Influence of flavin addition and removal on the formation of superoxide by NADPH-cytochrome P-450 reductase: A spin-trap study

Abstract

The spin trap 5,5-dimethyl-1-pyrroline-1-oxide was used to measure the formation of superoxide that occurs during the oxidation of NADPH by cytochrome P-450 reductase, a flavoprotein containing one molecule each of FMN and FAD per polypeptide chain. The enzyme's ability to reduce oxygen to superoxide appears to be equally dependent on its content of FMN and FAD. Selective removal of FMN from reductase decreases superoxide formation by an amount equivalent to the difference in total flavin (FMN and FAD) concentration between native and depleted preparations of the protein. In contrast, reductase-bound FMN is necessary for the transfer of electrons to another acceptor cytochrome c, but not to ferricyanide. NADPH oxidation and the concomitant formation of superoxide by native preparations of reductase are stimulated by the addition of riboflavin, FMN, and FAD with riboflavin being the most effective. The results obtained from this study suggest that added flavins are reduced by cytochrome P-450 reductase as electron acceptors and increased superoxide formation results from their autooxidation.