Kinetics of Intermediate Release Enhances P450 11B2-Catalyzed Aldosterone Synthesis.

The mitochondrial enzyme cytochrome P450 11B2 (aldosterone synthase) catalyzes the 3 terminal transformations in the biosynthesis of aldosterone from 11-deoxycorticosterone (DOC): 11ß-hydroxylation to corticosterone, 18-hydroxylation, and 18-oxidation. Prior studies have shown that P450 11B2 produces more aldosterone from DOC than from the intermediate corticosterone and that the reaction sequence is processive, with intermediates remaining bound to the active site between oxygenation reactions. In contrast, P450 11B1 (11ß-hydroxylase), which catalyzes the terminal step in cortisol biosynthesis, shares a 93% amino acid sequence identity with P450 11B2, converts DOC to corticosterone, but cannot synthesize aldosterone from DOC. The biochemical and biophysical properties of P450 11B2, which enable its unique 18-oxygenation activity and processivity, yet are not also represented in P450 11B1, remain unknown. To understand the mechanism of aldosterone biosynthesis, we introduced point mutations at residue 320, which partially exchange the activities of P450 11B1 and P450 11B2 (V320A and A320V, respectively). We then investigated NADPH coupling efficiencies, binding kinetics and affinities, and product formation of purified P450 11B1 and P450 11B2, wild-type, and residue 320 mutations in phospholipid vesicles and nanodiscs. Coupling efficiencies for the 18-hydroxylase reaction with corticosterone as the substrate failed to correlate with aldosterone synthesis, ruling out uncoupling as a relevant mechanism. Conversely, corticosterone dissociation rates correlated inversely with aldosterone production. We conclude that intermediate dissociation kinetics, not coupling efficiency, enable P450 11B2 to synthesize aldosterone via a processive mechanism. Our kinetic data also suggest that the binding of DOC to P450 11B enzymes occurs in at least two distinct steps, favoring an induced-fit mechanism.

Non-Ionic Inulin-Based Polymer Nanodiscs Enable Functional Reconstitution of a Redox Complex Composed of Oppositely Charged CYP450 and CPR in a Lipid Bilayer Membrane.

Although polymer-based lipid nanodiscs are increasingly used in the structural studies of membrane proteins, the charge of the belt-forming polymer is a major limitation for functional reconstitution of membrane proteins possessing an opposite net charge to that of the polymer. This limitation also rules out the reconstitution of a protein-protein complex composed of oppositely charged membrane proteins. In this study, we report the first successful functional reconstitution of a membrane-bound redox complex constituting a cationic cytochrome P450 (CYP450) and an anionic cytochrome P450 reductase (CPR) in non-ionic inulin-based lipid nanodiscs. The gel-to-liquid-crystalline phase-transition temperature (Tm) of DMPC:DMPG (7:3 w/w) lipids in polymer nanodiscs was determined by differential scanning calorimetry (DSC) and 31P NMR experiments. The CYP450-CPR redox complex reconstitution in polymer nanodiscs was characterized by size-exclusion chromatography (SEC), and the electron transfer kinetics was carried out using the stopped-flow technique under anaerobic conditions. The Tm of DMPC:DMPG (7:3 w/w) in polymer nanodiscs measured from 31P NMR agrees with that obtained from DSC and was found to be higher than that for liposomes due to the decreased cooperativity of lipids present in the nanodiscs. The stopped-flow measurements revealed the CYP450-CPR redox complex reconstituted in nanodiscs to be functional, and the electron transfer kinetics was found to be temperature-dependent. Based on the successful demonstration of the use of non-ionic inulin-based polymer nanodiscs reported in this study, we expect them to be useful in studying the function and structures of a variety of membrane proteins/complexes irrespective of the charge of the molecular components. Since the polymer nanodiscs were shown to align in an externally applied magnetic field, they can also be used to measure residual dipolar couplings (RDCs) and residual quadrupolar couplings (RQCs) for various molecules ranging from small molecules to soluble proteins and nucleic acids.

