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Mutation of the conserved I‐helix threonine in CYP2C9 eliminates heteroactivation

The I‐helix conserved threonine in P450 is thought to be critical in the proton transfer necessary to form iron‐oxo intermediate. Thr301 has been proposed to be the conserved threonine in CYP2C9. The role of Thr301 in (S)‐flurbiprofen 4′‐hydroxylation, substrate binding and heteroactivation was examined in a mutant (T301A) and compared to wild type enzyme. The mutation led to a 5‐fold decrease in Vmax, a 3‐fold lower substrate binding affinity but little effect on Km when compared with wild‐type enzyme. Accordingly, substrate‐induced spin state conversion was largely diminished by the mutation. Interestingly, heteroactivation by a series of model effectors was abolished in the T301A mutant as was any increase in spin state conversion typically produced by heteroactivation. In the presence of the alternative oxidant cumene hydroperoxide (CuOOH), the T301A mutant exhibited increased substrate turnover rate as compared to the wild type enzyme in the presence of CuOOH and to the T301A mutant utilizing NADPH and P450 reductase. Overall, these results suggest Thr301 in CYP2C9 is involved in stabilizing the displaced water ligand, maintaining proton delivery to the enzyme active site as well as controlling active site bulk solvent, all of which are essential for CYP2C9‐mediated catalysis and heteroactivation.