Biochemical characterization of rat P4502C11 fused to rat or bacterial NADPH-P450 reductase domains

cDNAs coding for rat P450 2C11 fused to either a bacterial (the NADPH-cytoc hrome P450 BM3 reductase domain of P450 BM3) or a truncated form of rat NAD PH-P450 reductases were expressed in Escherichia coli and characterized enz ymatically. Measurements of NADPH cytochrome c reductase activity showed fu sion-dependent increases in the rates of cytochrome c reduction by the bact erial or the mammalian flavoprotein (21 and 48%, respectively, of the rates observed with nonfused enzymes). Neither the bacterial flavoprotein nor th e truncated rat reductase supported arachidonic acid metabolism by P450 2C1 1. In contrast, fusion of P450 2C11 to either reductase yielded proteins th at metabolized arachidonic acid to products similar to those obtained with reconstituted systems containing P350 2C11 and native rat P450 reductase. A ddition of a 10-fold molar excess of rat P450 reductase markedly increased the rates of metabolism by both fused and nonfused P450s 2C11. These increa ses occurred with preservation of the regioselectivity of arachidonic acid metabolism. The fusion-independent reduction of P450 2C11 by bacterial P450 BM3 reductase was shown by measurements of NADPH-dependent H2O2 formation [73 +/- 10 and 10 +/- 1 nmol of H2O2 formed min(-1) (nmol of P450)(-1) for the reconstituted and fused protein systems, respectively]. These studies d emonstrate that (a) a self-sufficient, catalytically active arachidonate ep oxygenase can be constructed by using P450 2C11 to mammalian or bacterial P 450 reductases and (b) the P450 BM3 reductase interacts efficiently with ma mmalian P450 2C11 and catalyzes the reduction of the heme iron. However, fu sion is required for metabolism and product formation.