Bovine heart NADH-ubiquinone oxidoreductase contains one molecule of ubiquinone with ten isoprene units as one of the cofactors

摘要

NADH-ubiquinone oxidoreductase (Complex I) is located at the entrance of the mitochondrial electron transfer chain and transfers electrons from NADH to ubiquinone with 10 isoprene units (Q10) coupled with proton pumping. The composition of Complex I, the largest and most complex proton pump in the mitochondrial electron transfer system, especially the contents of Q_(10) and phospholipids, has not been well established. An improved purification method including solubilization of mitochondrial membrane with deoxycholate followed by sucrose gradient centrifugation and anion-exchange column chromatography provided reproducibly a heme-free preparation containing 1 Q10, 70 phosphorus atoms of phospholipids, 1 zinc ion, 1 FMN, 30 inorganic sulfur ions, and 30 iron atoms as the intrinsic constituents. The rotenone-sensitive enzymatic activity of the Complex I preparation was comparable to that of Complex I in the mitochondrial membrane. It has been proposed that Complex I has two Q10 binding sites, one involved in the proton pump and the other functioning as a converter between one and two electron transfer pathways [Ohnishi, T., Johnson, J. J. E., Yano, T., LoBrutto, R., and Widger, R. W. (2005) FEBS Lett. 579, 500-506]. The existence of one molecule of Q10 in the fully oxidized Complex I suggests that the affinity of Q10 to one of the two Q10 sites is greatly dependent on the oxidation state and/or the membrane potential and that theQ10 in the present preparation functions as the converter of the electron transfer pathways which should be present in any oxidation state.