Covalent binding of flavins to RnfG and RnfD in the Rnf complex from Vibrio cholerae.
Covalent binding of flavins to RnfG and RnfD in the Rnf complex from Vibrio cholerae.
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DOI：
10.1021/bi800920j
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发表时间：
2008-10-28
期刊：
影响因子：
2.9
通讯作者：
Barquera, Blanca
中科院分区：
文献类型：
--
作者：
Backiel, Julianne;Juarez, Oscar;Zagorevski, Dmitri V.;Wang, Zhenyu;Nilges, Mark J.;Barquera, Blanca
RNF家族的酶被认为是细菌氧化还原驱动的离子泵，将氧化还原过程耦合到Na+在细胞膜上的转移。在这里，我们首次证明了RNF是一种黄素蛋白，FMN在RnfG中与苏氨酸-175共价结合，第二个黄素在RnfD中与苏氨酸-187结合。RNF亚基D和G分别与Na+-NQR的B和C亚基同源。每个Na+-NQR亚基都包含一个保守的S(T)GAT基序，FMN共价结合到最后的苏氨酸上。RnfD和RnfG都包含相同的基序，表明它们以类似的方式结合黄素。为了研究这一点，我们克隆了霍乱弧菌的RnfD和RnfG基因，并在该菌中分别进行了表达。在这两种情况下，产生的蛋白质在十二烷基硫酸钠凝胶上的紫外光照射下发出荧光，进一步表明黄素的存在。对突变体RnfG-T175L、RnfD-T278L和RnfD-T187V的分析表明，RnfG-T175和RnfD-T187可能是黄素配体。这表明，在RnfD的情况下，黄素不是与SGAT序列结合，而是与TMAT序列的最终残基结合，TMAT序列是黄素结合基序的一种新变体。在RNFG的情况下，黄素分析，随后的MALDI-TOF-TOF质谱仪，表明FMN共价连接到苏氨酸-175，即S(T)GAT序列的最终苏氨酸。可见光、EPR和Endor光谱研究表明，部分还原后，分离的RnfG产生中性的半喹酮中间体。在完全还原时，半喹酮物种消失，变性蛋白中没有观察到半喹酮物种。结合报告蛋白融合和计算机预测的拓扑分析表明，RnfG和RnfD中的黄素定位于周质空间。相反，在NqrC和NqrB中，黄素位于细胞质环中。这一拓扑分析表明，RNF和Na+-NQR络合物之间可能存在机理上的差异。
Enzymes of the Rnf family are believed to be bacterial redox-driven ion pumps, coupling an oxidoreduction process to the translocation of Na+ across the cell membrane. Here we show for the first time that Rnf is a flavoprotein, with FMN covalently bound to threonine-175 in RnfG and a second flavin bound to threonine-187 in RnfD. Rnf subunits D and G are homologous to subunits B and C of Na+-NQR, respectively. Each of these Na+-NQR subunits includes a conserved S(T)GAT motif, with FMN covalently bound to the final threonine. RnfD and RnfG both contain the same motif, suggesting that they bind flavins in a similar way. In order to investigate this, the genes for RnfD and RnfG from Vibrio cholerae were cloned and expressed individually in that organism. In both cases the produced protein fluoresced under UV illumination on an SDS gel, further indicating the presence of flavin. However, analysis of the mutants RnfG-T175L, RnfD-T278L, and RnfD-T187V showed that RnfG-T175 and RnfD-T187 are the likely flavin ligands. This indicates that, in the case of RnfD, the flavin is bound, not to the SGAT sequence but to the final residues of a TMAT sequence, a novel variant of the flavin binding motif. In the case of RnfG, flavin analysis, followed by MALDI-TOF-TOF mass spectrometry, showed that an FMN is covalently attached to threonine-175, the final threonine of the S(T)GAT sequence. Studies by visible, EPR, and ENDOR spectroscopy showed that, upon partial reduction, the isolated RnfG produces a neutral semiquinone intermediate. The semiquinone species disappeared upon full reduction and was not observed in the denatured protein. A topological analysis combining reporter protein fusion and computer predictions indicated that the flavins in RnfG and RnfD are localized in the periplasmic space. In contrast, in NqrC and NqrB the flavins are located in a cytoplasmic loop. This topological analysis suggests that there may be mechanistic differences between the Rnf and Na+-NQR complexes.
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