Instructor, Anesthesiology, Perioperative and Pain Medicine
Conformational changes in NADPH-cytochrome P450 oxidoreductase (CYPOR) associated with electron transfer from NADPH to electron acceptors via FAD and FMN have been investigated via structural studies of the four-electron-reduced NADP+-bound enzyme and kinetic and structural studies of mutants that affect the conformation of the mobile Gly631-Asn635 loop (Asp632 loop). The structure of four-electron-reduced, NADP+-bound wild type CYPOR shows the plane of the nicotinamide ring positioned perpendicular to the FAD isoalloxazine with its carboxamide group forming H-bonds with N1 of the flavin ring and the Thr535 hydroxyl group. In the reduced enzyme, the C8-C8 atoms of the two flavin rings are ∼1 Å closer than in the fully oxidized and one-electron-reduced structures, which suggests that flavin reduction facilitates interflavin electron transfer. Structural and kinetic studies of mutants Asp632Ala, Asp632Phe, Asp632Asn, and Asp632Glu demonstrate that the carboxyl group of Asp632 is important for stabilizing the Asp632 loop in a retracted position that is required for the binding of the NADPH ribityl-nicotinamide in a hydride-transfer-competent conformation. Structures of the mutants and reduced wild type CYPOR permit us to identify a possible pathway for NADP(H) binding to and release from CYPOR. Asp632 mutants unable to form stable H-bonds with the backbone amides of Arg634, Asn635, and Met636 exhibit decreased catalytic activity and severely impaired hydride transfer from NADPH to FAD, but leave interflavin electron transfer intact. Intriguingly, the Arg634Ala mutation slightly increases the cytochrome P450 2B4 activity. We propose that Asp632 loop movement, in addition to facilitating NADP(H) binding and release, participates in domain movements modulating interflavin electron transfer.
View details for DOI 10.1021/acs.biochem.7b01102
View details for PubMedID 29308883
View details for PubMedCentralID PMC5967631
NADPH-cytochrome P450 oxidoreductase transfers electrons from NADPH to cytochromes P450 via its FAD and FMN. To understand the biochemical and structural basis of electron transfer from FMN-hydroquinone to its partners, three deletion mutants in a conserved loop near the FMN were characterized. Comparison of oxidized and reduced wild type and mutant structures reveals that the basis for the air stability of the neutral blue semiquinone is protonation of the flavin N5 and strong H-bond formation with the Gly-141 carbonyl. The ΔGly-143 protein had moderately decreased activity with cytochrome P450 and cytochrome c It formed a flexible loop, which transiently interacts with the flavin N5, resulting in the generation of both an unstable neutral blue semiquinone and hydroquinone. The ΔGly-141 and ΔG141/E142N mutants were inactive with cytochrome P450 but fully active in reducing cytochrome c In the ΔGly-141 mutants, the backbone amide of Glu/Asn-142 forms an H-bond to the N5 of the oxidized flavin, which leads to formation of an unstable red anionic semiquinone with a more negative potential than the hydroquinone. The semiquinone of ΔG141/E142N was slightly more stable than that of ΔGly-141, consistent with its crystallographically demonstrated more rigid loop. Nonetheless, both ΔGly-141 red semiquinones were less stable than those of the corresponding loop in cytochrome P450 BM3 and the neuronal NOS mutant (ΔGly-810). Our results indicate that the catalytic activity of cytochrome P450 oxidoreductase is a function of the length, sequence, and flexibility of the 140s loop and illustrate the sophisticated variety of biochemical mechanisms employed in fine-tuning its redox properties and function.
View details for DOI 10.1074/jbc.M116.724625
View details for PubMedID 27189945
View details for PubMedCentralID PMC4938185
Cytochrome P450 reductase (CPR) is a diflavin enzyme that transfers electrons to many protein partners. Electron transfer from CPR to cyt c has been extensively used as a model reaction to assess the redox activity of CPR. CPR is composed of multiple domains, among which the FMN binding domain (FBD) is the direct electron donor to cyt c. Here, electron transfer and complex formation between FBD and cyt c are investigated. Electron transfer from FBD to cyt c occurs at distinct rates that are dependent on the redox states of FBD. When compared with full-length CPR, FBD reduces cyt c at a higher rate in both the semiquinone and hydroquinone states. The NMR titration experiments reveal the formation of dynamic complexes between FBD and cyt c on a fast exchange time scale. Chemical shift mapping identified residues of FBD involved in the binding interface with cyt c, most of which are located in proximity to the solvent-exposed edge of the FMN cofactor along with other residues distributed around the surface of FBD. The structural model of the FBD-cyt c complex indicates two possible orientations of complex formation. The major complex structure shows a salt bridge formation between Glu-213/Glu-214 of FBD and Lys-87 of cyt c, which may be essential for the formation of the complex, and a predicted electron transfer pathway mediated by Lys-13 of cyt c. The findings provide insights into the function of CPR and CPR-cyt c interaction on a structural basis.
