Chemical modification studies of beef-heart mitochondrial b-c1 complex. Effect of modification by ethoxyformic anhydride.
The effect of the histidine-modifier ethoxyformic anhydride (EFA) on the enzymatic properties of the mitochondrial b-c1 complex (ubiquinol-cytochrome c reductase) has been investigated. Chemical modification by EFA inhibited to the same extent the reductase and the proton translocating activity of the complex. In particular EFA modification of the complex resulted in: strong inhibition of the antimycin-insensitive reduction of b cytochromes; inhibition of the antimycin-promoted oxidant-induced reduction of b cytochromes and inhibition of oxidation of pre-reduced b cytochromes. Analysis of the absorbance at 238 nm, indicative of N-(ethoxyformyl)histidine derivative, of the various polypeptide subunits separated by high-pressure liquid chromatography procedure, showed that EFA modified residues in core proteins and in the low-molecular-mass proteins. Both the inhibition of the redox and the protonmotive activity of the complex and the absorbance increase at 238 nm of the core protein fraction were readily reversed by hydroxylamine, indicating that modification of histidine residue(s) in core protein(s) is critical for the activity of the complex. This was supported by the finding that modification of the reductase with EFA prevented binding of fluorescein isothiocyanate to histidine residue(s) in core protein II. EFA modification of the reductase was without effect on the binding of N-(7-dimethylamino-4-methylcoumarinyl)maleimide to the various polypeptides of the complex except for the binding to the Fe-S protein which was greatly potentiated. Thus primary chemical modification of histidine residue(s) in core protein (II) appears to cause, in turn, a conformational change in the Rieske Fe-S protein.
D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology*; D004579:Electron Transport / Q000187:drug effects; D014450:Electron Transport Complex III / Q000037:antagonists & inhibitors*; D005292:Ferricyanides / Q000494:pharmacology; D005561:Formates / Q000494:pharmacology*; D006873:Hydroquinones / Q000378:metabolism; D007700:Kinetics; D008929:Mitochondria, Heart / Q000201:enzymology*; D010084:Oxidation-Reduction / Q000187:drug effects
D005292:Ferricyanides; D005561:Formates; D006873:Hydroquinones; C007931:hexacyanoferrate III; D014450:Electron Transport Complex III; D004047:Diethyl Pyrocarbonate; C016421:duroquinol