Role of deprotonation events in ubihydroquinone:cytochrome c oxidoreductase from bovine heart and yeast mitochondria.

The pH dependence of bovine and yeast cytochrome bc1 complex catalyzing electron transfer from ubi- and plastohydroquinone to cytochrome c have been analyzed. The pH dependence of the steady-state rate was found to be governed by two protonable groups, one of which (pK approximately 6.6) has to be deprotonated while the other (pK approximately 9.2) has to be protonated to allow catalysis. Using ubideuteroquinone instead of ubihydroquinone as a substrate resulted in 1.4- and 1.7-fold lower steady-state rates for the bovine and yeast enzymes, respectively. The activation energy at pH 8.0 was 33 kJ/mol for the bovine and 44 kJ/mol for the yeast enzyme and exhibited a linear decrease between pH 5.4 and 9.2. For ubihydroquinone the slope was very close to a value of -5.7 kJ/mol expected if the activation energy depended on a single deprotonation event. When plastohydroquinone was used instead, the slope more than doubled, indicating that a second deprotonation contributed to the activation barrier with this nonphysiological substrate. In contrast to previous kinetic models for the cytochrome bc1 complex, which propose that the activation barrier is associated with the formation of ubisemiquinone at the ubihydroquinone oxidation center, our results strongly suggest that the best approximation of the transition state is the singly deprotonated form of ubihydroquinone. This supports the recently proposed proton-gated charge transfer mechanism, which has control of catalysis by the first deprotonation of ubihydroquinone as one of its key features [Brandt, U. (1996) FEBS Lett. 387, 1-6]. All results reported here can be rationalized in a straightforward way based on other aspects of the same hypothesis.