REDOX PROPERTIES OF MICROSOMAL REDUCED NICOTINAMIDE ADENINE DINUCLEOTIDE CYTOCHROME-B5 REDUCTASE AND CYTOCHROME-B5

Pig liver NADH-cytochrome b5 reductase was reduced by 1 mol of dithionite or NADH/mol of enzyme/bound FAD, without forming a stable semiquinone or intermediate during the titrations. The addition of NAD+ to the partially reduced enzyme or illumination in the presence of both NAD+ and EDTA yielded a new intermediate. The intermediate had an absorption band at 375 nm and the optical spectrum resembled anionic semiquinones seen on reduction of other flavin enzymes. EPR measurements confirmed the free-radical nature of the species. To explain the results, a disproportionation reaction between the oxidized and reduced NAD+ complexes (E-FAD-NAD+ + E-FADH2-NAD+ .dblarw. 2E-FADH.cntdot.-NAD+) is assumed. Potentiometric titration of NADH-cytochrome b5 reductase at pH 7.0 with dithionite gave a midpoint potential of -258 mV; titration with NADH gave -160 mV. This difference may be due to a difference in the relative affinity of NAD+ for the reduced and oxidized forms of the enzyme. The effects of pH on the midpoint potential of the NAD+-free enzyme were very similar to those measured with free FAD. At pH 7.0, midpoint potentials of trypsin-solubilized and detergent-solubilized cytochrome b5 were 13 and 0 mV, respectively.