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

• Published in: The Journal of biological chemistry 2003-05, Vol.278 (18), p.15514-15522
• Main Authors: Yano, Takahiro, Sklar, Joseph, Nakamaru-Ogiso, Eiko, Takahashi, Yasuhiro, Yagi, Takao, Ohnishi, Tomoko
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Bacterial Proteins - chemistry; Electron Spin Resonance Spectroscopy; Electron Transport Complex I; Iron-Sulfur Proteins - chemistry; Molecular Sequence Data; Mutagenesis, Site-Directed; NADH, NADPH Oxidoreductases - chemistry; Paracoccus denitrificans - enzymology; Protein Subunits
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M212275200

The NADH-quinone oxidoreductase fromParacoccus 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 1×[2Fe-2S] and 2×[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 ∼ 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 His106 with Cys specifically affected the fast-relaxing [4Fe-4S]1+cluster, suggesting that this cluster is coordinated by His106. 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.