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

Abstract

Abstract Cytochrome b5 of rat liver microsomes is reduced by α-NADH; the extent of reduction is equal to that obtained with β-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 α-NADH are about 10% of the rates observed with β-NADH. Conditions have been established for the measurement of first order kinetic parameters in these reductions: Km of α- and β-NADH are 13.0 and 3.3 µm for cytochrome c reductase activity and 5.1 and 6.7 µm, respectively, for dichloroindophenol reduction. Three lines of evidence suggest that α-NADH and β-NADH may reduce cytochrome c and dichloroindophenol via the same microsomal flavoprotein. (a) There is competition between α- and β-NADH. (b) The relative rates of cytochrome c reduction are diminished equally for α- and β-NADH when the enzyme is inhibited or denatured. (c) The relative rates of reduction of both cytochrome c and dichloroindophenol are increased equally for α- and β-NADH when microsomal cytochrome b5 reductase is enriched. Both α- and β-NADH are electron donors to cytochrome P-450 in microsomes. The initial rates of reduction of cytochrome P-450 by α- and β-NADH are equal (K = 0.126 min-1) and considerably slower than reduction by NADPH. In a second, slower phase of cytochrome P-450 reduction, the rate is somewhat less rapid with α-NADH (K = 0.030 min-1) than with β-NADH (K = 0.042 min-1). Compared to β-NADH and NADPH, α-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 α-NADH is very slow (10% of β-NADH). To date, only diaphorase-like activities have been reported for oxidation of α-NADH. Although the present report shows clearly that α-NADH may function as an electron donor to microsomal mixed function oxidases, the physiological significance of these findings remains obscure because enzymic reduction of α-NAD+ has not yet been reported.