title: A conserved lysine residue controls iron-sulfur cluster redox chemistry in Escherichia coli fumarate reductase.
abstract: The Escherichia coli respiratory complex II paralogs succinate dehydrogenase (SdhCDAB) and fumarate reductase (FrdABCD) catalyze interconversion of succinate and fumarate coupled to quinone reduction or oxidation, respectively. Based on structural comparison of the two enzymes, equivalent residues at the interface between the highly homologous soluble domains and the divergent membrane anchor domains were targeted for study. This included the residue pair SdhB-R205 and FrdB-S203, as well as the conserved SdhB-K230 and FrdB-K228 pair. The close proximity of these residues to the [3Fe-4S] cluster and the quinone binding pocket provided an excellent opportunity to investigate factors controlling the reduction potential of the [3Fe-4S] cluster, the directionality of electron transfer and catalysis, and the architecture and chemistry of the quinone binding sites. Our results indicate that both SdhB-R205 and SdhB-K230 play important roles in fine tuning the reduction potential of both the [3Fe-4S] cluster and the heme. In FrdABCD, mutation of FrdB-S203 did not alter the reduction potential of the [3Fe-4S] cluster, but removal of the basic residue at FrdB-K228 caused a significant downward shift (>100mV) in potential. The latter residue is also indispensable for quinone binding and enzyme activity. The differences observed for the FrdB-K228 and Sdh-K230 variants can be attributed to the different locations of the quinone binding site in the two paralogs. Although this residue is absolutely conserved, they have diverged to achieve different functions in Frd and Sdh.
authors.affiliation: Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
authors.forename: Victor W T
authors.initials: VW
authors.lastname: Cheng
authors.forename: Quang M
authors.initials: QM
authors.lastname: Tran
authors.forename: Nasim
authors.initials: N
authors.lastname: Boroumand
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: D001665:Binding Sites; D002384:Catalysis; D004138:Dinitrocresols / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D007501:Iron / Q000737:chemistry*; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008239:Lysine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D013385:Succinate Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013455:Sulfur / Q000737:chemistry*
chemical_list: D004138:Dinitrocresols; D007506:Iron-Sulfur Proteins; C024132:4,6-dinitro-o-cresol; D013455:Sulfur; D007501:Iron; D013385:Succinate Dehydrogenase; D008239:Lysine
keywords: 2-n-heptyl-4-hyroxyquinoline-N-oxide; Complex II; E(m); EPR; ET; FAD; FQ; FrdABCD; G–F; HOQNO; Iron–sulfur cluster; LPCH; MQ/MQH(2); Q-site; Quinone binding site; SdhCDAB; Succinate dehydrogenase; UQ/UQH(2); electron paramagnetic resonance; electron transfer; flavin adenine dinucleotide; fluorescence quench; fumarate reductase; glycerol–fumarate; lapachol; midpoint potential; oxidized/reduced menaquinone; oxidized/reduced ubiquinone; quinone binding site; succinate dehydrogenase