A new flavin radical signal in the Na+-pumping NADH:quinone oxidoreductase from Vibrio cholerae -: An EPR/electron nuclear double resonance investigation of the role of the covalently bound flavins in subunits B and C
A new flavin radical signal in the Na+-pumping NADH:quinone oxidoreductase from Vibrio cholerae -: An EPR/electron nuclear double resonance investigation of the role of the covalently bound flavins in subunits B and C
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DOI：
10.1074/jbc.m605765200
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发表时间：
2006-12-01
影响因子：
4.8
通讯作者：
Nilges, Mark J.
中科院分区：
文献类型：
--
作者：
Barquera, Blanca;Ramirez-Silva, Leticia;Nilges, Mark J.
Na+泵送nadh -泛醌氧化还原酶具有6个多肽亚基（NqrA-F）和许多氧化还原辅助因子，包括非共价结合的FAD和位于F亚基的2Fe-2S中心，位于B和C亚基的共价结合的FMNs，以及位于未知位置的非共价结合的核黄素。亚基B和C中的FMN辅因子通过磷酸酯键与苏氨酸残基结合。在氧化酶中观察到中性黄酮-半醌自由基，而在还原酶中报道了阴离子黄酮-半醌。在这项工作中，我们通过用其他氨基酸取代苏氨酸配体来改变fmn在B和C亚基上的结合配体，并通过EPR和电子核双共振光谱研究了由此产生的突变体。我们得出结论，钠易位NADH：醌氧化还原酶形成三种光谱上不同的黄素自由基，如下：1)氧化酶中的中性自由基，在所有突变体中都观察到，最有可能来自核黄素；2)在完全还原酶中观察到阴离子自由基，在野生型和nqrc - t25y突变体中存在，而在NqrB-T236Y突变体中不存在；3)第二个阴离子自由基，主要出现在弱还原条件下，存在于野生型和NqrB-T236Y突变体中，而不存在于nqrc - t25y突变体中。因此，我们可以暂时将第一个阴离子自由基分配给亚基B的FMN，第二个阴离子自由基分配给亚基c的FMN。第二个阴离子自由基以前没有报道过。在电子核双共振谱中，与第一阴离子自由基相比，它表现出更大的谱线宽度和更大的8 -甲基质子分裂。
The Na+ pumping NADH-ubiquinone oxidoreductase has six polypeptide subunits (NqrA-F) and a number of redox cofactors, including a noncovalently bound FAD and a 2Fe-2S center in subunit F, covalently bound FMNs in subunits B and C, and a noncovalently bound riboflavin in an undisclosed location. The FMN cofactors in subunits B and C are bound to threonine residues by phosphoester linkages. A neutral flavin-semiquinone radical is observed in the oxidized enzyme, whereas an anionic flavin-semiquinone has been reported in the reduced enzyme. For this work, we have altered the binding ligands of the FMNs in subunits B and C by replacing the threonine ligands with other amino acids, and we studied the resulting mutants by EPR and electron nuclear double resonance spectroscopy. We conclude that the sodium-translocating NADH: quinone oxidoreductase forms three spectroscopically distinct flavin radicals as follows: 1) a neutral radical in the oxidized enzyme, which is observed in all of the mutants and most likely arises from the riboflavin; 2) an anionic radical observed in the fully reduced enzyme, which is present in wild type, and the NqrC-T225Y mutant but not the NqrB-T236Y mutant; 3) a second anionic radical, seen primarily under weakly reducing conditions, which is present in wild type, and the NqrB-T236Y mutant but not the NqrC-T225Y mutant. Thus, we can tentatively assign the first anionic radical to the FMN in subunit B and the second to the FMN in subunit C. The second anionic radical has not been reported previously. In electron nuclear double resonance spectra, it exhibits a larger line width and larger 8 alpha-methyl proton splittings, compared with the first anionic radical.