Hydroxylation Of Camphor Research Articles

Multireference Ab Initio Quantum Mechanics/Molecular Mechanics Study on Intermediates in the Catalytic Cycle of Cytochrome P450cam

We have investigated the elusive reactive species of cytochrome P450(cam) (Compound I), the hydroxo complex formed during camphor hydroxylation, and the ferric hydroperoxo complex (Compound 0) by combined quantum mechanical/molecular mechanical (QM/MM) calculations, employing both density functional theory (DFT) and correlated ab initio methods. The first two intermediates appear multiconfigurational in character, especially in the doublet state and less so in the quartet state. DFT(B3LYP)/MM calculations reproduce the relative energies from correlated ab initio QM/MM treatments quite well, except for the splitting of the lowest A(1u)-A(2u) radical states. The inclusion of dynamic correlation is crucial for the proper ab initio treatment of these intermediates.

A Functional Proline Switch in Cytochrome P450cam

The two-protein complex between putidaredoxin (Pdx) and cytochrome P450(cam) (CYP101) is the catalytically competent species for camphor hydroxylation by CYP101. We detected a conformational change in CYP101 upon binding of Pdx that reorients bound camphor appropriately for hydroxylation. Experimental evidence shows that binding of Pdx converts a single X-proline amide bond in CYP101 from trans or distorted trans to cis. Mutation of proline 89 to isoleucine yields a mixture of both bound camphor orientations, that seen in Pdx-free and that seen in Pdx-bound CYP101. A mutation in CYP101 that destabilizes the cis conformer of the Ile 88-Pro 89 amide bond results in weaker binding of Pdx. This work provides direct experimental evidence for involvement of X-proline isomerization in enzyme function.

P450cam biocatalysis in surfactant‐stabilized two‐phase emulsions

Cytochrome P450 monooxygenases (P450s) are powerful biocatalysts that have the ability to oxidize a broad range of substrates, often at non-reactive carbon centers. However, incorporation of P450s into synthetic schemes has so far been limited to a few whole-cell transformations. P450 substrates are often hydrophobic and have low water solubility, limiting the amount of product that can be produced. To help overcome this limitation, we have examined P450cam activity in two-phase hexane/water emulsions with and without the anionic surfactant, bis(2-ethylhexyl) sulfosuccinate sodium salt (AOT). Hydroxylation of camphor to hydroxycamphor by the three- component P450cam system was chosen as the model reaction, and regeneration of NADH was accomplished with yeast alcohol dehydrogenase (YADH). P450cam was activated in the surfactant-free emulsions, and addition of AOT improved the activity even further, at least over the range of camphor concentrations for which initial rates were readily measurable in all media. The largest observed rate enhancement was 4.5-fold. Nearly 50-times more product was formed in the surfactant-stabilized emulsions than was achieved in aqueous buffer, with total turnover numbers reaching 28,900 for P450cam and 11,800 for YADH. In the absence of surfactant, the two-phase reaction appeared to be mass-transfer limited, while inclusion of AOT alleviated transport limitations and/or afforded a larger interfacial area for P450 activation. The oxidation of hydroxycamphor to 2,5-diketocamphane was also observed, owing to the large concentration of hydroxycamphor relative to camphor in the aqueous phase of the two-phase emulsion. This competing reaction was accompanied by the uncoupled oxidation of NADH (i.e., NADH oxidation without formation of 2,5-diketocamphane), which reduced the availability of NADH for camphor oxidation and further limited the yield of hydroxycamphor in the two-phase emulsions. These results indicate that a surfactant-stabilized two-phase emulsion is a promising reaction medium for practical P450 biocatalysis, although its effectiveness for a given P450/substrate combination can depend on several factors, including competitive or sequential reactions, product inhibition, and NAD(P)H uncoupling.

