Activities and kinetic mechanisms of native and soluble NADPH-cytochrome P450 reductase

Native yeast NADPH-cytochrome P450 oxidoreductase (CPR; EC 1.6.2.4) and a s oluble derivative lacking 33 amino acids of the NH2-terminus have been over expressed as recombinant proteins in Escherichia coli. The presence of a he xahistidine sequence at the N-terminus allowed protein purification in a si ngle step using nickel-chelating affinity chromatography. Sodium dodecyl su lfate-polyacrylamide gel electrophoresis confirmed the predicted molecular weights of the proteins and indicated a purity of > 95%. Protein functional ity was demonstrated by cytochrome c reduction and reconstitution of CYP61- mediated sterol Delta (22)-desaturation. Steady-state kinetics of cytochrom e c reductase activity revealed a random Bi-Bi mechanism with NADPH donatin g electrons directly to CPR to produce a reduced intermediary form of the e nzyme. The kinetic mechanism studies showed no difference between the two y east CPRs in mechanism or after reconstitution with CYP61-mediated 22-desat uration, confirming that the retention of the NH2-terminable membrane ancho r is functionally dispensable. (C) 2001 Academic Press.