansing 48824, USA.
Sharpe
M
M
Qian
J
J
Mills
D
D
Ferguson-Miller
S
S
Nicholls
P
P
D000438:Alcohols / Q000494:pharmacology*; D000595:Amino Acid Sequence; D000818:Animals; D016718:Arachidonic Acid / Q000494:pharmacology; D002417:Cattle; D003576:Electron Transport Complex IV / Q000187:drug effects / Q000378:metabolism*; D005230:Fatty Acids, Nonesterified / Q000494:pharmacology*; D007700:Kinetics; D008564:Membrane Potentials; D008929:Mitochondria, Heart / Q000201:enzymology*; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011994:Recombinant Proteins / Q000187:drug effects / Q000378:metabolism; D012242:Rhodobacter sphaeroides / Q000201:enzymology*; D013329:Structure-Activity Relationship
D000438:Alcohols; D005230:Fatty Acids, Nonesterified; D011994:Recombinant Proteins; D016718:Arachidonic Acid; D003576:Electron Transport Complex IV