CARBOXIN RESISTANCE IN PARACOCCUS-DENITRIFICANS CONFERRED BY A MUTATION IN THE MEMBRANE-ANCHOR DOMAIN OF SUCCINATE-QUINONE REDUCTASE (COMPLEX II)

Succinate:quinone reductase is a membrane-bound enzyme of the citric a cid cycle and the respiratory chain. Carboxin is a potent inhibitor of the enzyme of certain organisms. The bacterium Paracoccus denitrifica ns was found to be sensitive to carboxin in vivo, and mutants that gro w in the presence of 3'-methyl carboxin were isolated. Membranes of th e mutants showed resistant succinate:quinone reductase activity. The m utation conferring carboxin resistance was identified in four mutants. They contained the same missense mutation in the sdhD gene, which enc odes one of two membrane-intrinsic polypeptides of the succinate:quino ne reductase complex. The mutation causes an Asp to Gly replacement at position 89 in the SdhD polypeptide. P. denitrificans strains that ov erproduced wild-type or mutant enzymes were constructed. Enzymic prope rties of the purified enzymes were analyzed. The apparent K-m for quin one (DPB) and the sensitivity to thenoyltrifluoroacetone was normal fo r the carboxin-resistant enzyme, but the succinate:quinone reductase a ctivity was lower than for the wild-type enzyme. Mutations conferring carboxin resistance indicate the region on the enzyme where the inhibi tor binds. A previously reported His to Leu replacement close to the [ 3Fe-4S] cluster in the iron-sulfur protein of Ustilago maydis succinat e:quinone reductase confers resistance to carboxin and thenoyltrifluor oacetone. The Asp to Gly replacement in the P. denitrificans SdhD poly peptide, identified in this study to confer resistance to carboxin but not to thenoyltrifluoroacetone, is in a predicted cytoplasmic loop co nnecting two transmembrane segments. It is likely that this loop is lo cated in the neighborhood of the [3Fe-4S] cluster.