The cytochrome b Zn binding amino acid residue histidine 291 is essential for ubihydroquinone oxidation at the Qo site of bacterial cytochrome bc1

Abstract

The ubiquinol:cytochrome (cyt) c oxidoreductase (or cyt bc₁) 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 c₁, which form two catalytic domains, the Q_o (hydroquinone (QH₂) oxidation) and Q_i (quinone (Q) reduction) sites. At the Q_o site, the pathways of bifurcated electron transfers emanating from QH₂ oxidation are known, but the associated proton release routes are not well defined. In energy transducing complexes, Zn²⁺ 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 bc₁. In this work, using the genetically tractable bacterial cyt bc₁, 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 QH₂ 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 QH₂ oxidation at the Q_o site of cyt bc₁.

Keywords: Bacterial photosynthesis and respiration; Cytochrome bc(1) complex; Q(o) site inactivation and proton release; Ubiquinol cytochrome c oxidoreductase; Zn binding.