title: Characterization of cluster N5 as a fast-relaxing [4Fe-4S] cluster in the Nqo3 subunit of the proton-translocating NADH-ubiquinone oxidoreductase from Paracoccus denitrificans.
abstract: The NADH-quinone oxidoreductase from Paracoccus denitrificans consists of 14 subunits (Nqo1-14) and contains one FMN and eight iron-sulfur clusters. The Nqo3 subunit possesses fully conserved 11 Cys and 1 His in its N-terminal region and is considered to harbor three iron-sulfur clusters; however, only one binuclear (N1b) and one tetranuclear (N4) were previously identified. In this study, the Nqo3 subunit containing 1x[2Fe-2S] and 2x[4Fe-4S] clusters was expressed in Escherichia coli. The second [4Fe-4S](1+) cluster is detected by EPR spectroscopy below 6 K, exhibiting very fast spin relaxation. The resolved EPR spectrum of this cluster is broad and nearly axial. The subunit exhibits an absorption-type EPR signal around g approximately 5 region below 6 K, most likely arising from an S = 3/2 ground state of the fast-relaxing [4Fe-4S](1+) species. The substitution of the conserved His(106) with Cys specifically affected the fast-relaxing [4Fe-4S](1+) cluster, suggesting that this cluster is coordinated by His(106). In the cholate-treated NDH-1-enriched P. denitrificans membranes, we observed EPR signals arising from a [4Fe-4S] cluster below 6 K, exhibiting properties similar to those of cluster N5 detected in other complex I/NDH-1 and of the fast-relaxing [4Fe-4S](1+) cluster in the expressed Nqo3 subunit. Hence, we propose that the His-coordinated [4Fe-4S] cluster corresponds to cluster N5.
authors.affiliation: Johnson Research Foundation, Department of Biochemistry and Biophysics, School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. yano@mail.med.upenn.edu
authors.forename: Takahiro
authors.initials: T
authors.lastname: Yano
authors.forename: Joseph
authors.initials: J
authors.lastname: Sklar
authors.forename: Eiko
authors.initials: E
authors.lastname: Nakamaru-Ogiso
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Takahashi
authors.forename: Takao
authors.initials: T
authors.lastname: Yagi
authors.forename: Tomoko
authors.initials: T
authors.lastname: Ohnishi
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy; D042967:Electron Transport Complex I; D007506:Iron-Sulfur Proteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry*; D010231:Paracoccus denitrificans / Q000201:enzymology*; D021122:Protein Subunits
chemical_list: D001426:Bacterial Proteins; D007506:Iron-Sulfur Proteins; D021122:Protein Subunits; D009247:NADH, NADPH Oxidoreductases; D042967:Electron Transport Complex I