Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 280, Issue 1, Pages 301-307Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M410377200
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- NIGMS NIH HHS [R01GM30736, R01GM33712] Funding Source: Medline
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The proton-pumping NADH-quinone oxidoreductase from Escherichia coli houses nine iron-sulfur clusters, eight of which are found in its mitochondrial counterpart, complex I. The extra putative iron-sulfur cluster binding site with a CXXCXXXCX27C motif in the NuoG subunit has been assigned to ligate a [2Fe-2S] (N1c). However, we have shown previously that the Thermus thermophilus N1c fragment containing this motif ligates a [4Fe-4S] (Nakamaru-Ogiso, E., Yano, T., Ohnishi, T., and Yagi, T. (2002) J. Biol. Chem. 277, 1680-1688). In the current study, we individually inactivated four sets of the iron-sulfur binding motifs in the E. coli NuoG subunit by replacing all four ligands with Ala. Each mutant subunit, designated DeltaN1b, DeltaN1c, DeltaN4, and DeltaN5, was expressed as maltose-binding protein fusion proteins. After in vitro reconstitution, all mutant subunits were characterized by EPR. Although EPR signals from cluster N1b were not detected in any preparations, we detected two [4Fe-4S] EPR signals with g values of g(x,y,z) = 1.89, 1.94, and 2.06, and g(x,y,z) = 1.91, 1.94, and 2.05 at 6-20 K in wild type, DeltaN1b, and DeltaN5. The former signal was assigned to cluster N4, and the latter signal was assigned to cluster N1c because of their disappearance in DeltaN4 and DeltaN1c. Confirming that a [4Fe-4S] cluster ligates to the N1c motif, we propose to replace its misleading [2Fe-2S] name, N1c, with cluster N7. In addition, because these mutations differently affected the assembly of peripheral subunits by in trans complementation analysis with the nuoG knock-out strain, the implicated structural importance of the iron-sulfur binding domains is discussed.
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