Crystal structures of the ferrous dioxygen complex of wild-type cytochrome P450eryF and its mutants, A245S and A245T - Investigation of the proton transfer system in P450eryF

Cytochrome P450eryF (CYP107A) from Saccaropolyspora ertherea catalyzes the hydroxylation of 6-deoxyerythronolide B, one of the early steps in the biosynthesis of erythromycin. P450eryF has an alanine rather than the conserved threonine that participates in the activation of dioxygen (O-2) in most other P450s. The initial structure of P450eryF (Cupp-Vickery, J. R., Han, O., Hutchinson, C. R., and Poulos, T. L. (1996) Nat. Struct. Biol. 3, 632-637) suggests that the substrate 5-OH replaces the missing threonine OH group and holds a key active site water molecule in position to donate protons to the iron-linked dioxygen, a critical step for the monooxygenase reaction. To probe the proton delivery system in P450eryF, we have solved crystal structures of ferrous wild-type and mutant (Fe2+) dioxygen-bound complexes. The catalytic water molecule that was postulated to provide protons to dioxygen is absent, although the substrate 5-OH group donates a hydrogen bond to the iron-linked dioxygen. The hydrogen bond network observed in the wild-type ferrous dioxygen complex, water 63-Glu(360)-Ser(246)-water 53-Ala(241) carbonyl in the I-helix cleft, is proposed as the proton transfer pathway. Consistent with this view, the hydrogen bond network in the O-2 center dot A245S and O-2 center dot A245T mutants, which have decreased or no enzyme activity, was perturbed or disrupted, respectively. The mutant Thr(245) side chain also perturbs the hydrogen bond between the substrate 5-OH and dioxygen ligand. Contrary to the previously proposed mechanism, these results support the direct involvement of the substrate in O-2 activation but raise questions on the role water plays as a direct proton donor to the iron-linked dioxygen.