What is the Active Species of Cytochrome P450 during Camphor Hydroxylation? QM/MM Studies of Different Electronic States of Compound I and of Reduced and Oxidized Iron−Oxo Intermediates

We have investigated C-H hydroxylation of camphor by Compound I (Cpd I) of cytochrome P450cam in different electronic states and by its one-electron reduced and oxidized forms, using QM/MM calculations in the native protein/solvent environment. Cpd I species with five unpaired electrons (pentaradicaloids) are ca. 12 kcal/mol higher in energy than the ground state Cpd I species with three unpaired electrons (triradicaloids). The H-abstraction transition states of pentaradicaloids lie ca. 21 (9) kcal/mol above the triradicaloid (pentaradicaloid) reactants. Hydroxylation via pentaradicaloids is thus facile provided that they can react before relaxing to the ground-state triradicaloids. Excited states of Cpd I with an Fe(V)-oxo moiety lie more than 20 kcal/mol above the triradicaloid ground state in single-point gas-phase calculations, but these electronic configurations are not stable upon including the point-charge protein environment which causes SCF convergence to the triradicaloid ground state. One-electron reduced species (Cpd II) show sluggish reactivity compared with Cpd I in agreement with experimental model studies. One-electron oxidized species are more reactive than Cpd I but seem too high in energy to be accessible. The barriers to hydrogen abstraction for the various forms of Cpd I are generally not affected much by the chosen protonation states of the Asp297 and His355 residues near the propionate side chains of the heme or by the appearance of radical character at Asp297, His355, or the propionates.

In Vitro Metabolism of (-)-Camphor Using Human Liver Microsomes and CYP2A6

The in vitro metabolism of (-)-camphor was examined in human liver microsomes and recombinant enzymes. Biotransformation of (-)-camphor was investigated by gas chromatography-mass spectrometry (GC-MS). (-)-Camphor was oxidized to 5-exo-hydroxyfenchone by human liver microsomal cytochrome (P450) enzymes. The formation of metabolites of (-)-camphor was determined by the relative abundance of mass fragments and retention time on gas chromatography (GC). CYP2A6 was the major enzyme involved in the hydroxylation of (-)-camphor by human liver microsomes, based on the following lines of evidence. First, of eleven recombinant human P450 enzymes tested, CYP2A6 catalyzed the oxidation of (-)-camphor. Second, oxidation of (-)-camphor was inhibited by (+)-menthofuran and anti-CYP2A6 antibody. Finally, there was a good correlation between CYP2A6 contents and (-)-camphor hydroxylation activities in liver microsomes of 9 human samples.

On the identity and reactivity patterns of the “second oxidant” of the T252A mutant of cytochrome P450 cam in the oxidation of 5-methylenenylcamphor

Density functional calculations show that in the absence of Compound I, the primary oxidant species of P450, the precursor species, Compound 0 (FeOOH), can effect double bond activation of 5-methylenylcamphor ( 1). The mechanism is initiated by homolytic cleavage of the O–O bond and formation of an OH radical bound to the Compound II species by hydrogen bonding interactions. Subsequently, the so-formed OH radical can either activate the double bond of 1 or attack the meso position of the heme en route to heme degradation. The calculations show that double bond activation is preferred over attack on the heme. Past the double bond activation, the intermediate can either lead to epoxidation or to a glycol formation. The glycol formation is predicted to be preferred, although in the P450 cam pocket the competition may be closer. Therefore, in the absence of Compound I, Compound 0 will be capable of epoxidizing double bonds. Previous studies [E. Derat, D. Kumar, H. Hirao, S. Shaik, J. Am. Chem. Soc. 128 (2006) 473–484] showed that in the case of a substrate that can undergo only C–H activation, the bound OH prefers heme degradation over hydrogen abstraction. Since the epoxidation barrier for Compound I is much smaller than that of Compound 0 (12.8 vs. 18.9 kcal/mol), when Compound I is present in the cycle, Compound 0 will be silent. As such, our mechanism explains lucidly why T252A P450 cam can epoxidize olefins like 5-methylenylcamphor but is ineffective in camphor hydroxylation [S. Jin, T.M. Makris, T. A. Bryson, S.G. Sligar, J.H. Dawson, J. Am. Chem. Soc. 125 (2003) 3406–3407]. Our calculations show that the glycol formation is a marker reaction of Compound 0 with 5-methylenylcamphor. If this product can be found in T252A P450 cam or in similar mutants of other P450 isozymes, this will constitute a more definitive proof for the action of Cpd 0 in P450 enzymes.