A Minimal Functional Complex of Cytochrome P450 and FBD of Cytochrome P450 Reductase in Nanodiscs.

Structural 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. Akin to cytochrome-b5 (cyt-b5 ), Arg 125 on the C-helix of CYP450s is found to be important for effective electron transfer, thus supporting the competitive behavior of redox partners for CYP450s. A general approach is presented to study protein-protein interactions combining the use of nanodiscs with NMR spectroscopy and SAXS. Linking structural details to the mechanism will help unravel the xenobiotic metabolism of diverse microsomal CYP450s in their native environment and facilitate the design of new drug entities.

Cytochrome-P450-Induced Ordering of Microsomal Membranes Modulates Affinity for Drugs.

Although 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. NMR experiments were used to identify and quantitatively determine the lipids present in nanodiscs. A combination of biophysical experiments and molecular dynamics simulations revealed a sphingomyelin binding region in CYP2B4. The protein-induced lipid raft formation increased the thermal stability of P450 and dramatically altered ligand binding kinetics of the hydrophilic ligand BHT. These results unveil membrane/protein dynamics that contribute to the delicate mechanism of redox catalysis in lipid membrane.

Cytochrome b5 Activates the 17,20-Lyase Activity of Human Cytochrome P450 17A1 by Increasing the Coupling of NADPH Consumption to Androgen Production.

Human 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. We determined the influence of b5 on coupling efficiency-defined as the ratio of product formation to NADPH consumption-in a reconstituted system using these 3 pairs of substrates for the 2 reactions. Rates of NADPH consumption ranged from 4 to 13 nmol/min/nmol P450 with wild-type P450 17A1. For the 17-hydroxylase reaction, progesterone oxidation was the most tightly coupled (∼50%) and negligibly changed upon addition of b5. Rates of NADPH consumption were similar for the 17-hydroxylase and corresponding 17,20-lyase reactions for each steroid series, and b5 only slightly increased NADPH consumption. For the 17,20-lyase reactions, b5 markedly increased product formation and coupling in parallel with all substrates, from 6% to 44% with the major substrate 17-hydroxypregnenolone. For the naturally occurring P450 17A1 mutations E305G and R347H, which impair 17,20-lyase activity, b5 failed to rescue the poor coupling with 17-hydroxypregnenolone (2-4%). When the conserved active-site threonine was mutated to alanine (T306A), both the activity and coupling were markedly decreased with all substrates. We conclude that b5 stimulation of the 17,20-lyase reaction primarily derives from more efficient use of NADPH for product formation rather than side products.

Effects of membrane mimetics on cytochrome P450-cytochrome b5 interactions characterized by NMR spectroscopy.

Mammalian 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. General line broadening and modest chemical shift perturbations of cytb5 resonances characterize CYP2B4-cytb5 interactions on the intermediate time scale. More significant intensity attenuation and a more specific protein-protein binding interface are observed in bicelles as compared with lipid-free solution, highlighting the importance of the lipid bilayer in stabilizing stronger and more specific interactions between CYP2B4 and cytb5, which may lead to a more efficient electron transfer. Similar results observed for the interactions between CYP2B4 lacking the transmembrane domain (tr-CYP2B4) and cytb5 imply interactions between tr-CYP2B4 and the membrane surface, which might assist in CYP2B4-cytb5 complex formation by orienting tr-CYP2B4 for efficient contact with cytb5. Furthermore, the observation of weak and nonspecific interactions between CYP2B4 and cytb5 in micelles suggests that lipid bilayer structures and low curvature membrane surface are preferable for CYP2B4-cytb5 complex formation. Results presented in this study provide structural insights into the mechanism behind the important role that the lipid bilayer plays in the interactions between P450s and their redox partners.

