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The thermodynamics and kinetics of electron transfer in the cytochrome P450(cam) enzyme system
Abstract
In anaerobic environments the first electron transfer in substrate-free P450cam is known to be thermodynamically unfavourable, but in the presence of dioxygen the reduction potential for the reaction shifts positively to make electron transfer thermodynamically favourable. Nevertheless a slower rate of electron transfer is observed in the substrate-free P450cam compared to substrate-bound P450cam. The ferric haem centre in substrate-free P450cam changes from six co-ordinate to five co-ordinate when reduced whereas in substrate-bound P450cam the iron centre remains five co-ordinate in both oxidation states. The slower rate of electron transfer in the substrate-free P450cam is therefore attributed to a larger reorganisation energy as predicted by Marcus theory.
... In the first place, reduction potentials do not, as thermodynamic parameters, correlate directly with FMN-to-haem electron transfer rates, since the latter are kinetic in nature and therefore also reflect the reorganisation energies associated with electron transfer, as described by Marcus theory. 192 To illustrate, the equilibrium constants associated with electron transfer through BMR differed from those calculated from equilibrium redox potentials by as much as two orders of magnitude. 190 Secondly, experimentally determined potentials do not reflect the impact of rapid dioxygen binding to the ferrous haem iron (which, like CO binding, alters the equilibrium potential), since they are necessarily measured under anaerobic conditions. ...
... 190 Secondly, experimentally determined potentials do not reflect the impact of rapid dioxygen binding to the ferrous haem iron (which, like CO binding, alters the equilibrium potential), since they are necessarily measured under anaerobic conditions. 178,192 Finally, P450 BM3 variants that consume NADPH at o1.5 nmol (nmol P450) À1 s À1 in the substrate-free form despite having first electron transfer rates of >200 s À1 have recently been identified 193,194 (section 5.2). A method of estimating the reduction potentials of P450 BM3 variants using UV-visible spectroscopy has been reported. ...
... Several of the earliest mutant crystal structures to be published were broadly identical to the SF WT structure, including those of SF T268A (PDB codes 1FAH 255 and 1YQO), 107 T268N (1YQP), 107 F393H (1JME), 259 F393A, F393W and F393Y (1P0V, 1P0W and 1P0X). 258 The highly plastic F/G loop and the final turns of the helices to either side (residues [189][190][191][192][193][194][195][196][197][198][199][200] were unresolved in all but one of these structures. In the T268A structure, the carbonyl oxygen of Ala264, though unable to hydrogen-bond to the side-chain of the mutated residue, remained hydrogen-bonded to the axial water molecule. ...
P450(BM3) (CYP102A1), a fatty acid hydroxylase from Bacillus megaterium, has been extensively studied over a period of almost forty years. The enzyme has been redesigned to catalyse the oxidation of non-natural substrates as diverse as pharmaceuticals, terpenes and gaseous alkanes using a variety of engineering strategies. Crystal structures have provided a basis for several of the catalytic effects brought about by mutagenesis, while changes to reduction potentials, inter-domain electron transfer rates and catalytic parameters have yielded functional insights. Areas of active research interest include drug metabolite production, the development of process-scale techniques, unravelling general mechanistic aspects of P450 chemistry, methane oxidation, and improving selectivity control to allow the synthesis of fine chemicals. This review draws together the disparate research themes and places them in a historical context with the aim of creating a resource that can be used as a gateway to the field.
... Despite the progressive aggregation of vernolate-bound CYP116B1 in this experiment, it is notable that the predominant species is now the P450 form, indicating substrate-dependent stabilization of the thiolate ligand, as observed previously in other systems [37,38,56]. Given the limited solubility of vernolate, its marginal effect on haem iron spin-state equilibrium and its influence on aggregation of CYP116B1, it is difficult to establish the extent to which electron transfer to the CYP116B1 haem iron is thermodynamically gated in the presence of this substrate and a kinetic gate to haem reduction in CYP116B1 cannot be ruled out [57]. Consistent with the monomeric form of CYP116B1 being the catalytically relevant species, specific rates of vernolate-dependent NADPH oxidation, determined across a range of CYP116B1 concentrations (10-500 nm), showed no significant variation. ...
... This makes it difficult to establish the extent to which substrate-dependent haem iron spinstate modulation is important for control of enzyme catalysis in CYP116B1, or whether an alternative mode of regulation may be involved [e.g. 57,58]. ...
