Two EPR-detectable [4Fe-4S] clusters, N2a and N2b, are bound to the NuoI (TYKY) subunit of NADH:ubiquinone oxidoreductase (Complex I) from Rhodobacter capsulatus.
NADH:ubiquinone oxidoreductases (Complex I) contain a subunit, TYKY in the bovine enzyme and NuoI in the enzyme from Rhodobacter capsulatus, which is assumed to bind two [4Fe-4S] clusters because it contains two sets of conserved cysteine motifs similar to those found in the 2[4Fe-4S] ferredoxins. It was recently shown that the TYKY subunit is not an ordinary 2[4Fe-4S] ferredoxin, but has a unique amino acid sequence, which is only found in NAD(P)H:quinone oxidoreductases and certain membrane-bound [NiFe]-hydrogenases expected to be involved in redox-linked proton translocation [FEBS Lett. 485 (2000) 1]. We have generated a set of R. capsulatus mutants in which five out of the eight conserved cysteine residues in NuoI were replaced by other amino acids. The resulting mutants fell into three categories with virtually no, intermediate or quite normal Complex I activities. EPR-spectroscopic analysis of the membranes of the C67S and C106S mutants, two mutants belonging to the second and third group, respectively, showed a specific 50% decrease of the EPR signal attributed to cluster N2. It is concluded that the NuoI (TYKY) subunit binds two clusters N2, called N2a and N2b, which exhibit very similar spectral features when analyzed by X-band EPR spectroscopy.
Chevallet
Mireille
M
BECP/DBMS/CEA Grenoble, EMI INSERM 9931, 17 Av des Martyrs, F-38054 Grenoble Cedex 09, France.
Dupuis
Alain
A
Issartel
Jean-Paul
JP
Lunardi
Joël
J
van Belzen
Ronald
R
Albracht
Simon P J
SP
D000900:Anti-Bacterial Agents; D002462:Cell Membrane / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D042967:Electron Transport Complex I; D007120:Immunochemistry; D007506:Iron-Sulfur Proteins / Q000032:analysis* / Q000378:metabolism; D008958:Models, Molecular; D009243:NAD / Q000737:chemistry; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010084:Oxidation-Reduction; D017354:Point Mutation; D011725:Pyridines; D016354:Rhodobacter capsulatus / Q000737:chemistry / Q000201:enzymology* / Q000254:growth & development
D000900:Anti-Bacterial Agents; D007506:Iron-Sulfur Proteins; D011725:Pyridines; D009243:NAD; C100213:piericidin A; D009247:NADH, NADPH Oxidoreductases; D042967:Electron Transport Complex I