Resonance surface plasmon spectroscopy: low molecular weight substrate binding to cytochrome p450

A new detection mechanism has been developed for low molecular weight substrate binding to heme proteins based on resonance localized surface plasmon spectroscopy. Cytochrome P450 has strong electronic transitions in the visible wavelength region. Upon binding of a substrate molecule (e.g., camphor), the absorption band of cytochrome P450 shifts to shorter wavelength. The event of camphor binding to a nanoparticle surface modified with cytochrome P450 protein receptors is monitored using UV-vis spectroscopy. It is observed for the first time that the binding of the substrate molecules to the protein receptor induces a blue-shift in the localized surface plasmon resonance (LSPR) of the nanosensors. The coupling between the molecular resonance of the substrate-free and substrate-bound cytochrome P450 proteins and the nanoparticles' LSPR leads to a highly wavelength-dependent LSPR response. When the LSPR of the nanoparticles is located at a wavelength distant from the cytochrome P450 resonance, an average of approximately 19 nm red-shift is observed upon cytochrome P450 binding to the nanoparticles and a approximately 6 nm blue-shift is observed upon camphor binding However, this response is significantly amplified approximately 3 to 5 times when the LSPR of the nanoparticles is located at a slightly longer wavelength than the cytochrome P450 resonance, that is, a 66.2 nm red-shift upon cytochrome P450 binding and a 34.7 nm blue-shift upon camphor binding. This is the first example of the detection of small molecules binding to a protein modified nanoparticle surface on the basis of LSPR.     

Molecular recognition in (+)-alpha-pinene oxidation by cytochrome P450cam

Oxygenated derivatives of the monoterpene (+)-alpha-pinene are found in plant essential oils and used as fragrances and flavorings. (+)-alpha-Pinene is structurally related to (+)-camphor, the natural substrate of the heme monooxygenase cytochrome P450(cam) from Pseudomonas putida. The aim of the present work was to apply the current understanding of P450 substrate binding and catalysis to engineer P450(cam) for the selective oxidation of (+)-alpha-pinene. Consideration of the structures of (+)-camphor and (+)-alpha-pinene lead to active-site mutants containing combinations of the Y96F, F87A, F87L, F87W, and V247L mutations. All mutants showed greatly enhanced binding and rate of oxidation of (+)-alpha-pinene. Some mutants had tighter (+)-alpha-pinene binding than camphor binding by the wild-type. The most active was the Y96F/V247L mutant, with a (+)-alpha-pinene oxidation rate of 270 nmol (nmol of P450(cam))(-)(1) min(-)(1), which was 70% of the rate of camphor oxidation by wild-type P450(cam). Camphor is oxidized by wild-type P450(cam) exclusively to 5-exo-hydroxycamphor. If the gem dimethyl groups of (+)-alpha-pinene occupied similar positions to those found for camphor in the wild-type structure, (+)-cis-verbenol would be the dominant product. All P450(cam) enzymes studied gave (+)-cis-verbenol as the major product but with much reduced selectivity compared to camphor oxidation by the wild-type. (+)-Verbenone, (+)-myrtenol, and the (+)-alpha-pinene epoxides were among the minor products. The crystal structure of the Y96F/F87W/V247L mutant, the most selective of the P450(cam) mutants initially examined, was determined to provide further insight into P450(cam) substrate binding and catalysis. (+)-alpha-Pinene was bound in two orientations which were related by rotation of the molecule. One orientation was similar to that of camphor in the wild-type enzyme while the other was significantly different. Analysis of the enzyme/substrate contacts suggested rationalizations of the product distribution. In particular competition rather than cooperativity between the F87W and V247L mutations and substrate movement during catalysis were proposed to be major factors. The crystal structure lead to the introduction of the L244A mutation to increase the selectivity of pinene oxidation by further biasing the binding orientation toward that of camphor in the wild-type structure. The F87W/Y96F/L244A mutant gave 86% (+)-cis-verbenol and 5% (+)-verbenone. The Y96F/L244A/V247L mutant gave 55% (+)-cis-verbenol but interestingly also 32% (+)-verbenone, suggesting that it may be possible to engineer a P450(cam) mutant that could oxidize (+)-alpha-pinene directly to (+)-verbenone. Verbenol, verbenone, and myrtenol are naturally occurring plant fragrance and flavorings. The preparation of these compounds by selective enzymatic oxidation of (+)-alpha-pinene, which is readily available in large quantities, could have applications in synthesis. The results also show that the protein engineering of P450(cam) for high selectivity of substrate oxidation is more difficult than achieving high substrate turnover rates because of the subtle and dynamic nature of enzyme-substrate interactions.     