A recombinant Escherichia coli whole cell biocatalyst harboring a cytochrome P450cam monooxygenase system coupled with enzymatic cofactor regeneration

A cytochrome P450cam monooxygenase (P450cam) system from the soil bacterium Pseudomonas putida requires electron transfer among three different proteins and a cofactor, nicotinamide adenine dinucleotide (NADH), for oxygenation of its natural substrate, camphor. Herein, we report a facile way to significantly enhance the catalytic efficiency of the P450cam system by the coupling of its native electron transfer system with enzymatic NADH regeneration catalyzed by glycerol dehydrogenase (GLD) in Escherichia coli whole cell biocatalysts. Recombinant E. coli harboring the P450cam system, but lacking GLD, exhibited little activity for camphor hydroxylation. In contrast, coexpression of GLD with the proteinaceous electron transfer components of P450cam resulted in about tenfold improvement in the substrate conversion, implying that the whole cell biocatalyst utilized molecular oxygen, endogenous NADH, and glycerol in the cell for catalysis. The addition of glycerol to the reaction media further promoted camphor hydroxylation, suggesting that exogenous glycerol is also available for GLD in the host cell and actively participates in the catalytic cycle. These results clearly show the utility of GLD towards functional reconstruction of the native P450cam system. The present approach may also be useful for E. coli whole cell biocatalysts with the other NADH-dependent oxygenases and oxidoreductases.

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.

Redox-Dependent Structural Differences in Putidaredoxin Derived from Homologous Structure Refinement via Residual Dipolar Couplings,

Structural differences in the [2Fe-2S] ferredoxin, putidaredoxin (Pdx), from the camphor hydroxylation pathway of Pseudomonas putida have been investigated as a function of oxidation state of the iron cluster. Pdx is involved in biological electron transfer to cytochrome P450(cam) (CYP101). Redox-dependent differences have been observed previously for Pdx in terms of binding affinities to CYP101, NMR spectral differences, and dynamic properties. To further characterize these differences, structure refinement of both oxidized and reduced Pdx has been carried out using a hybrid approach utilizing paramagnetic distance restraints and NMR orientational restraints in the form of backbone (15)N residual dipolar couplings. Use of these new restraints has improved the structure of oxidized Pdx considerably over the earlier solution NMR structure without RDC restraints, with the new structure now much closer in overall fold to the recently published X-ray crystal structures. We now observe better defined relative orientations of the major secondary structure elements as also of the conformation of the metal binding loop region. Extension of this approach to structure calculation of reduced Pdx has identified structural differences that are primarily localized for residues in the C-terminal interaction domain consisting of the functionally important residue Trp 106 and regions near the metal binding loop in Pdx. These redox-dependent structural differences in Pdx correlate to dynamic changes observed before and may be linked to differences in binding and electron transfer properties between oxidized and reduced Pdx.

Production and X-ray crystallographic analysis of fully deuterated cytochrome P450cam

Neutron protein crystallography allows H-atom positions to be located in biological structures at the relatively modest resolution of 1.5-2.0 A. A difficulty of this technique arises from the incoherent scattering from hydrogen, which considerably reduces the signal-to-noise ratio of the data. This can be overcome by preparing fully deuterated samples. Efficient protocols for routine and low-cost production of in vivo deuterium-enriched proteins have been developed. Here, the overexpression and crystallization of highly (>99%) deuterium-enriched cytochrome P450cam for neutron analysis is reported. Cytochrome P450cam from Pseudomonas putida catalyses the hydroxylation of camphor from haem-bound molecular O(2) via a mechanism that is thought to involve a proton-shuttle pathway to the active site. Since H atoms cannot be visualized in available X-ray structures, neutron diffraction is being used to determine the protonation states and water structure at the active site of the enzyme. Analysis of both hydrogenated and perdeuterated P450cam showed no significant changes between the X-ray structures determined at 1.4 and 1.7 A, respectively. This work demonstrates that the fully deuterated protein is highly isomorphous with the native (hydrogenated) protein and is appropriate for neutron protein crystallographic analysis.

Functionalization of the cytochrome P450cam monooxygenase system in the cell-like aqueous compartments of water-in-oil emulsions

The functionalization of the cytochrome P450cam monooxygenase system, which requires electron transfer among three different proteins, was investigated in the micro-scale aqueous compartments of stable water-in-oil (W/O) emulsions formed with the nonionic surfactant tetraethylene glycol dodecyl ether. Neither an organic-aqueous biphasic system nor a non-emulsified organic-aqueous solution containing the same amount of surfactant showed substantial hydroxylation of camphor, a natural substrate of P450cam, whereas substantial monooxygenation activity was detected when stable aqueous compartments were provided by the formation of W/O emulsions. Since the camphor hydroxylation in W/O emulsions was modest, we explored the integration of an enzymatic NADH regeneration system in order to effectively provide a reducing equivalent. Two different dehydrogenases, bacterial glycerol dehydrogenase (GLD) and yeast alcohol dehydrogenase (ADH), were selected, and each of these was coupled with the P450cam catalytic cycle in W/O emulsions. As a result, the camphor hydroxylation rate was successfully improved by approximately 5-fold when GLD was employed under optimized conditions. These results reveal the potential utility of the micro-scale cell-like aqueous compartments of W/O emulsions for multicomponent enzymatic reactions especially for substrates with low aqueous solubility.

