title: Mutations in cytochrome b that affect kinetics of the electron transfer reactions at center N in the yeast cytochrome bc1 complex.
abstract: We have examined the pre-steady-state kinetics and thermodynamic properties of the b hemes in variants of the yeast cytochrome bc1 complex that have mutations in the quinone reductase site (center N). Trp-30 is a highly conserved residue, forming a hydrogen bond with the propionate on the high potential b heme (bH heme). The substitution by a cysteine (W30C) lowers the redox potential of the heme and an apparent consequence is a lower rate of electron transfer between quinol and heme at center N. Leu-198 is also in close proximity to the b(H) heme and a L198F mutation alters the spectral properties of the heme but has only minor effects on its redox properties or the electron transfer kinetics at center N. Substitution of Met-221 by glutamine or glutamate results in the loss of a hydrophobic interaction that stabilizes the quinone ligands. Ser-20 and Gln-22 form a hydrogen-bonding network that includes His-202, one of the carbonyl groups of the ubiquinone ring, and an active-site water. A S20T mutation has long-range structural effects on center P and thermodynamic effects on both b hemes. The other mutations (M221E, M221Q, Q22E and Q22T) do not affect the ubiquinol oxidation kinetics at center P, but do modify the electron transfer reactions at center N to various extents. The pre-steady reduction kinetics suggest that these mutations alter the binding of quinone ligands at center N, possibly by widening the binding pocket and thus increasing the distance between the substrate and the bH heme. These results show that one can distinguish between the contribution of structural and thermodynamic factors to center N function.
authors.affiliation: Department of Biochemistry, Dartmouth Medical School, 7200 Vail, Hanover, New Hampshire 03755, USA.
authors.forename: Frederik A J
authors.initials: FA
authors.lastname: Rotsaert
authors.forename: Raul
authors.initials: R
authors.lastname: Covian
authors.forename: Bernard L
authors.initials: BL
authors.lastname: Trumpower
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D045303:Cytochromes b / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D045304:Cytochromes c / Q000378:metabolism; D004579:Electron Transport; D014450:Electron Transport Complex III / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006418:Heme / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D008928:Mitochondria / Q000201:enzymology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010084:Oxidation-Reduction; D011485:Protein Binding; D011487:Protein Conformation; D018892:Proton-Motive Force; D011808:Quinone Reductases / Q000235:genetics / Q000378:metabolism; D013816:Thermodynamics; D014451:Ubiquinone / Q000031:analogs & derivatives / Q000378:metabolism; D015003:Yeasts / Q000201:enzymology* / Q000235:genetics
chemical_list: D008024:Ligands; D014451:Ubiquinone; D006418:Heme; D045304:Cytochromes c; D045303:Cytochromes b; D011808:Quinone Reductases; D014450:Electron Transport Complex III; C003741:ubiquinol