How do azoles inhibit cytochrome P450 enzymes? A density functional study

To examine how azole inhibitors interact with the heme active site of the cytochrome P450 enzymes, we have performed a series of density functional theory studies on azole binding. These are the first density functional studies on azole interactions with a heme center and give fundamental insight into how azoles inhibit the catalytic function of P450 enzymes. Since azoles come in many varieties, we tested three typical azole motifs representing a broad range of azole and azole-type inhibitors: methylimidazolate, methyltriazolate, and pyridine. These structural motifs represent typical azoles, such as econazole, fluconazole, and metyrapone. The calculations show that azole binding is a stepwise mechanism whereby first the water molecule from the resting state of P450 is released from the sixth binding site of the heme to create a pentacoordinated active site followed by coordination of the azole nitrogen to the heme iron. This process leads to the breaking of a hydrogen bond between the resting state water molecule and the approaching inhibitor molecule. Although, formally, the water molecule is released in the first step of the reaction mechanism and a pentacoordinated heme is created, this does not lead to an observed spin state crossing. Thus, we show that release of a water molecule from the resting state of P450 enzymes to create a pentacoordinated heme will lead to a doublet to quartet spin state crossing at an Fe-OH(2) distance of approximately 3.0 A, while the azole substitution process takes place at shorter distances. Azoles bind heme with significantly stronger binding energies than a water molecule, so that these inhibitors block the catalytic cycle of the enzyme and prevent oxygen binding and the catalysis of substrate oxidation. Perturbations within the active site (e.g., a polarized environment) have little effect on the relative energies of azole binding. Studies with an extra hydrogen-bonded ethanol molecule in the model, mimicking the active site of the CYP121 P450, show that the resting state and azole binding structures are close in energy, which may lead to chemical equilibrium between the two structures, as indeed observed with recent protein structural studies that have demonstrated two distinct azole binding mechanisms to P450 heme.     

Computation of binding free energy with molecular dynamics and grand canonical Monte Carlo simulations

The binding of a ligand to a receptor is often associated with the displacement of a number of bound water molecules. When the binding site is exposed to the bulk region, this process may be sampled adequately by standard unbiased molecular dynamics trajectories. However, when the binding site is deeply buried and the exchange of water molecules with the bulk region may be difficult to sample, the convergence and accuracy in free energy perturbation (FEP) calculations can be severely compromised. These problems are further compounded when a reduced system including only the region surrounding the binding site is simulated. To address these issues, we couple molecular dynamics (MD) with grand canonical Monte Carlo (GCMC) simulations to allow the number of water to fluctuate during an alchemical FEP calculation. The atoms in a spherical inner region around the binding pocket are treated explicitly while the influence of the outer region is approximated using the generalized solvent boundary potential (GSBP). At each step during thermodynamic integration, the number of water in the inner region is equilibrated with GCMC and energy data generated with MD is collected. Free energy calculations on camphor binding to a deeply buried pocket in cytochrome P450cam, which causes about seven water molecules to be expelled, are used to test the method. It concluded that solvation free energy calculations with the GCMC/MD method can greatly improve the accuracy of the computed binding free energy compared to simulations with fixed number of water.     

