Abstract
The ubiquinol:cytochrome (cyt) c oxidoreductase (or cyt bc(1)) is an important membrane protein complex in photosynthetic and respiratory energy transduction. In bacteria such as Rhodobacter capsulatus it is constituted of three subunits: the iron-sulfur protein, cyt b and cyt c(1), which form two catalytic domains, the Q(o) (hydroquinone (QH(2)) oxidation) and Q(i) (quinone (Q) reduction) sites. At the Q(o) site, the pathways of bifurcated electron transfers emanating from QH(2) oxidation are known, but the associated proton release routes are not well defined. In energy transducing complexes, Zn(2+) binding amino acid residues often correlate with proton uptake or release pathways. Earlier, using combined EXAFS and structural studies, we identified Zn coordinating residues of mitochondrial and bacterial cyt bc(1). In this work, using the genetically tractable bacterial cyt bc(1), we substituted each of the proposed Zn binding residues with non-protonatable side chains. Among these mutants, only the His291Leu substitution destroyed almost completely the Q(o) site catalysis without perturbing significantly the redox properties of the cofactors or the assembly of the complex. In this mutant, which is unable to support photosynthetic growth, the bifurcated electron transfer reactions that result from QH(2) oxidation at the Q(o) site, as well as the associated proton(s) release, were dramatically impaired. Based on these findings, on the putative role of His291 in liganding Zn, and on its solvent exposed and highly conserved position, we propose that His291 of cyt b is critical for proton release associated to QH(2) oxidation at the Q(o) site of cyt bc(1).