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.

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.

Temperature-induced structural changes in putidaredoxin: a circular dichroism and UV?VIS absorption study1

Putidaredoxin (Pdx) is an 11,400-Da iron–sulfur protein that sequentially transfers two electrons to the cytochrome P450cam during the enzymatic cycle of the stereospecific camphor hydroxylation. We report two transitions in the Pdx UV–VIS absorption and circular dichroism (CD) temperature dependencies, occurring at 16.3±0.5 °C and 28.4±0.5 °C. The 16.3 °C transition is attributed to the disruption of the hydrogen bonding of the active center bridging sulfur atom with cysteine 45 and alanine 46. The transition at 28.4 °C occurs exclusively in the Pdx ox at very nearly the same temperature as the earlier reported biphasicity in the redox potential. The formal potential temperature slope constancy reflects the relative stability of the concentration ratio of both oxidation states. The lower temperature transition affects both Pdx red and Pdx ox to a comparable extent, and their concentration ratio remains constant. In contrast, the 28.4 °C transition preferentially destabilizes Pdx ox thereby accelerating the formal potential negative shift and lower redox reaction entropy. There is evidence to suggest that disrupting hydrogen bonding of the iron ligating cysteines 45, 39 with residues threonine 47, serine 44, glycine 41, and serine 42 causes the 28.4 °C transition. The sensitivity of the UV–VIS absorption and CD spectroscopy to subtle structural protein backbone transitions is demonstrated.

Electron-Transfer Reactions and Functionalization of Cytochrome P450cam Monooxygenase System in Reverse Micelles

Enzyme-based electron-transfer reactions involved in the cytochrome P450 monooxygenase system were investigated in nanostructural reverse micelles. A bacterial flavoprotein, putidaredoxin reductase (PdR), was activated and shown to be capable of catalyzing the electron transport from NADH to electron-carrier proteins such as cytochrome b5 (tCyt-b5) and putidaredoxin (Pdx) in reverse micelles. Ferric tCyt-b5 in reverse micelles was effectively converted to its ferrous form by the exogenous addition of separately prepared reverse micellar solution harboring PdR and NADH. The fact that direct interactions of macromolecular proteins should be possible in the reverse micellar system encouraged us to functionalize a multicomponent monooxygenase system composed of the bacterial cytochrome P450cam (P450cam), putidaredoxin (Pdx), and PdR in reverse micelles. The successful camphor hydroxylation reaction catalyzed by P450cam was significantly dependent on the coexistence of Pdx, PdR, and NADH but not H2O2, suggesting that the oxygen-transfer reactions proceeded via a "monooxygenation" mechanism. This is the first report of a multicomponent cytochrome P450 system exhibiting enzymatic activity in organic media.

Temperature-induced structural changes in putidaredoxin: a circular dichroism and UV–VIS absorption study

Putidaredoxin (Pdx) is an 11,400-Da iron–sulfur protein that sequentially transfers two electrons to the cytochrome P450cam during the enzymatic cycle of the stereospecific camphor hydroxylation. We report two transitions in the Pdx UV–VIS absorption and circular dichroism (CD) temperature dependencies, occurring at 16.3±0.5 °C and 28.4±0.5 °C. The 16.3 °C transition is attributed to the disruption of the hydrogen bonding of the active center bridging sulfur atom with cysteine 45 and alanine 46. The transition at 28.4 °C occurs exclusively in the Pdx ox at very nearly the same temperature as the earlier reported biphasicity in the redox potential. The formal potential temperature slope constancy reflects the relative stability of the concentration ratio of both oxidation states. The lower temperature transition affects both Pdx red and Pdx ox to a comparable extent, and their concentration ratio remains constant. In contrast, the 28.4 °C transition preferentially destabilizes Pdx ox thereby accelerating the formal potential negative shift and lower redox reaction entropy. There is evidence to suggest that disrupting hydrogen bonding of the iron ligating cysteines 45, 39 with residues threonine 47, serine 44, glycine 41, and serine 42 causes the 28.4 °C transition. The sensitivity of the UV–VIS absorption and CD spectroscopy to subtle structural protein backbone transitions is demonstrated.

