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

作者：Bowen, Alice M.; Johnson, Eachan O. D.; Mercuri, Francesco; Hoskins, Nicola J.; Qiao, Ruihong; McCullagh, James S. O.; Lovett, Janet E.; Bell, Stephen G.; Zhou, Weihong^*; Timmel, Christiane R.; Wong, Luet Lok^*; Harmer, Jeffrey R.^*

来源：Journal of the American Chemical Society, 2018, 140(7): 2514-2527.

DOI：10.1021/jacs.7b11056

• CYTOCHROME P450CAM
• CONFORMATIONAL DYNAMICS
• CRYSTAL-STRUCTURE
• TRANSFER SYSTEM
• HYDROXYLATION
• DISTANCE
• BINDING
• MONOOXYGENASES
• PUTIDAREDOXIN
• CYP199A2

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 alpha(3) 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.

• 出版日期2018-2-21
• 单位MIT; 南开大学