Modification of bovine heart succinate dehydrogenase with ethoxyformic anhydride and rose bengal: evidence for essential histidyl residues protectable by substrates.
Purified and membrane-bound succinate dehydrogenase (SDH) from bovine heart mitochondria was inhibited by the histidine-modifying reagents ethoxyformic anhydride (EFA) and Rose Bengal in the presence of light. Succinate and competitive inhibitors protected against inhibition, and decreased the number of histidyl residues modified by EFA. The essential residue modified by EFA was not the essential thiol of SDH, but modification of the essential thiol abolished the protective effect of malonate against inhibition of SDH by EFA. The EFA inhibition was reversed by hydroxylamine nearly completely when the inhibition was less than or equal to 35%, and only partially when the inhibition was more extensive. The uv spectrum of EFA-modified SDH before and after hydroxylamine treatment suggested that extensive inhibition of SDH with EFA may result in ethoxyformylation at both imidazole nitrogens of histidyl residues. Such a modification is not reversed by hydroxylamine. Succinate dehydrogenases and fumarate reductases from several different sources have similar compositions, and the two enzymes from Escherichia coli have considerable homology in the amino acid composition of their respective flavoprotein and iron-sulfur protein subunits. In the former, there is a short stretch containing conserved histidine, cysteine, and arginine residues. These residues, if also conserved in the bovine enzyme, may be the essential active site residues suggested by this work (histidine) and previously (cysteine, arginine).
D000818:Animals; D002417:Cattle; D004047:Diethyl Pyrocarbonate / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D005561:Formates / Q000494:pharmacology*; D006639:Histidine / Q000378:metabolism*; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000494:pharmacology; D008314:Malonates / Q000494:pharmacology; D008929:Mitochondria, Heart / Q000201:enzymology*; D010084:Oxidation-Reduction; D010777:Photochemistry; D012395:Rose Bengal / Q000494:pharmacology*; D013385:Succinate Dehydrogenase / Q000037:antagonists & inhibitors* / Q000378:metabolism / Q000528:radiation effects
D005561:Formates; D006898:Hydroxylamines; D008314:Malonates; D012395:Rose Bengal; D019811:Hydroxylamine; D006639:Histidine; C030290:malonic acid; D013385:Succinate Dehydrogenase; D004047:Diethyl Pyrocarbonate