Cellular solid-state NMR investigation of a membrane protein using dynamic nuclear polarization.

While 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. Research in our laboratory is focused on developing a combination of solid-state NMR and biological approaches to overcome these challenges in order to obtain high-resolution structural insights into electron transfer processes mediated by membrane-bound proteins like mammalian cytochrome-b5, cytochrome-P450 and cytochrome-P450-reductase. In this study, we demonstrate the feasibility of using dynamic nuclear polarization (DNP) magic angle spinning (MAS) NMR spectroscopy for in-cell studies on a membrane-anchored protein. Our experimental results obtained from ¹³C-labeled membrane-anchored cytochrome-b5 in native Escherichia coli cells show a ~16-fold DNP signal enhancement. Further, results obtained from a 2D ¹³C/¹³C chemical shift correlation MAS experiment demonstrate the feasibility of suppressing the background signals from other cellular contents for high-resolution structural studies on membrane proteins. We believe that this study would pave new avenues for high-resolution structural studies on a variety of membrane-associated proteins and their complexes in the cellular context to fully understand their functional roles in physiological processes.

Reconstitution of the Cytb5-CytP450 Complex in Nanodiscs for Structural Studies using NMR Spectroscopy.

Cytochrome 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. To overcome these challenges, reconstitution of the P450-cytb5 complex in peptide-based nanodiscs, containing no detergents, has been demonstrated, which are characterized by size exclusion chromatography and NMR spectroscopy. In addition, NMR experiments are used to identify the binding interface of the P450-cytb5 complex in the nanodisc. This is the first successful demonstration of a protein-protein complex in a nanodisc using NMR structural studies and should be useful to obtain valuable structural information on membrane-bound protein complexes.

Temperature-resistant bicelles for structural studies by solid-state NMR spectroscopy.

Three-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. For structural studies on such proteins either by nuclear magnetic resonance (NMR) spectroscopy or X-ray crystallography, bicelles have been demonstrated to be superior to conventional micelles, yet their temperature restrictions attributed to their thermal instabilities are a major disadvantage. Here, we report an approach to overcome this drawback through searching for an optimum combination of bicellar compositions. We demonstrate that bicelles composed of 1,2-didecanoyl-sn-glycero-3-phosphocholine (DDPC) and 1,2-diheptanoyl-sn-glycero-3-phosphocholin (DHepPC), without utilizing additional stabilizing chemicals, are quite stable and are resistant to temperature variations. These temperature-resistant bicelles have a robust bicellar phase and magnetic alignment over a broad range of temperatures, between -15 and 80 °C, retain the native structure of a membrane protein, and increase the sensitivity of solid-state NMR experiments performed at low temperatures. Advantages of two-dimensional separated-local field (SLF) solid-state NMR experiments at a low temperature are demonstrated on magnetically aligned bicelles containing an electron carrier membrane protein, cytochrome b5. Morphological information on different DDPC-based bicellar compositions, varying q ratio/size, and hydration levels obtained from (31)P NMR experiments in this study is also beneficial for a variety of biophysical and spectroscopic techniques, including solution NMR and magic-angle-spinning (MAS) NMR for a wide range of temperatures.

Probing the transmembrane structure and dynamics of microsomal NADPH-cytochrome P450 oxidoreductase by solid-state NMR.

NADPH-cytochrome P450 oxidoreductase (CYPOR) is an essential redox partner of the cytochrome P450 (cyt P450) superfamily of metabolic enzymes. In the endoplasmic reticulum of liver cells, such enzymes metabolize ~75% of the pharmaceuticals in use today. It is known that the transmembrane domain of CYPOR plays a crucial role in aiding the formation of a complex between CYPOR and cyt P450. Here we present the transmembrane structure, topology, and dynamics of the FMN binding domain of CYPOR in a native membrane-like environment. Our solid-state NMR results reveal that the N-terminal transmembrane domain of CYPOR adopts an α-helical conformation in the lipid membrane environment. Most notably, we also show that the transmembrane helix is tilted ~13° from the lipid bilayer normal, and exhibits motions on a submillisecond timescale including rotational diffusion of the whole helix and fluctuation of the helical director axis. The approaches and the information reported in this study would enable further investigations on the structure and dynamics of the full-length NADPH-cytochrome P450 oxidoreductase and its interaction with other membrane proteins in a membrane environment.