View details for DOI 10.1074/jbc.M114.582700
View details for PubMedID 25512382
View details for PubMedCentralID PMC4335224
The structural basis of the regulation of microsomal cytochrome P450 (P450) activity was investigated by mutating the highly conserved heme binding motif residue, Phe429, on the proximal side of cytochrome P450 2B4 to a histidine. Spectroscopic, pre-steady-state and steady-state kinetic, thermodynamic, theoretical, and structural studies of the mutant demonstrate that formation of an H-bond between His429 and the unbonded electron pair of the Cys436 axial thiolate significantly alters the properties of the enzyme. The mutant lost >90% of its activity; its redox potential was increased by 87 mV, and the half-life of the oxyferrous mutant was increased ∼37-fold. Single-crystal electronic absorption and resonance Raman spectroscopy demonstrated that the mutant was reduced by a small dose of X-ray photons. The structure revealed that the δN atom of His429 forms an H-bond with the axial Cys436 thiolate whereas the εN atom forms an H-bond with the solvent and the side chain of Gln357. The amide of Gly438 forms the only other H-bond to the tetrahedral thiolate. Theoretical quantification of the histidine-thiolate interaction demonstrates a significant electron withdrawing effect on the heme iron. Comparisons of structures of class I-IV P450s demonstrate that either a phenylalanine or tryptophan is often found at the location corresponding to Phe429. Depending on the structure of the distal pocket heme, the residue at this location may or may not regulate the thermodynamic properties of the P450. Regardless, this residue appears to protect the thiolate from solvent, oxidation, protonations, and other deleterious reactions.
View details for DOI 10.1021/bi5003794
View details for PubMedID 25029089
View details for PubMedCentralID PMC4131899
Resonance Raman spectroscopy is employed to characterize heme site structural changes arising from conformational heterogeneity in deoxyMb and ligated derivatives, i.e., the ferrous CO (MbCO) and ferric cyanide (MbCN) complexes. The spectra for the reversed forms of these derivatives have been extracted from the spectra of reconstituted samples. Dramatic changes in the low-frequency spectra are observed, where newly observed RR modes of the reversed forms are assigned using protohemes that are selectively deuterated at the four methyl groups or at the four methine carbons. Interestingly, while substantial changes in the disposition of the peripheral vinyl and propionate groups can be inferred from the dramatic spectral shifts, the bonds to the internal histidyl imidazole ligand and those of the Fe-CO and Fe-CN fragments are not significantly affected by the heme rotation, as judged by lack of significant shifts in the nu(Fe-N(His)), nu(Fe-C), and nu(C-O) modes. In fact, the apparent lack of an effect on these key vibrational parameters of the Fe-N(His), Fe-CO, and Fe-CN fragments is entirely consistent with previously reported equilibrium and kinetic studies that document virtually identical functional properties for the native and reversed forms.
View details for DOI 10.1021/bi801779d
View details for PubMedID 18986170
View details for PubMedCentralID PMC2654223
Heme proteins that have been reconstituted with certain hemins may contain substantial fractions of a minor component in which the orientation of the heme in the folded pocket differs from the major ("native") conformation by a 180 degrees rotation about the alpha-gamma meso axis. In fact, this minor component has also been shown to exist in some native proteins, including several mammalian globins. While resonance Raman spectroscopy has emerged as a powerful probe of active site structure of heme proteins, no systematic study has yet been undertaken to elucidate the specific spectral changes associated with this disorder. In the present work, combined analyses of the temporal behavior of both NMR and RR data sets have been completed to permit the extraction of a unique RR spectrum for the disoriented form, documenting rather dramatic changes associated with this rotational disorder. In addition, the use of protohemes bearing selectively deuterated peripheral methyl groups has permitted the association of the observed modes with specific fragments of the heme residing in the reversed orientation. The studies conducted here clearly illustrate the exquisite sensitivity of low frequency heme deformation modes to altered protein-heme interactions.
View details for DOI 10.1002/bip.20887
View details for PubMedID 18008322
Latest information on COVID-19