The mechanism of cytochrome b₅ reduction by NADPH-cytochrome c reductase

Microsomal suspensions of liver can catalyze the reduction of cytochrome b₅ by the membrane-bound NADPH-cytochrome c reductase but the highly purified NADPH-cytochrome c reductase obtained by lipase solubilization does not reduce cytochrome b₅ under normal assay conditions. However, the purified reductase does reduce cytochrome b₅ in solutions containing high concentrations of salt. Although the flavoprotein rapidly generates superoxide anion in the presence of high concentrations of salt, the reduction of cytochrome b₅ is not dependent upon superoxide anion generation but possibly occurs via a direct interaction between the flavoprotein and the cytochrome. Further, the mechanism of cytochrome b₅ reduction involves the cycling of the enzyme between its fully reduced and half-reduced forms during the steady-state reduction of cytochrome. The reduction of cytochrome b₅ by detergent- and proteolytically solubilized NADPH-cytochrome c reductase is similar in the presence of high concentrations of KCl. However, with low salt concentrations, only the detergent-solubilized NADPH-cytochrome c reductase reduces cytochrome b₅, albeit slowly. No apparent differences in rate of reduction or steady-state level of cytochrome b₅ reduction is seen when either the detergent- or proteolytically prepared cytochrome b₅ is employed.