Electron Transfer from a-Reduced Nicotinamide Adenine Dinucleotide to Flavoprotein, Cytochromes, and Mixed Function Oxidases of Rat Liver Microsomes*

Cytochrome b5 of rat liver microsomes is reduced by a-NADH ; the extent of reduction is equal to that obtained with P-NADH. With both nucleotides, the rates of reduction of cytochrome b5 are very fast ; alternatively, rates of reduction of cytochrome c and dichloroindophenol have been measured. The rates observed with a+NADH are about 10% of the rates observed with P-NADH. Conditions have been established for the measurement of first order kinetic parameters in these reductions: K, of o(and /3-NADH are 13.0 and 3.3 pM for cytochrome c reductase activity and 5.1 and 6.7 PM, respectively, for dichloroindophenol reduction. Three lines of evidence suggest that LU-NADH and P-NADH may reduce cytochrome c and dichloroindophenol via the same microsomal flavoprotein. (a) There is competition between LYand /3-NADH. (b) The relative rates of cytochrome c reduction are diminished equally for o(and ,RNADH when the enzyme is inhibited or denatured. (c) The relative rates of reduction of both cytochrome c and dichloroindophenol are increased equally for czand /3-NADH when microsomal cytochrome b5 reductase is enriched. Both o(and /3-NADH are electron donors to cytochrome P-450 in microsomes. The initial rates of reduction of cytochrome P-450 by 01and P-NADH are equal (K = 0.126 min-I) and considerably slower than reduction by NADPH. In a second, slower phase of cytochrome P-450 reduction, the rate is somewhat less rapid with a-NADH (K = 0.030 min-‘) than with /3-NADH (K = 0.042 min-l). Compared to /3NADH and NADPH, a(-NADH is a more efficacious donor of electrons for microsomal mixed function oxidation of a methyl sterol intermediate of cholesterol biosynthesis. Furthermore, the substrate-independent rate of oxidation of LYNADH is very slow (10% of P-NADH). To date, only diaphorase-like activities have been reported