title: The cytochrome b Zn binding amino acid residue histidine 291 is essential for ubihydroquinone oxidation at the Qosite 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 Qo(hydroquinone (QH2) oxidation) and Qi(quinone (Q) reduction) sites. At the Qosite, the pathways of bifurcated electron transfers emanating from QH2oxidation 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 Qosite 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 QH2oxidation at the Qosite, 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 QH2oxidation at the Qosite of cyt bc1.
authors.affiliation: Laboratorio di Biochimica e Biofisica Molecolare, Dipartimento di Farmacia e Biotecnologie, FaBiT, Università di Bologna, 40126 Bologna, Italy.
authors.forename: Francesco
authors.initials: F
authors.lastname: Francia
authors.affiliation: Laboratorio di Biochimica e Biofisica Molecolare, Dipartimento di Farmacia e Biotecnologie, FaBiT, Università di Bologna, 40126 Bologna, Italy.
authors.forename: Marco
authors.initials: M
authors.lastname: Malferrari
authors.affiliation: Department of Biology, University of Pennsylvania, Philadelphia, PA 19104, USA.
authors.forename: Pascal
authors.initials: P
authors.lastname: Lanciano
authors.affiliation: Department of Biology, University of Pennsylvania, Philadelphia, PA 19104, USA.
authors.forename: Stefan
authors.initials: S
authors.lastname: Steimle
authors.affiliation: Department of Biology, University of Pennsylvania, Philadelphia, PA 19104, USA. Electronic address: fdaldal@sas.upenn.edu.
authors.forename: Fevzi
authors.initials: F
authors.lastname: Daldal
authors.affiliation: Laboratorio di Biochimica e Biofisica Molecolare, Dipartimento di Farmacia e Biotecnologie, FaBiT, Università di Bologna, 40126 Bologna, Italy; Consorzio Nazionale Interuniversitario per le Scienze Fisiche della Materia (CNISM), Dipartimento di Fisica, Università di Bologna, 40127 Bologna, Italy.
authors.forename: Giovanni
authors.initials: G
authors.lastname: Venturoli
mesh_terms: D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D014450:Electron Transport Complex III / Q000737:chemistry* / Q000378:metabolism; D006639:Histidine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010084:Oxidation-Reduction; D016354:Rhodobacter capsulatus / Q000201:enzymology / Q000378:metabolism; D014451:Ubiquinone / Q000378:metabolism; D015032:Zinc / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D014451:Ubiquinone; D006639:Histidine; D014450:Electron Transport Complex III; D015032:Zinc
keywords: Bacterial photosynthesis and respiration; Cytochrome bc(1) complex; Q(o) site inactivation and proton release; Ubiquinol cytochrome c oxidoreductase; Zn binding