title: Iron-sulfur cluster N2 of the Escherichia coli NADH:ubiquinone oxidoreductase (complex I) is located on subunit NuoB.
abstract: The proton-pumping NADH:ubiquinone oxidoreductase, also called respiratory complex I, couples the transfer of electrons from NADH to ubiquinone with the translocation of protons across the membrane. One FMN and up to 9 iron-sulfur (Fe/S) clusters participate in the redox reaction. There is discussion that the EPR-detectable Fe/S cluster N2 is involved in proton pumping. However, the assignment of this cluster to a distinct subunit of the complex as well as the number of Fe/S clusters giving rise to the EPR signal are still under debate. Complex I from Escherichia coli consists of 13 polypeptides called NuoA to N. Either subunit NuoB or NuoI could harbor Fe/S cluster N2. Whereas NuoB contains a unique motif for the binding of one Fe/S cluster, NuoI contains a typical ferredoxin motif for the binding of two Fe/S clusters. Individual mutation of all four conserved cysteine residues in NuoB resulted in a loss of complex I activity and of the EPR signal of N2 in the cytoplasmic membrane as well as in the isolated complex. Individual mutations of all eight conserved cysteine residues of NuoI revealed a variable phenotype. Whereas cluster N2 was lost in most NuoI mutants, it was still present in the cytoplasmic membranes of the mutants NuoI C63A and NuoI C102A. N2 was also detected in the complex isolated from the mutant NuoI C102A. From this we conclude that the Fe/S cluster N2 is located on subunit NuoB.
authors.affiliation: Institut für Organische Chemie und Biochemie, Albert-Ludwigs-Universität Albertstr. 21, 79104 Freiburg, Germany.
authors.forename: Dirk
authors.initials: D
authors.lastname: Flemming
authors.forename: Angela
authors.initials: A
authors.lastname: Schlitt
authors.forename: Volker
authors.initials: V
authors.lastname: Spehr
authors.forename: Tobias
authors.initials: T
authors.lastname: Bischof
authors.forename: Thorsten
authors.initials: T
authors.lastname: Friedrich
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D015153:Blotting, Western; D002455:Cell Division; D003545:Cysteine / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D042967:Electron Transport Complex I / Q000737:chemistry*; D004926:Escherichia coli / Q000201:enzymology* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry*; D005288:Ferredoxins / Q000737:chemistry; D017353:Gene Deletion; D005822:Genetic Vectors; D007506:Iron-Sulfur Proteins / Q000737:chemistry*; D008024:Ligands; D008280:Magnetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry*; D009249:NADP / Q000737:chemistry; D010084:Oxidation-Reduction; D010455:Peptides / Q000737:chemistry; D011522:Protons; D017386:Sequence Homology, Amino Acid
chemical_list: D029968:Escherichia coli Proteins; D005288:Ferredoxins; D007506:Iron-Sulfur Proteins; D008024:Ligands; D010455:Peptides; D011522:Protons; D009249:NADP; D009247:NADH, NADPH Oxidoreductases; C480162:NuoB protein, E coli; D042967:Electron Transport Complex I; D003545:Cysteine