The reaction of NADPH with bovine mitochondrial NADH:ubiquinone oxidoreductase revisited: II. Comparison of the proposed working hypothesis with literature data.
The first purification of bovine NADH:ubiquinone oxidoreductase (Complex I) was reported nearly half a century ago (Hatefi et al. J Biol Chem 237:1676-1680, 1962). The pathway of electron-transfer through the enzyme is still under debate. A major obstacle is the assignment of EPR signals to the individual iron-sulfur clusters in the subunits. The preceding paper described a working model based on the kinetics with NADPH. This model is at variance with current views in the field. The present paper provides a critical overview on the possible causes for the discrepancies. It is concluded that the stability of all purified preparations described thus far, including Hatefi's Complex I, is compromised due to removal of the enzyme from the protective membrane environment. In addition, most preparations described during the last two decades are purified by methods involving synthetic detergents and column chromatography. This results in delipidation, loss of endogenous quinones and loss of reactions with (artificial) quinones in a rotenone-sensitive way. The Fe:FMN ratio's indicate that FMN-a is absent, but that all Fe-S clusters may be present. In contrast to the situation in bovine SMP and Hatefi's Complex I, three of the six expected [4Fe-4S] clusters are not detected in EPR spectra. Qualitatively, the overall EPR lineshape of the remaining three cubane signals may seem similar to that of Hatefi's Complex I, but quantitatively it is not. It is further proposed that point mutations in any of the TYKY, PSST, 49-kDa or 30-kDa subunits, considered to make up the delicate structural heart of Complex I, may have unpredictable effects on any of the other subunits of this quartet. The fact that most point mutations led to inactive enzymes makes a correct interpretation of such mutations even more ambiguous. In none of the Complex-I-containing membrane preparations from non-bovine origin, the pH dependencies of the NAD(P)H-->O(2) reactions and the pH-dependent reduction kinetics of the Fe-S clusters with NADPH have been determined. This excludes a proper discussion on the absence or presence of FMN-a in native Complex I from other organisms.
Albracht
Simon P J
SP
Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, The Netherlands. s.p.j.albracht@uva.nl
D000818:Animals; D002417:Cattle; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D042967:Electron Transport Complex I / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D008929:Mitochondria, Heart / Q000201:enzymology* / Q000378:metabolism; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism*
D009243:NAD; D009249:NADP; D042967:Electron Transport Complex I