Evidence of an ubisemiquinone radical(s) from the NADH-ubiquinone reductase of the mitochondrial respiratory chain.
NADH-ubiquinone (Q) reductase isolated from beef heart mitochondria exhibited, upon reduction by NADH, a prominent EPR signal at room temperature attributable to stable ubisemiquinone radical(s). The concentration of the ubisemiquinone radical reached as high as 40% of the total Q content in the reductase. The radical was virtually abolished by adding rotenone, whereas rotenone had no effect on the reduction of FMN by NADH. The radical showed an EPR signal of g = 2.0042 at approximately 9.5 GHz with no resolved hyperfine structure and had a line width of 6.8 Gauss at 23 degrees C. The Q-band EPR spectra at 35 GHz showed well resolved g-anisotropy and had a field separation between derivative extrema of 24 Gauss. These results substantiate the fact that this radical was bound to a protein; we call it ubiquinone protein-N (QP-N). The pH dependence of the EPR signals demonstrated that the species of the ubisemiquinone radical(s) consisted of not only an anionic form but also a neutral form. Only about half of the QP-N radical formed by NADH reduction was abolished by p-chloromercuric sulfonate. The microwave power saturation curve of the radical was biphasic; the first phase leveled off at about 5 milliwatts and then at about 20 milliwatts. These results suggested that the ubisemiquinone radical from QP-N was heterogenous, consisting of at least two populations of stable ubisemiquinone radical(s). It is suggested that two kinds of QP-N exist in NADH-Q reductase. Each mole of protein may bind two mol of Q.
D000818:Animals; D002417:Cattle; D003067:Coenzymes; D004578:Electron Spin Resonance Spectroscopy; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008872:Microwaves; D008929:Mitochondria, Heart / Q000201:enzymology*; D016660:NAD(P)H Dehydrogenase (Quinone); D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D011808:Quinone Reductases / Q000378:metabolism*; D012402:Rotenone / Q000494:pharmacology; D014451:Ubiquinone / Q000031:analogs & derivatives* / Q000032:analysis
D003067:Coenzymes; D012402:Rotenone; D014451:Ubiquinone; D009247:NADH, NADPH Oxidoreductases; D016660:NAD(P)H Dehydrogenase (Quinone); D011808:Quinone Reductases; C024989:coenzyme Q10