van Belzen R, Albracht S P
University of Amsterdam, Amsterdam, The Netherlands.
Biochim Biophys Acta. 1989 May 30;974(3):311-20. doi: 10.1016/s0005-2728(89)80249-x.
The pre-steady-state reduction by NADPH of NADH:Q oxidoreductase, as present in submitochondrial particles, has been further investigated with the rapid-mixing, rapid-freezing technique. It was found that trypsin treatment, that had previously been used to inactivate the transhydrogenase activity (Bakker, P.T.A. and Albracht, S.P.J. (1986) Biochim. Biophys. Acta 850, 413-422), considerably affected the stability at pH 6.2 of the NAD(P)H oxidation activity of submitochondrial particles. Use of the inhibitor butadione circumvented this problem, thus allowing a more careful investigation of the kinetics at pH 6.2. In the presence of the inhibitor rotenone it was found that 50% of the Fe-S clusters 3 and all of the Fe-S clusters 2 and 4 could be reduced by NADPH within 30 ms at pH 6.2. The remainder of the Fe-S clusters 3 and all of the Fe-S clusters 1 were reduced slowly (complete reduction only after more than 60 s). It was concluded that these latter Fe-S clusters play no role in the NADPH oxidation activity. In the absence of rotenone at pH 6.2 only 50% of the Fe-S clusters 2-4 could be reduced within 30 ms, while Fe-S cluster 1 was again not reduced. This difference was attributed to the fast reoxidation of part of the Fe-S clusters 2 and 4 by ubiquinone. At pH 8.0, where the NADPH oxidation activity is almost zero, 50% of the Fe-S clusters 2-4 could still be reduced by NADPH within 30 ms, while Fe-S cluster 1 was not reduced. The presence of rotenone had no effect on this reduction. From these observations it is concluded that the Fe-S clusters 2 and 4, which were rapidly reduced by NADPH and reoxidised by ubiquinone at pH 6.2, could not be reduced by NADPH at 8.0. This provides an explanation why NADH:Q oxidoreductase was not able to oxidise NADPH at pH 8.0, while part of the Fe-S clusters were still rapidly reduced. As a working hypothesis a dimeric structure for NADH:Q oxidoreductase is proposed. One protomer (B) contains FMN and Fe-S clusters 1-4 in equal amounts; the other protomer (A) is identical except for the absence of Fe-S cluster 1. NADH is able to react with both protomers, while NADPH only reacts with protomer A. A pH-dependent electron transfer from protomer A to protomer B is proposed, which would allow the reduction of Fe-S clusters 2 and 4 of protomer B by NADPH at pH 6.2, which is required for NADPH:Q oxidoreductase activity.
利用快速混合、快速冷冻技术，对亚线粒体颗粒中存在的NADH：Q氧化还原酶被NADPH预稳态还原的情况进行了进一步研究。结果发现，先前用于使转氢酶活性失活的胰蛋白酶处理（Bakker，P.T.A.和Albracht，S.P.J.（1986年），《生物化学与生物物理学报》850，413 - 422），极大地影响了亚线粒体颗粒在pH 6.2时NAD（P）H氧化活性的稳定性。使用抑制剂丁二酮可避免此问题，从而能够更仔细地研究pH 6.2时的动力学。发现在抑制剂鱼藤酮存在的情况下，在pH 6.2时，50%的铁硫簇3以及所有的铁硫簇2和4可在30毫秒内被NADPH还原。其余的铁硫簇3和所有的铁硫簇1还原缓慢（仅在60多秒后才完全还原）。得出的结论是，后一组铁硫簇在NADPH氧化活性中不起作用。在pH 6.2且不存在鱼藤酮的情况下，30毫秒内只有50%的铁硫簇2 - 4可被还原，而铁硫簇1再次未被还原。这种差异归因于部分铁硫簇2和4被泛醌快速再氧化。在pH 8.0时，NADPH氧化活性几乎为零，NADPH仍可在30毫秒内将50%的铁硫簇2 - 4还原，而铁硫簇1未被还原。鱼藤酮的存在对此还原没有影响。从这些观察结果可以得出结论，在pH 6.2时被NADPH快速还原并被泛醌再氧化的铁硫簇2和4，在pH 8.0时不能被NADPH还原。这就解释了为什么NADH：Q氧化还原酶在pH 8.0时不能氧化NADPH，而部分铁硫簇仍能被快速还原。作为一个工作假设，提出了NADH：Q氧化还原酶的二聚体结构。一个原体（B）含有等量的FMN和铁硫簇1 - 4；另一个原体（A）除了没有铁硫簇1外与之相同。NADH能够与两个原体反应，而NADPH只与原体A反应。提出了一个依赖于pH的从原体A到原体B的电子转移，这将使在pH 6.2时NADPH能够还原原体B的铁硫簇2和4，这是NADPH：Q氧化还原酶活性所必需的。
