title: Investigating the function of [2Fe-2S] cluster N1a, the off-pathway cluster in complex I, by manipulating its reduction potential.
abstract: NADH:quinone oxidoreductase (complex I) couples NADH oxidation and quinone reduction to proton translocation across an energy-transducing membrane. All complexes I contain a flavin to oxidize NADH, seven iron-sulfur clusters to transfer electrons from the flavin to quinone and an eighth cluster (N1a) on the opposite side of the flavin. The role of cluster N1a is unknown, but Escherichia coli complex I has an unusually high-potential cluster N1a and its reduced flavin produces H2O2, not superoxide, suggesting that cluster N1a may affect reactive oxygen species production. In the present study, we combine protein film voltammetry with mutagenesis in overproduced N1a-binding subunits to identify two residues that switch N1a between its high- (E. coli, valine and asparagine) and low- (Bos taurus and Yarrowia lipolytica, proline and methionine) potential forms. The mutations were incorporated into E. coli complex I: cluster N1a could no longer be reduced by NADH, but H2O2 and superoxide production were unaffected. The reverse mutations (that increase the potential by ~0.16 V) were incorporated into Y. lipolytica complex I, but N1a was still not reduced by NADH. We conclude that cluster N1a does not affect reactive oxygen species production by the complex I flavin; it is probably required for enzyme assembly or stability.
authors: *MRC Mitochondrial Biology Unit, Wellcome Trust/MRC Building, Hills Road, Cambridge CB2 0XY, U.K.
authors: James A
authors: JA
authors: Birrell
authors: Klaudia
authors: K
authors: Morina
authors: Hannah R
authors: HR
authors: Bridges
authors: Thorsten
authors: T
authors: Friedrich
authors: Judy
authors: J
authors: Hirst
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D002417:Cattle; D004138:Dinitrocresols / Q000737:chemistry; D042967:Electron Transport Complex I / Q000737:chemistry* / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006860:Hydrogen Bonding; D007700:Kinetics; D009154:Mutation; D010084:Oxidation-Reduction; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics; D017382:Reactive Oxygen Species / Q000737:chemistry; D025062:Yarrowia / Q000201:enzymology / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D004138:Dinitrocresols; D021122:Protein Subunits; D017382:Reactive Oxygen Species; C024132:4,6-dinitro-o-cresol; D042967:Electron Transport Complex I
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