Reference: Bruel C, et al. (1995) Role of the evolutionarily conserved cytochrome b tryptophan 142 in the ubiquinol oxidation catalyzed by the bc1 complex in the yeast Saccharomyces cerevisiae. J Biol Chem 270(38):22321-8

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Abstract


Trp-142 is a highly conserved residue of the cytochrome b subunit in the bc1 complexes. To study the importance of this residue in the quinol oxidation catalyzed by the bc1 complex, we characterized four yeast mutants with arginine, lysine, threonine, and serine at position 142. The mutant W142R was isolated previously as a respiration-deficient mutant unable to grow on non-fermentable carbon sources (Lemesle-Meunier, D., Brivet-Chevillotte, P., di Rago, J.-P, Slonimski, P.P., Bruel, C., Tron, T., and Forget, N. (1993) J. Biol. Chem. 268, 15626-15632). The mutants W142K, W142T, and W142S were obtained here as respiration-sufficient revertants from mutant W142R. Mutant W142R exhibited a decreased complex II turnover both in the presence and absence of antimycin A; this suggests that the structural effect of W142R in the bc1 complex probably interferes with the correct assembly of the succinate-ubiquinone reductase complex. The mutations resulted in a parallel decrease in turnover number and apparent Km, with the result that there was no significant change in the second-order rate constant for ubiquinol oxidation. Mutants W142K and W142T exhibited some resistance toward myxothiazol, whereas mutant W142R showed increased sensitivity. The cytochrome cc1 reduction kinetics were found to be severely affected in mutants W142R, W142K, and W142T. The respiratory activities and the amounts of reduced cytochrome b measured during steady state suggest that the W142S mutation also modified the quinol-cytochrome c1 electron transfer pathway. The cytochrome b reduction kinetics through center P were affected when Trp-142 was replaced with arginine or lysine, but not when it was replaced with threonine or serine. Of the four amino acids tested at position 142, only arginine resulted in a decrease in cytochrome b reduction through center N. These findings are discussed in terms of the structure and function of the quinol oxidation site and seem to indicate that Trp-142 is not critical to the kinetic interaction of ubiquinol with the reductase, but plays an important role in the electron transfer reactions that intervene between ubiquinol oxidation and cytochrome c1 reduction.

Reference Type
Journal Article | Research Support, Non-U.S. Gov't
Authors
Bruel C, di Rago JP, Slonimski PP, Lemesle-Meunier D
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