A model of the membrane-bound cytochrome b5-cytochrome P450 complex from NMR and mutagenesis data.

Microsomal cytochrome b5 (cytb5) is a membrane-bound protein that modulates the catalytic activity of its redox partner, cytochrome P4502B4 (cytP450). Here, we report the first structure of full-length rabbit ferric microsomal cytb5 (16 kDa), incorporated in two different membrane mimetics (detergent micelles and lipid bicelles). Differential line broadening of the cytb5 NMR resonances and site-directed mutagenesis data were used to characterize the cytb5 interaction epitope recognized by ferric microsomal cytP450 (56 kDa). Subsequently, a data-driven docking algorithm, HADDOCK (high ambiguity driven biomolecular docking), was used to generate the structure of the complex between cytP4502B4 and cytb5 using experimentally derived restraints from NMR, mutagenesis, and the double mutant cycle data obtained on the full-length proteins. Our docking and experimental results point to the formation of a dynamic electron transfer complex between the acidic convex surface of cytb5 and the concave basic proximal surface of cytP4502B4. The majority of the binding energy for the complex is provided by interactions between residues on the C-helix and ß-bulge of cytP450 and residues at the end of helix α4 of cytb5. The structure of the complex allows us to propose an interprotein electron transfer pathway involving the highly conserved Arg-125 on cytP450 serving as a salt bridge between the heme propionates of cytP450 and cytb5. We have also shown that the addition of a substrate to cytP450 likely strengthens the cytb5-cytP450 interaction. This study paves the way to obtaining valuable structural, functional, and dynamic information on membrane-bound complexes.

Adrenodoxin allosterically alters human cytochrome P450 11B enzymes to accelerate substrate binding and decelerate release.

Two human mitochondrial membrane CYP11B enzymes play a pivotal role in steroidogenesis. CYP11B1 generates the major glucocorticoid cortisol, while CYP11B2 catalysis yields the primary mineralocorticoid aldosterone. Catalysis by both requires electron delivery by a soluble iron-sulfur adrenodoxin redox partner. However recent studies have shown that adrenodoxin/CYP11B interaction alone allosterically increases substrate and inhibitor affinity as exhibited by decreased dissociation constant (K d) values. The current study moves beyond such equilibrium studies, by defining adrenodoxin effects on the rates of P450 ligand binding and release separately. Stopped-flow data clearly demonstrate that adrenodoxin interaction with the P450 proximal surfaces increases ligand binding in both P450 CYP11B active sites by increasing the on rate constant and decreasing the off rate constant. As substrate entry and exit from the sequestered P450 active site requires conformational changes on the distal side of the P450 enzyme, a likely explanation is that adrenodoxin binding allosterically modulates CYP11B conformational changes. The 93% identical CYP11B enzymes can bind and hydroxylate each other's native substrates differing only by a hydroxyl. However, CYP11B1 exhibits monophasic substrate binding and CYP11B2 biphasic substrate binding, even when the substrates are swapped. This indicates that small differences in amino acid sequence between human CYP11B1 and CYP11B2 enzymes are more functionally important in ligand binding and could suggest avenues for more selective inhibition of these drug targets. Both protein/protein interactions and protein/substrate interactions are most likely to act by modulating CYP11B conformational dynamics.

Characterization of nanodisc-forming peptides for membrane protein studies.