Fluorescent probes for cytochrome p450 structural characterization and inhibitor screening

We have synthesized two luminescent probes (D-4-Ad and D-8-Ad) that target cytochrome P450cam. D-4-Ad luminescence is quenched by F?rster energy transfer upon binding (Kd = 0.83 muM) but is restored when the probe is displaced from the active site by camphor. In contrast, D-8-Ad (Kd approximately 0.02 muM) is not displaced from the enzyme, even in the presence of a large excess of camphor. The 2.2 A resolution crystal structure of the D-8-Ad:P450cam complex reveals extensive hydrophobic contacts between the probe and the enzyme, which result from the conformational flexibility of the B', F, and G helices. Probes with properties similar to those of D-4-Ad potentially could be useful for screening P450 inhibitors.     

Substrate binding favors enhanced NO binding to P450cam

Ferric cytochrome P450cam from Pseudomonas putida (P450cam) in buffer solution at physiological pH 7.4 reversibly binds NO to yield the nitrosyl complex P450cam(NO). The presence of 1R-camphor affects the dynamics of NO binding to P450cam and enhances the association and dissociation rate constants significantly. In the case of the substrate-free form of P450cam, subconformers are evident and the NO binding kinetics are much slower than in the presence of the substrate. The association and dissociation processes were investigated by both laser flash photolysis and stopped-flow techniques at ambient and high pressure. Large and positive values of S and V observed for NO binding to and release from the substrate-free P450cam complex are consistent with the operation of a limiting dissociative ligand substitution mechanism, where the lability of coordinated water dominates the reactivity of the iron(III)-heme center with NO. In contrast, NO binding to P450cam in the presence of camphor displays negative activation entropy and activation volume values that support a mechanism dominated by a bond formation process. Volume profiles for the binding of NO appear to be a valuable approach to explain the differences observed for P450cam in the absence and presence of the substrate and enable the clarification of the underlying reaction mechanisms at a molecular level. Changes in spin state of the iron center during the binding/release of NO contribute significantly to the observed volume effects. The results are discussed in terms of relevance for the biological function of cytochrome P450 and in context to other investigations of the related reactions between NO and imidazole- and thiolate-ligated iron(III) hemoproteins.     

A predictive pattern of computed barriers for C-h hydroxylation by compound I of cytochrome p450

The communication presents DFT calculations of 10 different C-H hydroxylation barriers by the active species of the enzyme cytochrome P450. The work demonstrates the existence of an excellent barrier-bond energy correlation. The so-obtained equation of the straight line is demonstrated to be useful for predicting barriers of related C-H activation processes, as well as for assessing barrier heights within the protein environment. This facility is demonstrated be estimating the barrier of camphor hydroxylation by P450cam.     

Redox control of the P450cam catalytic cycle: effects of Y96F active site mutation and binding of a non-natural substrate

Spectroelectrochemistry measurements are used to demonstrate that active site mutation and binding of an non-natural substrate to P450cam (CYP101) reduces the shift in the redox potential caused by substrate-binding, and thereby results in slower catalytic turnover rate relative to wild-type enzyme with the natural camphor substrate.     

The relationship between the redox reaction of camphor‐induced cytochrome p‐450 and its activity

The relationship between the redox reaction of camphor‐induced cytochrome P‐450 (P‐450_cam) and its activity was measured by using cyclic voltammetry. The redox potential of P‐450_cam solution shifted to the lower side of the potential by binding of substrate, and the change was proportional to the amount of the substrate binding to the protein. The substrate binding was inhibited at the low concentration of oxygen in the reaction solution. The reaction product, hydroxycamphor, was observed in the reaction mixture by gas chromatography/mass spectroscopy. However, hydroxycamphor was not observed at an oxygen concentration of about a tenth part of the saturated one. The shift of redox potential of P‐450_cam solution corresponded to the substrate specificity of the activity. These results suggest that the redox reaction of P‐450_cam related to the substrate‐binding to the protein and its activity. Furthermore, the present system was very simple and speedy for the measurement of the activity. Copyright © 1999 John Wiley & Sons, Ltd.     

