A Structural Model of a P450-Ferredoxin Complex from Orientation-Selective Double Electron–Electron Resonance Spectroscopy

Cytochrome
P450 (CYP) monooxygenases catalyze the oxidation of
chemically inert carbon–hydrogen bonds in diverse endogenous
and exogenous organic compounds by atmospheric oxygen. This C–H
bond oxy-functionalization activity has huge potential in biotechnological
applications. Class I CYPs receive the two electrons required for
oxygen activation from NAD­(P)H via a ferredoxin reductase and ferredoxin.
The interaction of Class I CYPs with their cognate ferredoxin is specific.
In order to reconstitute the activity of diverse CYPs, structural
characterization of CYP-ferredoxin complexes is necessary, but little
structural information is available. Here we report a structural model
of such a complex (CYP199A2-HaPux) in frozen solution derived from
distance and orientation restraints gathered by the EPR technique
of orientation-selective double electron–electron resonance
(os-DEER). The long-lived oscillations in the os-DEER spectra were
well modeled by a single orientation of the CYP199A2-HaPux complex.
The structure is different from the two known Class I CYP-Fdx structures:
CYP11A1-Adx and CYP101A1-Pdx. At the protein interface, HaPux residues
in the [Fe2S2] cluster-binding loop and the
α3 helix and the C-terminus residue interact with CYP199A2 residues
in the proximal loop and the C helix. These residue contacts are consistent
with biochemical data on CYP199A2-ferredoxin binding and electron
transfer. Electron-tunneling calculations indicate an efficient electron-transfer
pathway from the [Fe2S2] cluster to the heme.
This new structural model of a CYP-Fdx complex provides the basis
for tailoring CYP enzymes for which the cognate ferredoxin is not
known, to accept electrons from HaPux and display monooxygenase activity.