Lipid-bilayer nanodiscs provide a stable, native-like membrane environment for the functional and structural studies of membrane proteins and other membrane-binding molecules. Peptide-based nanodiscs having unique properties are developed for membrane protein studies and other biological applications. While the self-assembly process rendering the formation of peptide-nanodiscs is attractive, it is important to understand the stability and suitability of these nanodisc systems for membrane protein studies. In this study, we investigated the nanodiscs formation by the anti-inflammatory and tumor-suppressing peptide AEM28. AEM28 is a chimeric peptide containing a cationic-rich heparan sulfate proteoglycan- (HSPG)-binding domain from human apolipoprotein E (hapoE) (141-150) followed by the 18A peptide's amino acid sequence. AEM28-based nanodiscs made with different types of lipids were characterized using various biophysical techniques and compared with the nanodiscs formed using 2F or 4F peptides. Variable temperature dynamic light-scattering and 31P NMR experiments indicated the fusion and size heterogeneity of nanodiscs at high temperatures. The suitability of AEM28 and Ac-18A-NH2- (2F-) based nanodiscs for studying membrane proteins is demonstrated by reconstituting and characterizing a drug-metabolizing enzyme, cytochrome-P450 (CYP450), or the redox complex CYP450-CYP450 reductase. AEM28 and 2F were also tested for their efficacies in solubilizing E. coli membranes to understand the possibility of using them for detergent-free membrane protein isolation. Our experimental results suggest that AEM28 nanodiscs are suitable for studying membrane proteins with a net positive charge, whereas 2F-based nanodiscs are compatible with any membrane proteins and their complexes irrespective of their charge. Furthermore, both peptides solubilized E. coli cell membranes, indicating their use in membrane protein isolation and other applications related to membrane solubilization.

Influence of cholesterol on kinetic parameters for human aromatase (P450 19A1) in phospholipid nanodiscs.

Cholesterol, a significant constituent of the endoplasmic reticulum membrane, exerts a substantial effect on the membrane's biophysical and mechanical properties. Cholesterol, however, is often neglected in model systems used to study membrane-bound proteins. For example, the influence of cholesterol on the enzymatic functions of type 2 cytochromes P450, which require a phospholipid bilayer and the redox partner P450-oxidoreductase (POR) for activity, are rarely investigated. Human aromatase (P450 19A1) catalyzes three sequential oxygenations of 19­carbon steroids to estrogens and is widely expressed across various tissues, which are characterized by varying cholesterol compositions. Our study examined the impact of cholesterol on the functionality of the P450 19A1 complex with POR. Nanodiscs containing P450 19A1 with 20% cholesterol/80% phospholipid had similar rates and affinity of androstenedione binding as phospholipid-only P450 19A1 nanodiscs, and rates of product formation were indistinguishable among these conditions. In contrast, the rate of the first electron transfer from POR to P450 19A1 was 3-fold faster in cholesterol-containing nanodiscs than in phospholipid-only nanodiscs. These results suggest that cholesterol influences some aspects of POR interaction with P450 19A1 and might serve as an additional regulatory mechanism in this catalytic system.

Bacterial expression, purification, and reconstitution of human steroid 5α-reductases in phospholipid liposomes and nanodiscs.

The two human steroid 5α-reductase (5αR) enzymes catalyze the conversion 3-keto-Δ4-steroids to their 5α-reduced congeners. In the genital skin and prostate, the type 2 isoenzyme converts testosterone (T) to the more potent androgen 5α-dihydrotestosterone (DHT), and intracellular DHT is essential for the morphogenesis of the undifferentiated external genitalia to the male phenotype. Both isoenzymes also metabolize other 19- and 21-carbon 3-keto-Δ4-steroids, both endogenous compounds and some steroid-based drugs. Rigorous biochemical studies have been limited due to the extremely hydrophobic nature of these proteins. We have described the heterologous expression of these enzymes in bacteria, their purification with affinity chromatography, and the reconstitution of activity in liposomes. This article details these procedures, as well as reconstitution in phospholipid nanodiscs and enzyme assay.