A negative cooperativity mechanism of human CYP2E1 inferred from molecular dynamics simulations and free energy calculations

Human cytochrome P450 2E1 (CYP2E1) participates in the metabolism of over 2% of all the oral drugs. A hallmark peculiar feature of this enzyme is that it exhibits a pronounced negative cooperativity in substrate binding. However the mechanism by which the negative cooperativity occurs is unclear. Here, we performed molecular dynamics simulations and free energy calculations on human CYP2E1 to examine the structural differences between the substrate-free and the enzymes with one and two aniline molecules bound. Our results indicate that although the effector substrate does not bind in the active site cavity, it still can directly interact with the active site residues of human CYP2E1. The interaction of the effector substrate with the active site leads to a reorientation of active site residues, which thereby weakens the interactions of the active substrate with this site. We also identify a conserved residue T303 that plays a crucial role in the negative cooperative binding on the short-range effects. This residue is a key factor in the positioning of substrates and in proton delivery to the active site. Additionally, a long-range effect of the effector substrate is identified in which F478 is proposed to play a key role. As located in the interface between the active and effector sites, this residue structurally links the active and effector sites and is found to play a significant role in affecting substrate access and ligand positioning within the active site. In the negative cooperative binding, this residue can decrease the interactions of the active substrate with the active site by π-π stacking which then lowers the hydroxylation activity for the active substrate. These findings are in agreement with previous experimental observations and thus provide detailed atomistic insight into the poorly understood mechanism of the negative cooperativity in human CYP2E1.     

Cryoreduction EPR and 13C, 19F ENDOR study of substrate-bound substates and solvent kinetic isotope effects in the catalytic cycle of cytochrome P450cam and its T252A mutant

We recently used cryoreduction EPR/ENDOR techniques to show that a substrate can modulate the properties of both the monooxygenase active-oxygen intermediates and of the proton-delivery network which encompasses them. In the present report we use Q-band pulsed 19F ENDOR (Mims 3-pulse sequence) to examine the substrate binding geometries of camphor, through use of the 5,5'--difluorocamphor, and 13C ENDOR to examine the binding of 5-methylenyl camphor labeled with 13C at C11. These probes are examined in multiple states of the catalytic cycle of P450cam and its T252A mutant. As part of this investigation we further report a new cryoreduction reaction, the reduction of a ferroheme to the EPR-visible Fe(I) state, and use it to probe the substrate binding to the EPR-silent ferroheme state. Finally we report the solvent kinetic isotope effect on the decay of the camphor complex of the hydroperoxo-ferric intermediate, the first such measurement on an individual step within the P450cam reaction cycle. Following reduction of oxyferrous-P450cam, this step is the rate-limiting step in camphor hydroxylation, and its solv-KIE of 1.8 at 190 K establishes that it involves activation of the hydroperoxo moiety by transfer of the 'second' proton of catalysis. We suggest that the finding that the heme pocket can exist in multiple substates, including multiple substrate binding locations, even in P450cam, along with the established possibility that the hydroperoxo-ferriheme intermediate can react with substrate, may explain the formation of multiple products by P450s.     

Roles of the proximal heme thiolate ligand in cytochrome p450(cam).

To examine the roles of the proximal thiolate iron ligand, the C357H mutant of P450(cam) (CYP101) was characterized by resonance Raman, UV, circular dichroism, and activity measurements. The C357H mutant must be reconstituted with hemin for activity to be observed. The reconstituted enzyme is a mixture of high and low spin species. Low temperature (10 degrees C), low enzyme concentration (1 microM), high camphor concentration (1 mM), and 5--50 mM buffer concentrations increase the high to low spin ratio, but under no conditions examined was the protein more than 60% high spin. The C357H mutant has a poorer K(m) for camphor (23 vs 2 microM) and a poorer K(d) for putidaredoxin (50 vs 20 microM) than wild-type P450(cam). The mutant also exhibits a greatly decreased camphor oxidation rate, elevated uncoupling rate, and much greater peroxidase activity. Electron transfer from putidaredoxin to the mutant is much slower than to the wild-type even though redox potential measurements show that the electron transfer remains thermodynamically favored. These experiments confirm that the thiolate ligand facilitates the O--O bond cleavage by P450 enzymes and also demonstrate that this ligand satisfies important roles in protein folding, substrate binding, and electron transfer.     

