THE ROLE OF THR268 IN OXYGEN ACTIVATION OF CYTOCHROME P450(BM-3)

Cytochrome P450(BM-3), a catalytically self-sufficient monooxygenase from Bacillus megaterium, catalyzes the omega-n (n = 1-3) hydroxylation of fatty acids in the presence of O-2 and NADPH. Like most other P450s, cytochrome P450(BM-3) contains a threonine residue (Thr268) in the distal I helix thought to be important for O-2 binding and activation. Thr268 has been converted to alanine and the enzymatic properties and heme domain crystal structure determined. Using sodium laurate as the substrate, the mutant exhibited slower rates of O-2 and NADPH consumption. In addition, electron transfer is uncoupled from substrate hydroxylation as evidenced by the greater production of water and peroxide in the mutant compared to the wild-type enzyme. The crystal structure of the mutant reveals that the only changes in structure are confined to the site of mutation. These data indicate an important role for Thr268 in O-2 binding and activation in the metabolism of sodium laurate by cytochrome P450(BM-3).