Conformational changes of NADPH-cytochrome P450 oxidoreductase are essential for catalysis and cofactor binding.

The crystal structure of NADPH-cytochrome P450 reductase (CYPOR) implies that a large domain movement is essential for electron transfer from NADPH via FAD and FMN to its redox partners. To test this hypothesis, a disulfide bond was engineered between residues Asp(147) and Arg(514) in the FMN and FAD domains, respectively. The cross-linked form of this mutant protein, designated 147CC514, exhibited a significant decrease in the rate of interflavin electron transfer and large (≥90%) decreases in rates of electron transfer to its redox partners, cytochrome c and cytochrome P450 2B4. Reduction of the disulfide bond restored the ability of the mutant to reduce its redox partners, demonstrating that a conformational change is essential for CYPOR function. The crystal structures of the mutant without and with NADP(+) revealed that the two flavin domains are joined by a disulfide linkage and that the relative orientations of the two flavin rings are twisted ∼20° compared with the wild type, decreasing the surface contact area between the two flavin rings. Comparison of the structures without and with NADP(+) shows movement of the Gly(631)-Asn(635) loop. In the NADP(+)-free structure, the loop adopts a conformation that sterically hinders NADP(H) binding. The structure with NADP(+) shows movement of the Gly(631)-Asn(635) loop to a position that permits NADP(H) binding. Furthermore, comparison of these mutant and wild type structures strongly suggests that the Gly(631)-Asn(635) loop movement controls NADPH binding and NADP(+) release; this loop movement in turn facilitates the flavin domain movement, allowing electron transfer from FMN to the CYPOR redox partners.

Stabilization and spectroscopic characterization of the dioxygen complex of wild-type cytochrome P4502B4 (CYP2B4) and its distal side E301Q, T302A and proximal side F429H mutants at subzero temperatures.

Mammalian cytochrome P450 2B4 (CYP2B4) is a phenobarbital-inducible rabbit hepatic monooxygenase that catalyzes the N-demethylation of benzphetamine and metabolism of numerous other compounds. To probe the interactions of the heme environment and bound benzphetamine with the dioxygen (O2) complex of CYP2B4, homogeneous O2 complexes of the wild-type enzyme and three mutants at sites of conserved amino acids, two on the heme distal side (T302A and E301Q) and one on the proximal side (F429H), have been prepared and stabilized at ~-50°C in mixed solvents (60-70% v/v glycerol). We report that the magnetic circular dichroism and electronic absorption spectra of wild-type oxyferrous CYP2B4, in the presence and absence of substrate, are quite similar to those of the dioxygen complex of bacterial cytochrome P450-CAM (CYP101). However, the oxyferrous complexes of the T302A and E301Q CYP2B4 mutants have significantly perturbed electronic structure (~4 nm and ~3 nm red-shifted Soret features, respectively) compared to that of the wild-type oxyferrous complex. On the other hand, the heme proximal side mutant, CYP2B4 F429H, undergoes relatively facile conversion to a partially (~50%) denatured (P420) form upon reduction. The structural changes in the heme pocket environments of the CYP2B4 mutants that lead to the spectroscopic distinctions reported herein can be related to the differences in oxidation activities of wild-type CYP2B4 and its E301Q, T302A and F429H mutants.

The interaction of microsomal cytochrome P450 2B4 with its redox partners, cytochrome P450 reductase and cytochrome b(5).

