title: Identifying involvement of Lys251/Asp252 pair in electron transfer and associated proton transfer at the quinone reduction site of Rhodobacter capsulatus cytochrome bc1.
abstract: Describing dynamics of proton transfers in proteins is challenging, but crucial for understanding processes which use them for biological functions. In cytochrome bc1, one of the key enzymes of respiration or photosynthesis, proton transfers engage in oxidation of quinol (QH2) and reduction of quinone (Q) taking place at two distinct catalytic sites. Here we evaluated by site-directed mutagenesis the contribution of Lys251/Asp252 pair (bacterial numbering) in electron transfers and associated with it proton uptake to the quinone reduction site (Qi site). We showed that the absence of protonable group at position 251 or 252 significantly changes the equilibrium levels of electronic reactions including the Qi-site mediated oxidation of heme bH, reverse reduction of heme bH by quinol and heme bH/Qi semiquinone equilibrium. This implicates the role of H-bonding network in binding of quinone/semiquinone and defining thermodynamic properties of Q/SQ/QH2 triad. The Lys251/Asp252 proton path is disabled only when both protonable groups are removed. With just one protonable residue from this pair, the entrance of protons to the catalytic site is sustained, albeit at lower rates, indicating that protons can travel through parallel routes, possibly involving water molecules. This shows that proton paths display engineering tolerance for change as long as all the elements available for functional cooperation secure efficient proton delivery to the catalytic site.
authors.affiliation: Department of Molecular Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University in Krakow, Gronostajowa 7, 30-387 Kraków, Poland.
authors.forename: Patryk
authors.initials: P
authors.lastname: Kuleta
authors.affiliation: Department of Molecular Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University in Krakow, Gronostajowa 7, 30-387 Kraków, Poland.
authors.forename: Marcin
authors.initials: M
authors.lastname: Sarewicz
authors.affiliation: Structural Bioinformatics Laboratory, Biochemistry, Faculty of Science and Engineering, Åbo Akademi University, Turku, Finland.
authors.forename: Pekka
authors.initials: P
authors.lastname: Postila
authors.affiliation: Department of Physics, Tampere University of Technology, P.O. Box 692, FI-33101 Tampere, Finland; Department of Physics, University of Helsinki, P.O. Box 64, FI-00014 Helsinki, Finland.
authors.forename: Tomasz
authors.initials: T
authors.lastname: Róg
authors.affiliation: Department of Molecular Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University in Krakow, Gronostajowa 7, 30-387 Kraków, Poland. Electronic address: artur.osyczka@uj.edu.pl.
authors.forename: Artur
authors.initials: A
authors.lastname: Osyczka
mesh_terms: D000596:Amino Acids / Q000378:metabolism; D001665:Binding Sites / Q000502:physiology; D004579:Electron Transport / Q000502:physiology*; D014450:Electron Transport Complex III / Q000378:metabolism*; D004583:Electrons; D006418:Heme / Q000378:metabolism; D007700:Kinetics; D016297:Mutagenesis, Site-Directed / Q000379:methods; D010084:Oxidation-Reduction; D011522:Protons; D011809:Quinones / Q000378:metabolism*; D016354:Rhodobacter capsulatus / Q000378:metabolism*
chemical_list: D000596:Amino Acids; D011522:Protons; D011809:Quinones; D006418:Heme; D014450:Electron Transport Complex III
keywords: Cytochrome bc(1); Electron transfer; Mitochondrial complex III; Proton transfer; Quinone