The effect of deletion of the genes encoding the 40 kDa subunit II or the 17 kDa subunit VI on the steady-state kinetics of yeast ubiquinol-cytochrome-c oxidoreductase.
Yeast ubiquinol-cytochrome c oxidoreductase is still active after inactivation of the genes encoding the 40 kDa Core II protein or the 17 kDa subunit VI (Oudshoorn et al. (1987) Eur. J. Biochem. 163, 97-103 and Schoppink et al. (1988) Eur. J. Biochem. 173, 115-122). The steady-state levels of several other subunits of Complex III are severely reduced in the 40 kDa0 mutant. The level of spectrally detectable Complex III cytochrome b in the mutant submitochondrial particles is about 5% of that of the wild type. However, when the steady-state activity of Complex III with respect to the cytochrome c reduction was examined, similar maximal turnover numbers and Km values were found for the mutated and the wild-type complexes, both when yeast cytochrome c and when horse-heart cytochrome c was used as electron acceptor. We therefore conclude that the Core II subunit of yeast Complex III plays no role in the binding of cytochrome c and that it has no major influence of the overall electron transport and on the binding of ubiquinol by the enzyme. Absence of the 17 kDa subunit VI of yeast Complex III, the homologous counterpart of the hinge protein of the bovine heart enzyme, resulted in a decrease in the rate of reduction of both horse-heart cytochrome c and yeast cytochrome c by Complex III under conditions of relatively high ionic strength. However, under conditions of optimal ionic strength, no difference could be seen in the maximal turnover numbers and Km values, neither with horse-heart cytochrome c nor with yeast cytochrome c between Complex III deficient in the 17 kDa protein and the wild-type complex. Binding of ATP to ferricytochrome c inhibits its reduction by Complex III under conditions of relatively high ionic strength. But when the 17 kDa protein is absent, this inhibition is also observed under optimal ionic-strength conditions. These results can be explained by assuming a stimulating role for the acidic 17 kDa protein in the association of basic cytochrome c with Complex III. This association is (part of) the rate-limiting step in the reduction of cytochrome c by Complex III under conditions of relatively high ionic strength or when this association is hindered, for instance, by binding of ATP.(ABSTRACT TRUNCATED AT 400 WORDS)
Schoppink
P J
PJ
Laboratory of Biochemistry, Faculty of Chemistry, University of Amsterdam, The Netherlands.
Hemrika
W
W
Berden
J A
JA
D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D002417:Cattle; D002872:Chromosome Deletion; D003574:Cytochrome c Group / Q000378:metabolism; D014450:Electron Transport Complex III / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology; D009994:Osmolar Concentration; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D013053:Spectrophotometry; D013367:Submitochondrial Particles / Q000201:enzymology
D003574:Cytochrome c Group; D000255:Adenosine Triphosphate; D014450:Electron Transport Complex III