Mechanisms of reaction in cytochrome P450: Hydroxylation of camphor in P450cam

The fundamental nature of reactivity in cytochrome P450 enzymes is currently controversial. Modelling of bacterial P450cam has suggested an important role for the haem propionates in the catalysis, though this finding has been questioned. Understanding the mechanisms of this enzyme family is important both in terms of basic biochemistry and potentially in the prediction of drug metabolism. We have modelled the hydroxylation of camphor by P450cam, using combined quantum mechanics/molecular mechanics (QM/MM) methods. A set of reaction pathways in the enzyme was determined. We were able to pinpoint the source of the discrepancies in the previous results. We show that when a correct ionization state is assigned to Asp297, no spin density appears on the haem propionates and the protein structure in this region remains preserved. These results indicate that the haem propionates are not involved in catalysis.     

Electronic ground states of iron porphyrin and of the first species in the catalytic reaction cycle of cytochrome P450s

Electronic structures of iron(II) and iron(III) porphyrins are studied with density functional theory (DFT) using the GGA exchange functional OPTX in combination with the correlation functional PBE (OPBE) and with the correlation functional Perdew (OPerdew) together with a triple zeta-type basis set. These functionals, known for accurately predicting the spin ground state of iron complexes, are evaluated against other functionals for their performance in calculating relative energies for the various electronic states of both the iron porphyrins. The calculated energy orderings are triplet < quintet < singlet for the iron(II) porphyrin and quartet < sextet < doublet for the iron(III) porphyrin cation. Complexation by a thiolate ion (SH-) changes the preferred ground state for both species to high spin. This thiolate complex is used as a mimic for the cytochrome P450s active site to model the first step of the catalytic cycle of this enzyme. This first step is believed to concern the removal of an axial oxygen donating ligand from the hexacoordinated aqua-thiolate-porphyrin-iron(III) resting state. The DFT results suggest that this is not a free water molecule, because of its repulsive nature, but that it has instead hydroxy anion character. These calculations are in line with the experimentally observed change in the spin state from low to high spin upon this removal of the axial hydroxo ligand by binding of the substrate in the heme pocket of cytochrome P450.     

Surface-enhanced Raman scattering as a tool to probe cytochrome P450-catalysed substrate oxidation

Surface-enhanced Raman scattering was used as a spectroscopic tool to investigate the changes brought upon cytochrome P450BSß after fatty acid binding. Differences in the spectra of substrate-free and substrate-bound enzyme were observed indicating the potential for this method to be used in the screening of P450 substrates. In particular, the binding characteristics of myristic acid, an inherent substrate, and hydroxylauric acid, a product of fatty acid oxidation, towards P450BSß in the presence of H₂O₂ were investigated. Specific spectral changes could be assigned to changes in the heme environment only for myristic acid, indicating an occurrence of oxidation process characteristic for the enzymatic substrate.     

Enzyme–Substrate Complementarity Governs Access to a Cationic Reaction Manifold in the P450BM3‐Catalysed Oxidation of Cyclopropyl Fatty Acids

The products of cytochrome P450_BM3‐catalysed oxidation of cyclopropyl‐containing dodecanoic acids are consistent with the presence of a cationic reaction intermediate, which results in efficient dehydrogenation of the rearranged probes by the enzyme. These results highlight the importance of enzyme–substrate complementarity, with a cationic intermediate occurring only when the probes used begin to diverge from ideal substrates for this enzyme. This also aids in reconciling literature reports supporting the presence of cationic intermediates with certain cytochrome P450 enzyme/substrate pairs.     

