The pathway of electron transfer in NADH:Q oxidoreductase.
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.
van Belzen
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R
University of Amsterdam, Amsterdam, The Netherlands.
Albracht
S P
SP
D000818:Animals; D002417:Cattle; D004579:Electron Transport; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D008929:Mitochondria, Heart; D009243:NAD / Q000378:metabolism; D016660:NAD(P)H Dehydrogenase (Quinone); D009249:NADP / Q000378:metabolism; D010653:Phenylbutazone / Q000494:pharmacology; D011808:Quinone Reductases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013367:Submitochondrial Particles / Q000378:metabolism*; D014357:Trypsin / Q000494:pharmacology; D014451:Ubiquinone / Q000378:metabolism
D009243:NAD; D014451:Ubiquinone; D009249:NADP; C041831:NADH dehydrogenase (quinone); D016660:NAD(P)H Dehydrogenase (Quinone); D011808:Quinone Reductases; D014357:Trypsin; D010653:Phenylbutazone