From a Monooxygenase to an Oxidase: The P-450 BM3 Mutant T268A

Abstract

P-450s are heme monooxygenases that are widely distributed among living organisms. The P-450 catalytic cycle requires an input of two electrons and a molecule of oxygen. The generic reaction is the insertion of an oxygen atom in a substrate and the release of a water molecule. If the catalytic cycle does not complete successfully, the available electrons can generate reduced oxygen species like peroxide, superoxide, or water. These reactions are defined as uncoupled. Understanding how uncoupling can occur and the role of the side-chain amino acids around the heme is of great importance for describing the catalytic cycle. P-450 BM3 is a soluble P-450 isolated from Bacillus megaterium. It catalyzes hydroxylation of various fatty acids, as well as epoxidations of double bonds. We have constructed the mutant T268A and analyzed the effect on arachidonic acid (AA) and palmitic acid (PA) binding and metabolism. Data indicate that the mutation changes the binding and the coupling for both AA and PA metabolism. Cumene hydroperoxide-driven reactions are unaffected in the mutant. These data support the hypothesis of a role of T-268 in maintaining substrate position so that the reaction can be fully coupled. The role of substrate movement in generating active oxygen species is discussed.