The mitochondrial and prokaryotic proton-trans locating NADH:ubiquinone oxidoreductases:: similarities and dissimilarities of the quinone-junction sites

The catalytic properties of the rotenone-sensitive NADH:ubiquinone reductase (Complex 1) in bovine heart submitochondrial particles and in inside-out vesicles derived from Paracoccus denitrificans and Rhodobacter capsulatus were compared. The prokaryotic enzymes catalyze the NADH oxidase and NADH:quinone reductase reactions with similar kinetic parameters as those for the mammalian Complex 1, except for lower apparent affinities for the substrates-nucleotides. Unidirectional competitive inhibition of NADH oxidation by ADPribose, previously discovered for submitochondrial particles, was also evident for tightly coupled R denitrificans vesicles, thus suggesting that a second, NAD(+)-specific site is present in the simpler prokaryotic enzyme. The inhibitor sensitivity of the forward and reverse electron transfer reactions was compared. In P denitrificans and Bos taurus vesicles different sensitivities to rotenone and Triton X-100 for the forward and reverse electron transfer reactions were found. In bovine heart preparations, both reactions showed the same sensitivity to piericidin, and the inhibition was titrated as a straight line. In P denitrificans, the forward and reverse reactions show different sensitivity to piericidin and the titrations of both activities were curvilinear with apparent I-50 (expressed as mole of inhibitor per mole of enzyme) independent of the enzyme concentration. This behavior is explained by a model involving two different sites rapidly interacting with piericidin within the hydrophobic phase. (C) 2003 Elsevier B.V. All rights reserved.