ON THE MECHANISM OF ROTENONE-INSENSITIVE REDUCTION OF QUINONES BY MITOCHONDRIAL NADH - UBIQUINONE REDUCTASE - THE HIGH-AFFINITY BINDING OF NAD+ AND NADH TO THE REDUCED ENZYME FORM

NADH acts as an incomplete competitive inhibitor for 5,8-dioxy-1,4-naphthoquinone during its rotenone-insensitive reduction by mitochondrial NADH:ubiquinone reductase. NAD+ and ADP-ribose act as incomplete mixed-type inhibitors. K(i) of NAD+ and NADH towards quinone are about one order less than towards ferricyanide. The bimolecular rate constant of the reduction of the enzyme by NADH in the quinone reductase reaction is about 2 times less than that of ferricyanide reductase reaction. These data indicate that the reduction site of 5,8-dioxy-1,4-naphthoquinone is close to NAD+/NADH and ferricyanide binding site. It seems that during the steady-state reduction of ferricyanide and 5,8-dioxy-1,4-naphthoquinone these oxidizers react with NADH:ubiquinone reductase reduced to different extents.