Albracht S P, Bakker P T
Biochim Biophys Acta. 1986 Jul 23;850(3):423-8. doi: 10.1016/0005-2728(86)90110-6.
The pre-steady-state kinetics of reoxidation of NADH:Q oxidoreductase present in submitochondrial particles has been studied by the freeze-quench method. It was found that at pH 8 only 50% of the Fe-S clusters 2 and 4 and 75% of the clusters 3 were rapidly reoxidised after transient and complete reduction by a pulse of NADH in the presence of excess NADPH. Thus, NADPH keeps 50% of the clusters 2 and 4 and 25% of the clusters 3 permanently reduced at this pH. Since NADH oxidation is nearly optimal at this pH, whereas NADPH oxidation is virtually absent, it was concluded that these permanently reduced clusters were not involved in the NADH oxidation activity. Incomplete reoxidation of the clusters 2, 3 and 4 after a pulse of NADH was also found in the absence of NADPH, both at pH 6.5 and at pH 8. A pulse of NADPH given at pH 6.5, where NADPH oxidation by oxygen is nearly optimal, caused a slow reduction of 50% of clusters 2 and 4 and 30% of the clusters 3, which persisted for a period of at least 15 s. It was concluded that these clusters were not involved in the oxidation of NADPH by oxygen, as catalysed by the particles. As a working hypothesis a dimeric model for NAD(P)H:Q oxidoreductase is proposed, consisting of two different protomers. One of the protomers, containing FMN and the Fe-S clusters 1-4 in stoichiometric amounts, only reacts with NADH, and its oxidation by ubiquinone is rapid at pH but slow at pH 6.5. The other protomer, containing FMN and the clusters 2, 3 and 4, reacts with both NADH and NADPH and has a pH optimum at 6-6.5 for the reaction with ubiquinone.
采用冷冻淬灭法研究了亚线粒体颗粒中NADH:Q氧化还原酶再氧化的预稳态动力学。发现在pH 8时，在过量NADPH存在下，经NADH脉冲短暂完全还原后，只有50%的铁硫簇2和4以及75%的簇3能快速再氧化。因此，在该pH值下，NADPH使50%的簇2和4以及25%的簇3永久还原。由于NADH氧化在该pH值下接近最佳状态，而NADPH氧化几乎不存在，因此得出结论，这些永久还原的簇不参与NADH氧化活性。在没有NADPH的情况下，无论是在pH 6.5还是pH 8时，NADH脉冲后簇2、3和4的再氧化也不完全。在pH 6.5时给予NADPH脉冲，此时氧气对NADPH的氧化接近最佳状态，导致50%的簇2和4以及30%的簇3缓慢还原，这种状态持续至少15秒。得出结论，这些簇不参与颗粒催化的氧气对NADPH的氧化。作为一个工作假设，提出了NAD(P)H:Q氧化还原酶的二聚体模型，该模型由两个不同的原体组成。其中一个原体含有化学计量的FMN和铁硫簇1-4，仅与NADH反应，其被泛醌氧化在pH时快速，但在pH 6.5时缓慢。另一个原体含有FMN和簇2、3和4，与NADH和NADPH都反应，与泛醌反应的最适pH为6-6.5。