Quantum mechanical/molecular mechanical investigation of the mechanism of C-H hydroxylation of camphor by cytochrome P450cam: theory supports a two-state rebound mechanism.

The stereospecific cytochrome P450-catalyzed hydroxylation of the C(5)-H((5-exo)) bond in camphor has been studied theoretically by a combined quantum mechanical/molecular mechanical (QM/MM) approach. Density functional theory is employed to treat the electronic structure of the active site (40-100 atoms), while the protein and solvent environment (ca. 24,000 atoms) is described by the CHARMM force field. The calculated energy profile of the hydrogen-abstraction oxygen-rebound mechanism indicates that the reaction takes place in two spin states (doublet and quartet), as has been suggested earlier on the basis of calculations on simpler models ("two-state reactivity"). While the reaction on the doublet potential energy surface is nonsynchronous, yet effectively concerted, the quartet pathway is truly stepwise, including formation of a distinct intermediate substrate radical and a hydroxo-iron complex. Comparative calculations in the gas phase demonstrate the effect of the protein environment on the geometry and relative stability of intermediates (in terms of spin states and redox electromers) through steric constraints and electronic polarization.

A theoretical study on the mechanism of camphor hydroxylation by compound I of cytochrome P450

Mechanistic and energetic aspects for the conversion of camphor to 5-exo-hydroxycamphor by the compound I iron-oxo species of cytochrome P450 are discussed from B3LYP DFT calculations. This reaction occurs in a two-step manner along the lines that the oxygen rebound mechanism suggests. The activation energy for the first transition state of the H atom abstraction at the C5 atom of camphor is computed to be more than 20 kcal/mol. This H atom abstraction is the rate-determining step in this hydroxylation reaction, leading to a reaction intermediate that involves a carbon radical species and the iron-hydroxo species. The second transition state of the rebound step that connects the reaction intermediate and the product alcohol complex lies a few kcal/mol below that for the H atom abstraction on the doublet and quartet potential energy surfaces. This energetic feature allows the virtually barrierless recombination in both spin states, being consistent with experimentally observed high stereoselectivity and br...

Peripheral heme substituents control the hydrogen-atom abstraction chemistry in cytochromes P450.

We elucidate the hydroxylation of camphor by cytochrome P450 with the use of density functional and mixed quantum mechanics/molecular mechanics methods. Our results reveal that the enzyme catalyzes the hydrogen-atom abstraction step with a remarkably low free-energy barrier. This result provides a satisfactory explanation for the experimental failure to trap the proposed catalytically competent high-valent heme Fe(IV) oxo (oxyferryl) species responsible for this hydroxylation chemistry. The primary and previously unappreciated contribution to stabilization of the transition state is the interaction of positively charged residues in the active-site cavity with carboxylate groups on the heme periphery. A similar stabilization found in dioxygen binding to hemerythrin, albeit with reversed polarity, suggests that this mechanism for controlling the relative energetics of redox-active intermediates and transition states in metalloproteins may be widespread in nature.

A model for effector activity in a highly specific biological electron transfer complex: the cytochrome P450(cam)-putidaredoxin couple.

The camphor hydroxylase cytochrome P450(cam) (CYP101) catalyzes the 5-exo hydroxylation of camphor in the first step of camphor catabolism by Pseudomonas putida. CYP101 forms a specific electron transfer complex with its physiological reductant, the Cys(4)Fe(2)S(2) ferredoxin putidaredoxin (Pdx). Pdx, along with other proteins and small molecules, has also been shown to be an effector for turnover by CYP101. Multidimensional nuclear magnetic resonance (NMR) techniques have been used to make extensive sequential (1)H, (15)N, and (13)C resonance assignments in CYP101 that permit a more complete characterization of the complex formed by CYP101 and Pdx. NMR-detected perturbations in CYP101 upon Pdx binding encompass regions of the CYP101 remote from the putative Pdx binding site, including in particular a region of the CYP101 molecule that has been implicated in substrate access to the active site via dynamical processes. A model for effector activity is proposed in which the primary role of the effector is to prevent uncoupling (formation of reduced oxo species without formation of hydroxycamphor) by enforcing conformations of CYP101 that prevent loss of substrate and/or intermediates prior to turnover. A secondary role could also be to enforce conformations that permit efficient proton transfer into the active site for coupled proton/electron transfer.