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

Abstract

Microsomal suspensions of liver can catalyze the reduction of cytochrome b 5 by the membrane-bound NADPH-cytochrome c reductase but the highly purified NADPH-cytochrome c reductase obtained by lipase solubilization does not reduce cytochrome b 5 under normal assay conditions. However, the purified reductase does reduce cytochrome b 5 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 b 5 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 b 5 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 b 5 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 b 5, albeit slowly. No apparent differences in rate of reduction or steady-state level of cytochrome b 5 reduction is seen when either the detergent- or proteolytically prepared cytochrome b 5 is employed.