The diflavin NADPH-cytochrome P450 reductase (CYPOR) plays a critical role in human cytochrome P450 (CYP) activity by sequentially delivering two electrons from NADPH to CYP enzymes during catalysis. Although electron transfer to forty-eight human CYP enzymes by the FMN hydroquinone of CYPOR is well-known, the role of the linker between the NH-terminus membrane-binding domain (MBD) and FMN domain in supporting the activity of P450 enzymes remains poorly understood. Here we demonstrate that a linker with at least eight residues is required to form a functional CYPOR-CYP2B4 complex.
View Article and Find Full Text PDFCytochrome P450 reductase (CYPOR) provides electrons to all human microsomal cytochrome P450s (cyt P450s). The length and sequence of the "140s" FMN binding loop of CYPOR has been shown to be a key determinant of its redox potential and activity with cyt P450s. Shortening the "140s loop" by deleting glycine-141(ΔGly141) and by engineering a second mutant that mimics flavo-cytochrome P450 BM3 (ΔGly141/Glu142Asn) resulted in mutants that formed an unstable anionic semiquinone.
View Article and Find Full Text PDFMicrosomal cytochrome b5 (cytb5) is a membrane-bound protein capable of donating the second electron to cytochrome P450s (cytP450s) in the cytP450s monooxygenase reactions. Recent studies have demonstrated the importance of the transmembrane domain of cytb5 in the interaction with cytP450 by stabilizing its monomeric structure. While recent NMR studies have provided high-resolution insights into the structural interactions between the soluble domains of ~16-kDa cytb5 and ~57-kDa cytP450 in a membrane environment, there is need for studies to probe the residues in the transmembrane region as well as to obtain intermolecular distance constraints to better understand the very large size cytb5-cytP450 complex structure in a near native membrane environment.
View Article and Find Full Text PDFAlthough there is a growing interest in using polymer lipid-nanodiscs, the polymer charge poses limitations for studies on membrane proteins. Here, we demonstrate the functional reconstitution of a large soluble-domain containing positively-charged ∼57 kDa cytochrome-P450 and negatively-charged ∼16 kDa cytochrome-b5 in lipid-nanodiscs, and the role of the polymer charge for high-resolution studies on membrane proteins.
View Article and Find Full Text PDFLipids are critical for the function of membrane proteins. NADPH-cytochrome-P450-reductase, the sole electron transferase for microsomal oxygenases, possesses a conformational dynamics entwined with its topology. Here, we use peptide-nanodiscs to unveil cytochrome-P450-reductase's lipid boundaries, demonstrating a protein-driven enrichment of ethanolamine lipids (by 25%) which ameliorates by 3-fold CPR's electron-transfer ability.
View Article and Find Full Text PDFInvestigating the interplay between cytochrome-P450 and its redox partners (CPR and cytochrome-b5) is vital for understanding the metabolism of most hydrophobic drugs. Dynamic structural interactions with the ternary complex, with and without substrates, captured by NMR reveal a gating mechanism for redox partners to promote P450 function.
View Article and Find Full Text PDFStructural interactions that enable electron transfer to cytochrome-P450 (CYP450) from its redox partner CYP450-reductase (CPR) are a vital prerequisite for its catalytic mechanism. The first structural model for the membrane-bound functional complex to reveal interactions between the full-length CYP450 and a minimal domain of CPR is now reported. The results suggest that anchorage of the proteins in a lipid bilayer is a minimal requirement for CYP450 catalytic function.
View Article and Find Full Text PDFAlthough membrane environment is known to boost drug metabolism by mammalian cytochrome P450s, the factors that stabilize the structural folding and enhance protein function are unclear. In this study, we use peptide-based lipid nanodiscs to "trap" the lipid boundaries of microsomal cytochrome P450 2B4. We report the first evidence that CYP2B4 is able to induce the formation of raft domains in a biomimetic compound of the endoplasmic reticulum.
View Article and Find Full Text PDFConformational 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.
View Article and Find Full Text PDFHeme's spin-multiplicity is key in determining the enzymatic function of cytochrome P450 (cytP450). The origin of the low-spin state in ferric P450 is still under debate. Here, we report the first experimental demonstration of P450's membrane interaction altering its spin equilibrium which is accompanied by a stronger affinity for cytochrome b.
View Article and Find Full Text PDFCytochrome b (cytb ) is a membrane protein vital for the regulation of cytochrome P450 (cytP450) metabolism and is capable of electron transfer to many redox partners. Here, using cyt c as a surrogate for cytP450, we report the effect of membrane on the interaction between full-length cytb and cyt c for the first time. As shown through stopped-flow kinetic experiments, electron transfer capable cytb - cyt c complexes were formed in the presence of bicelles and nanodiscs.
View Article and Find Full Text PDFThe dynamic protein-protein and protein-ligand interactions of integral bitopic membrane proteins with a single membrane-spanning helix play a plethora of vital roles in the cellular processes associated with human health and diseases, including signaling and enzymatic catalysis. While an increasing number of high-resolution structural studies of membrane proteins have successfully manifested an in-depth understanding of their biological functions, intact membrane-bound bitopic protein-protein complexes pose tremendous challenges for structural studies by crystallography or solution NMR spectroscopy. Therefore, there is a growing interest in developing approaches to investigate the functional interactions of bitopic membrane proteins embedded in lipid bilayers at atomic-level.