Cytochrome P450 Catalyzes Benzene Ring Formation in the Biosynthesis of Trialkyl-Substituted Aromatic Polyketides

Lorneic acid and related natural products are characterized by a trialkyl-substituted benzene ring. The formation of the aromatic core in the middle of the polyketide chain is unusual. We characterized a cytochrome P450 enzyme that can catalyze the hallmark benzene ring formation from an acyclic polyene substrate through genetic and biochemical analysis. Using this P450 as a beacon for genome mining, we obtained 12 homologous type I polyketide synthase (PKS) gene clusters, among which two gene clusters are activated and able to produce trialkyl-substituted aromatic polyketides. Quantum chemical calculations were performed to elucidate the plausible mechanism for P450-catalyzed benzene ring formation. Our work expands our knowledge of the catalytic diversity of cytochrome P450.     

An evaluation of molecular models of the cytochrome P450 Streptomyces griseolus enzymes P450SU1 and P450SU2

Summary P450SU1 and P450SU2 are herbicide-inducible bacterial cytochrome P450 enzymes from Streptomyces griseolus. They have two of the highest sequence identities to camphor hydroxylase (P450cam from Pseudomonas putida), the cytochrome P450 with the first known crystal structure. We have built several models of these two proteins to investigate the variability in the structures that can occur from using different modeling protocols. We looked at variability due to alignment methods, backbone loop conformations and refinement methods. We have constructed two models for each protein using two alignment algorithms, and then an additional model using an identical alignment but different loop conformations for both buried and surface loops. The alignments used to build the models were created using the Needleman-Wunsch method, adapted for multiple sequences, and a manual method that utilized both a dotmatrix search matrix and the Needleman-Wunsch method. After constructing the initial models, several energy minimization methods were used to explore the variability in the final models caused by the choice of minimization techniques. Features of cytochrome P450cam and the cytochrome P450 superfamily, such as the ferredoxin binding site, the heme binding site and the substrate binding site were used to evaluate the validity of the models. Although the final structures were very similar between the models with different alignments, active-site residues were found to be dependent on the conformations of buried loops and early stages of energy minimization. We show which regions of the active site are the most dependent on the particular methods used, and which parts of the structures seem to be independent of the methods.     

Ligand-induced conformational heterogeneity of cytochrome P450 CYP119 identified by 2D NMR spectroscopy with the unnatural amino acid (13)C-p-methoxyphenylalanine

Conformational dynamics are thought to play an important role in ligand binding and catalysis by cytochrome P450 enzymes, but few techniques exist to examine them in molecular detail. Using a unique isotopic labeling strategy, we have site specifically inserted a (13)C-labeled unnatural amino acid residue, (13)C-p-methoxyphenylalanine (MeOF), into two different locations in the substrate binding region of the thermophilic cytochrome P450 enzyme CYP119. Surprisingly, in both cases the resonance signal from the ligand-free protein is represented by a doublet in the (1)H,(13)C-HSQC spectrum. Upon binding of 4-phenylimidazole, the signals from the initial resonances are reduced in favor of a single new resonance, in the case of the F162MeOF mutant, or two new resonances, in the case of the F153MeOF mutant. This represents the first direct physical evidence for the ligand-dependent existence of multiple P450 conformers simultaneously in solution. This general approach may be used to further illuminate the role that conformational dynamics plays in the complex enzymatic phenomena exhibited by P450 enzymes.     

Observation of ligand binding to cytochrome P450 BM-3 by means of solid-state NMR spectroscopy

A broad understanding of the binding modes of ligands and inhibitors to cytochrome P450 is vital for the development of new drugs. We investigated ligand binding in a site-specific fashion on cytochrome P450 BM-3 from Bacillus megaterium, a 119 kDa paramagnetic enzyme, using solid-state magic angle spinning nuclear magnetic resonance methods. Selective labeling and longitudinal relaxation effects were utilized to identify the peaks in a site-specific fashion and to provide evidence for binding. Well-resolved one-dimensional and two-dimensional NMR spectra of cytochrome P450 BM-3 reveal shifts upon binding of its substrate, N-palmitoylglycine. These data are consistent with the crystallographic result that a biochemically important amino acid residue, Phe87, moves upon ligation. This experimental scheme provides a tool for probing ligand binding for complex systems.