title: Out of plane distortions of the heme b of Escherichia coli succinate dehydrogenase.
abstract: The role of the heme b in Escherichia coli succinate dehydrogenase is highly ambiguous and its role in catalysis is questionable. To examine whether heme reduction is an essential step of the catalytic mechanism, we generated a series of site-directed mutations around the heme binding pocket, creating a library of variants with a stepwise decrease in the midpoint potential of the heme from the wild-type value of +20 mV down to -80 mV. This difference in midpoint potential is enough to alter the reactivity of the heme towards succinate and thus its redox state under turnover conditions. Our results show both the steady state succinate oxidase and fumarate reductase catalytic activity of the enzyme are not a function of the redox potential of the heme. As well, lower heme potential did not cause an increase in the rate of superoxide production both in vitro and in vivo. The electron paramagnetic resonance (EPR) spectrum of the heme in the wild-type enzyme is a combination of two distinct signals. We link EPR spectra to structure, showing that one of the signals likely arises from an out-of-plane distortion of the heme, a saddled conformation, while the second signal originates from a more planar orientation of the porphyrin ring.
authors.affiliation: Membrane Protein Research Group, Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada.
authors.forename: Quang M
authors.initials: QM
authors.lastname: Tran
authors.forename: Carmen
authors.initials: C
authors.lastname: Fong
authors.forename: Richard A
authors.initials: RA
authors.lastname: Rothery
authors.forename: Elena
authors.initials: E
authors.lastname: Maklashina
authors.forename: Gary
authors.initials: G
authors.lastname: Cecchini
authors.forename: Joel H
authors.initials: JH
authors.lastname: Weiner
mesh_terms: D002384:Catalysis; D002462:Cell Membrane / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000201:enzymology*; D006418:Heme / Q000737:chemistry*; D006801:Humans; D008968:Molecular Conformation; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010084:Oxidation-Reduction; D011166:Porphyrins / Q000737:chemistry; D017382:Reactive Oxygen Species; D013056:Spectrophotometry, Ultraviolet / Q000379:methods; D013385:Succinate Dehydrogenase / Q000737:chemistry*
chemical_list: D011166:Porphyrins; D017382:Reactive Oxygen Species; D006418:Heme; D013385:Succinate Dehydrogenase