View Article and Find Full Text PDFMicrosomal cytochromes P450 (P450) require two electrons and two protons for the oxidation of substrates. Although the two electrons can be provided by cytochrome P450 reductase, the second electron can also be donated by cytochrome b5 (b5). The steady-state activity of P450 2B4 is increased up to 10-fold by b5.
View Article and Find Full Text PDFHuman cytochrome P450 17A1 is required for all androgen biosynthesis and is the target of abiraterone, a drug used widely to treat advanced prostate cancer. P450 17A1 catalyzes both 17-hydroxylation and subsequent 17,20-lyase reactions with pregnenolone, progesterone, and allopregnanolone. The presence of cytochrome b5 (b5) markedly stimulates the 17,20-lyase reaction, with little effect on 17-hydroxylation; however, the mechanism of this b5 effect is not known.
View Article and Find Full Text PDFNADPH-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.
View Article and Find Full Text PDFCytochrome P450s (P450s) are a superfamily of enzymes responsible for the catalysis of a wide range of substrates. Dynamic interactions between full-length membrane-bound P450 and its redox partner cytochrome b5 (cytb5 ) have been found to be important for the enzymatic activity of P450. However, the stability of the circa 70 kDa membrane-bound complex in model membranes renders high-resolution structural NMR studies particularly difficult.
View Article and Find Full Text PDFCrystallographic studies have shown that the F429H mutation of cytochrome P450 2B4 introduces an H-bond between His429 and the proximal thiolate ligand, Cys436, without altering the protein fold but sharply decreases the enzymatic activity and stabilizes the oxyferrous P450 2B4 complex. To characterize the influence of this hydrogen bond on the states of the catalytic cycle, we have used radiolytic cryoreduction combined with electron paramagnetic resonance (EPR) and (electron-nuclear double resonance (ENDOR) spectroscopy to study and compare their characteristics for wild-type (WT) P450 2B4 and the F429H mutant. (i) The addition of an H-bond to the axial Cys436 thiolate significantly changes the EPR signals of both low-spin and high-spin heme-iron(III) and the hyperfine couplings of the heme-pyrrole (14)N but has relatively little effect on the (1)H ENDOR spectra of the water ligand in the six-coordinate low-spin ferriheme state.
View Article and Find Full Text PDFMammalian cytochrome P450 (P450) is a membrane-bound monooxygenase whose catalytic activities require two electrons to be sequentially delivered from its redox partners: cytochrome b5 (cytb5) and cytochrome P450 reductase, both of which are membrane proteins. Although P450 functional activities are known to be affected by lipids, experimental evidence to reveal the effect of membrane on P450-cytb5 interactions is still lacking. Here, we present evidence for the influence of phospholipid bilayers on complex formation between rabbit P450 2B4 (CYP2B4) and rabbit cytb5 at the atomic level, utilizing NMR techniques.
View Article and Find Full Text PDFMammalian cytochrome b5 (cyt b5) is a membrane-bound protein capable of donating an electron to cytochrome P450 (P450) in the P450 catalytic cycle. The interaction between cyt b5 and P450 has been reported to be affected by the substrates of P450; however, the mechanism of substrate modulation on the cyt b5-P450 complex formation is still unknown. In this study, the complexes between full-length rabbit cyt b5 and full-length substrate-free/substrate-bound cytochrome P450 2B4 (CYP2B4) are investigated using NMR techniques.
View Article and Find Full Text PDFThree-dimensional structure determination of membrane proteins is important to fully understand their biological functions. However, obtaining a high-resolution structure has been a major challenge mainly due to the difficulties in retaining the native folding and function of membrane proteins outside of the cellular membrane environment. These challenges are acute if the protein contains a large soluble domain, as it needs bulk water unlike the transmembrane domains of an integral membrane protein.
View Article and Find Full Text PDFCytochrome 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.
View Article and Find Full Text PDFMultidomain enzymes often rely on large conformational motions to function. However, the conformational setpoints, rates of domain motions and relationships between these parameters and catalytic activity are not well understood. To address this, we determined and compared the conformational setpoints and the rates of conformational switching between closed unreactive and open reactive states in four mammalian diflavin NADPH oxidoreductases that catalyze important biological electron transfer reactions: cytochrome P450 reductase, methionine synthase reductase and endothelial and neuronal nitric oxide synthase.
View Article and Find Full Text PDFThe 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.
View Article and Find Full Text PDFWhile an increasing number of structural biology studies successfully demonstrate the power of high-resolution structures and dynamics of membrane proteins in fully understanding their function, there is considerable interest in developing NMR approaches to obtain such information in a cellular setting. As long as the proteins inside the living cell tumble rapidly in the NMR timescale, recently developed in-cell solution NMR approaches can provide 3D structural information. However, there are numerous challenges to study membrane proteins inside a cell.
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