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

Abstract

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 [FeS] 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 [FeS] 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.

Structural insight into the electron transfer pathway of a self-sufficient P450 monooxygenase

“…For CYP116 family, two crystal structures of the heme domain have been reported and their substrate-binding modes were proposed accordingly 26,27 . The complex structures of a heme domain and ferredoxin from P450 systems that acquire electrons from separate redox partners have also been reported [28][29][30][31] , which demonstrated how the electrons are injected from [2Fe-2S] cluster to the heme (Supplementary Fig. 2b).…”

Novel approach to improve progesterone hydroxylation selectivity by CYP106A2 via rational design of adrenodoxin binding

“…For the related P450–ferredoxin complexes of CYP199A2‐HaPux (top ranked complex from structure determination by orientation‐selective double electron–electron) and of CYP101A1‐Pdx (pdb entry http://www.rcsb.org/pdb/search/structidSearch.do?structureId=4JWS.pdb) the obtained RMSD values are much higher (14.1 Å and 13.6 Å, respectively). Visual inspection, however, showed that the binding site is matched, but the ferredoxins are somewhat skewed.…”

Pulsed EPR Dipolar Spectroscopy under the Breakdown of the High‐Field Approximation: The High‐Spin Iron(III) Case

“…The latter case has severala dvantages and applications: 1) the use of metal centers forP DS allows to reduce the number of spin labels and, consequently,t he number of protein mutations, 2) it enables orthogonal spin labeling [8] as the metal center has different spectroscopic properties than the majority of spin labels, 3) the distance constraintso btained from such PDS measurementse nable the localization of metal ions within the protein fold by trilateration [9,10] and the docking of different parts of protein complexes using metal ions as anchor points. [11] To date, PDS measurements have been applied to av ariety of different intrinsic metal centers, such as Cu 2 + , [12][13][14][15][16][17][18][19][20] Mn 2 + , [21][22][23][24][25] Co 2 + , [26,27] and low-spin Fe 3 + , [6,[28][29][30][31] as well as ironsulfur [17,[32][33][34] and manganese [35,36] clusters. In all these cases, the protocols for PSD measurements are well-established and the extractiono fd istance constraints from the corresponding PDS data can be readily achieved under the high-field approximation.…”