Velazquez Isabel, Nakamaru-Ogiso Eiko, Yano Takahiro, Ohnishi Tomoko, Yagi Takao
Division of Biochemistry, Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA 92037, USA.
FEBS Lett. 2005 Jun 6;579(14):3164-8. doi: 10.1016/j.febslet.2005.05.005.
The NuoF subunit, which harbors NADH-binding site, of Escherichia coli NADH-quinone oxidoreductase (NDH-1) contains five conserved cysteine residues, four of which are predicted to ligate cluster N3. To determine this coordination, we overexpressed and purified the NuoF subunit and NuoF+E subcomplex in E. coli. We detected two distinct EPR spectra, arising from a [4Fe-4S] cluster (g(x,y,z)=1.90, 1.95, and 2.05) in NuoF, and a [2Fe-2S] cluster (g(x,y,z)=1.92, 1.95, and 2.01) in NuoE subunit. These clusters were assigned to clusters N3 and N1a, respectively. Based on the site-directed mutagenesis experiments, we identified that cluster N3 is ligated to the 351Cx2Cx2Cx40C398 motif.
大肠杆菌NADH-醌氧化还原酶（NDH-1）的NuoF亚基含有NADH结合位点，该亚基包含五个保守的半胱氨酸残基，其中四个预计与簇N3配位。为了确定这种配位情况，我们在大肠杆菌中过表达并纯化了NuoF亚基和NuoF+E亚复合物。我们检测到两种不同的电子顺磁共振光谱，一种来自NuoF中的[4Fe-4S]簇（g(x,y,z)=1.90、1.95和2.05），另一种来自NuoE亚基中的[2Fe-2S]簇（g(x,y,z)=1.92、1.95和2.01）。这些簇分别被指定为簇N3和N1a。基于定点诱变实验，我们确定簇N3与351Cx2Cx2Cx40C398基序配位。