Cytochrome P450 2B4 is a microsomal protein with a multi-step reaction cycle similar to that observed in the majority of other cytochromes P450. The cytochrome P450 2B4-substrate complex is reduced from the ferric to the ferrous form by cytochrome P450 reductase. After binding oxygen, the oxyferrous protein accepts a second electron which is provided by either cytochrome P450 reductase or cytochrome b(5). In both instances, product formation occurs. When the second electron is donated by cytochrome b(5), catalysis (product formation) is ∼10- to 100-fold faster than in the presence of cytochrome P450 reductase. This allows less time for side product formation (hydrogen peroxide and superoxide) and improves by ∼15% the coupling of NADPH consumption to product formation. Cytochrome b(5) has also been shown to compete with cytochrome P450 reductase for a binding site on the proximal surface of cytochrome P450 2B4. These two different effects of cytochrome b(5) on cytochrome P450 2B4 reactivity can explain how cytochrome b(5) is able to stimulate, inhibit, or have no effect on cytochrome P450 2B4 activity. At low molar ratios (<1) of cytochrome b(5) to cytochrome P450 reductase, the more rapid catalysis results in enhanced substrate metabolism. In contrast, at high molar ratios (>1) of cytochrome b(5) to cytochrome P450 reductase, cytochrome b(5) inhibits activity by binding to the proximal surface of cytochrome P450 and preventing the reductase from reducing ferric cytochrome P450 to the ferrous protein, thereby aborting the catalytic reaction cycle. When the stimulatory and inhibitory effects of cytochrome b(5) are equal, it will appear to have no effect on the enzymatic activity. It is hypothesized that cytochrome b(5) stimulates catalysis by causing a conformational change in the active site, which allows the active oxidizing oxyferryl species of cytochrome P450 to be formed more rapidly than in the presence of reductase.

INEPT-based separated-local-field NMR spectroscopy: a unique approach to elucidate side-chain dynamics of membrane-associated proteins.

Despite recent advances in NMR approaches for structural biology, determination of membrane protein dynamics in its native environment continues to be a monumental challenge, as most NMR structural studies of membrane proteins are commonly carried out either in micelles or in vesicle systems under frozen conditions. To overcome this difficulty, we propose a solid-state NMR technique that allows for the determination of side-chain dynamics from membrane proteins in lipid bilayers. This new technique, namely dipolar enhanced polarization transfer (DREPT), allows for a wide range of dipolar couplings to be encoded, providing high resolution and sensitivity for systems that undergo motional averaging such as that of amino acid side chains. NMR observables such as dipolar couplings and chemical shift anisotropy, which are highly sensitive to molecular motions, provide a direct way of probing protein dynamics over a wide range of time scales. Therefore, using an appropriate model, it is possible to determine side-chain dynamics and provide additional information on the topology and function of a membrane protein in its native environment.

Proton-evolved local-field solid-state NMR studies of cytochrome b5 embedded in bicelles, revealing both structural and dynamical information.

Structural biology of membrane proteins has rapidly evolved into a new frontier of science. Although solving the structure of a membrane protein with atomic-level resolution is still a major challenge, separated local field (SLF) NMR spectroscopy has become an invaluable tool in obtaining structural images of membrane proteins under physiological conditions. Recent studies have demonstrated the use of rotating-frame SLF techniques to accurately measure strong heteronuclear dipolar couplings between directly bonded nuclei. However, in these experiments, all weak dipolar couplings are suppressed. On the other hand, weak heteronuclear dipolar couplings can be measured using laboratory-frame SLF experiments, but only at the expense of spectral resolution for strongly dipolar coupled spins. In the present study, we implemented two-dimensional proton-evolved local-field (2D PELF) pulse sequences using either composite zero cross-polarization (COMPOZER-CP) or windowless isotropic mixing (WIM) for magnetization transfer. These PELF sequences can be used for the measurement of a broad range of heteronuclear dipolar couplings, allowing for a complete mapping of protein dynamics in a lipid bilayer environment. Experimental results from magnetically aligned bicelles containing uniformly (15)N-labeled cytochrome b(5) are presented and theoretical analyses of the new PELF sequences are reported. Our results suggest that the PELF-based experimental approaches will have a profound impact on solid-state NMR spectroscopy of membrane proteins and other membrane-associated molecules in magnetically aligned bicelles.