(508a) A Study of Mutations That Cooperatively Increase Leakage in the Cytochrome P450 BM3 Reaction Cycle

Many redox enzymes of biotechnological utility consist of oxidase and reductase domains encoded on a single polypeptide chain. Coupling between the oxidase and reductase domains is important for efficient use of cofactors and for maximal enzyme lifetime in cases where uncoupling leads to undesirable reactive species, and yet the physical basis for coupling remains poorly understood.  We report a series of experiments that explores the relationship between mutations and coupling in a member of the cytochrome P450 family of enzymes.  Three different mutants of cytochrome P450 BM3 have been previously reported in the literature to enhance the leak rate, that is, oxidation of nicotinamide adenine phosphate (NADPH) in the absence of any other substrate besides oxygen. An increased leak rate corresponds to a lesser degree of coupling between the oxidase and reductase domains in this enzyme.  We have combined the mutations in all possible combinations, resulting in four additional mutants with even higher NADPH oxidation activity in the absence of native fatty acid substrates. These new mutants also had decreased thermostabilities, as determined by T₅₀ measurements, relative to their parents. For each mutant, we used horseradish peroxidase and 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) to measure the hydrogen peroxide content of the reaction product. Finally, we measured the total turnover number of each mutant, that is, the total amount of NADPH consumed by a mutant within the enzyme’s lifetime.