Characterization of the iron-sulfur cluster coordinated by a cysteine cluster motif (CXXCXXXCX27C) in the Nqo3 subunit in the proton-translocating NADH-quinone oxidoreductase (NDH-1) of Thermus thermophilus HB-8.
The proton-translocating NADH-quinone oxidoreductase (NDH-1) of Thermus thermophilus HB-8 is composed of 14 subunits (designated Nqo1-14). This NDH-1 houses nine putative iron-sulfur binding sites, eight of which are generally found in bacterial NDH-1 and its mitochondrial counterpart (complex I). The extra site contains a CXXCXXXCX(27)C motif and is located in the Nqo3 subunit. This motif was originally found in Escherichia coli NDH-1 and was assigned to a binuclear cluster (g(z, y, x) = 2.00, 1.95, 1.92) and named N1c. In this report, the Thermus Nqo3 fragment containing this motif was heterologously overexpressed, using a glutathione S-transferase fusion system. This fragment contained a small amount of iron-sulfur cluster, whose content was significantly increased by in vitro reconstitution. The UV-visible and EPR spectroscopic properties of this fragment indicate that the ligated iron-sulfur cluster is tetranuclear with nearly axial symmetry (g( parallel, perpendicular) = 2.045, approximately 1.94). Site-directed mutants show that all four cysteines participate in the ligation of a [4Fe-4S] cluster. Considering the fact that the same motif coordinates only tetranuclear clusters in other enzymes so far known, we propose that the CXXCXXXCX(27)C motif in the Nqo3 subunit most likely ligates the [4Fe-4S] cluster.
Nakamaru-Ogiso
Eiko
E
Department of Molecular and Experimental Medicine, Division of Biochemistry, The Scripps Research Institute, La Jolla, California 92037, USA.
Yano
Takahiro
T
Ohnishi
Tomoko
T
Yagi
Takao
T
D000595:Amino Acid Sequence; D001483:Base Sequence; D003545:Cysteine / Q000378:metabolism*; D017931:DNA Primers; D004578:Electron Spin Resonance Spectroscopy; D005982:Glutathione Transferase / Q000378:metabolism; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011522:Protons; D011808:Quinone Reductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D016963:Thermus thermophilus / Q000201:enzymology*
D017931:DNA Primers; D007506:Iron-Sulfur Proteins; D011522:Protons; D011993:Recombinant Fusion Proteins; C041831:NADH dehydrogenase (quinone); D011808:Quinone Reductases; D005982:Glutathione Transferase; D003545:Cysteine