The novel cytochrome P450/redox partner fusion enzyme CYP116B1 from Cupriavidus metallidurans was expressed in and purified from Escherichia coli. Isolated CYP116B1 exhibited a characteristic Fe(II)CO complex with Soret maximum at 449 nm. EPR and resonance Raman analyses indicated low-spin, cysteinate-coordinated ferric haem iron at both 10 K and ambient temperature, respectively, for oxidized CYP116B1. The EPR of reduced CYP116B1 demonstrated stoichiometric binding of a 2Fe-2S cluster in the reductase domain. FMN binding in the reductase domain was confirmed by flavin fluorescence studies. Steady-state reduction of cytochrome c and ferricyanide were supported by both NADPH/NADH, with NADPH used more efficiently (K(m[NADPH]) = 0.9 ± 0.5 μM and K(m[NADH]) = 399.1 ± 52.1 μM). Stopped-flow studies of NAD(P)H-dependent electron transfer to the reductase confirmed the preference for NADPH. The reduction potential of the P450 haem iron was -301 ± 7 mV, with retention of haem thiolate ligation in the ferrous enzyme. Redox potentials for the 2Fe-2S and FMN cofactors were more positive than that of the haem iron. Multi-angle laser light scattering demonstrated CYP116B1 to be monomeric. Type I (substrate-like) binding of selected unsaturated fatty acids (myristoleic, palmitoleic and arachidonic acids) was shown, but these substrates were not oxidized by CYP116B1. However, CYP116B1 catalysed hydroxylation (on propyl chains) of the herbicides S-ethyl dipropylthiocarbamate (EPTC) and S-propyl dipropylthiocarbamate (vernolate), and the subsequent N-dealkylation of vernolate. CYP116B1 thus has similar thiocarbamate-oxidizing catalytic properties to Rhodoccocus erythropolis CYP116A1, a P450 involved in the oxidative degradation of EPTC.
... We wished to assess if the activity would correlate with the magnitude of the shift observed in the UV-vis absorbance spectrum on substrate binding. The mixed high-spin/lowspin ferric state of the CYP125A6 and CYP125A7 enzymes may result in them not being as constrained by substrate gating as other CYP enzymes are [59][60][61][62]. The catalytic oxidation of the steroids by these enzymes was supported using spinach ferredoxin and spinach ferredoxin reductase electron transfer partners a system which is known to support the activity of these enzymes [63]. ...
... This can be rationalized via either thermodynamic (more negative redox potential compared to 4-methoxybenzoic acid) or kinetic gating of the P450. 65,66,90 The positive shifts in the redox potentials with 4-methoxybenzoic acid and VBA are in agreement with those reported previously for type I substrates. The relationship between the magnitude of the redox potential shift, the measured spin state conversion, the extent of aqua ligand occupancy, and the rate of turnover of the catalytic cycle are as expected. ...
The cytochrome P450 (CYP) family of heme monooxygenase enzymes commonly catalyzes enantioselective hydroxylation and epoxidation reactions. Epoxidation reactions have been hypothesized to proceed via multiple mechanisms involving different reactive intermediates. Here, we use activity, spectroscopic, structural, and molecular dynamics data to investigate the activity and stereoselectivity of 4-vinylbenzoic acid epoxidation by the bacterial enzyme CYP199A4 from Rhodopseudomonas palustris HaA2. The epoxidation of 4-vinylbenzoic acid by CYP199A4 proceeded with high enantioselectivity, giving the (S)-epoxide in 99% ee at an activity of 220 nmol nmol-CYP-1 min-1. Optical and EPR spectroscopy, redox potential measurements, and the crystal structure of 4-vinylbenzoic acid-bound CYP199A4 indicated the partial retention of an aqua ligand at the heme center in the presence of the substrate, providing a justification of the lower activity (∼20%) compared to the oxidative demethylation of 4-methoxybenzoic acid. Mutagenesis at the conserved acid.alcohol pair (D251/T252), which perturbs the generation of the reactive oxygen intermediates, was employed to investigate their role in epoxidation reactions. The T252A mutant increased the rate of turnover of the catalytic cycle, but an elevation in hydrogen peroxide generation via uncoupling resulted in a similar rate of epoxide formation. The activity of epoxidation significantly reduced with the D251N mutant. The chemoselectivity and stereoselectivity of the epoxidation reaction were maintained in the turnovers by these mutants. Overall, there was little evidence that other intermediates, aside from the archetypal reactive ferryl porphyrin cation radical, Compound I, contributed significantly to the epoxidation reaction. The observation of the high selectivity for the (S)-enantiomer was rationalized by molecular dynamics simulations. When the arrangement of the alkene and the active intermediate approached an ideal transition state structure for epoxidation, one face of the alkene was more often exposed to the iron oxo unit.
... [7c, 8d] Fort his reason, and in consistence with experimental observations, [7g] H 2 O 2 binding is not expected to trigger the long-range ET from CYT to LPMO observed for O 2 binding. These findings may have farreaching implication for the O 2 or CO-binding triggered longrange electron transfer in metalloenzymes, [15,16,28] where the exergonic O 2 or CO binding may act as an important driving force for long-range ET. [29] ...
Long‐range electron transfer (ET) in metalloenzymes is a general and fundamental process governing O2 activation and reduction. Lytic polysaccharide monooxygenases (LPMOs) are key enzymes for the oxidative cleavage of insoluble polysaccharides, but their reduction mechanism by cellobiose dehydrogenase (CDH), one of the most commonly used enzymatic electron donors, via long‐range ET is still an enigma. Using multiscale simulations, we reveal that interprotein ET between CDH and LPMO is mediated by the heme propionates of CDH and solvent waters. We also show that oxygen binding to the copper center of LPMO is coupled with the long‐range interprotein ET. This process, which is spin‐regulated and enhanced by the presence of O2, directly leads to LPMO−CuII−O2⁻, bypassing the formation of the generally assumed LPMO−CuI species. The uncovered ET mechanism rationalizes experimental observations and might have far‐reaching implications for LPMO catalysis as well as the O2‐ or CO‐binding‐enhanced long‐range ET processes in other metalloenzymes.
... Both 3-formyl-and 3-methylamino-benzoic acid slightly red shifted the Soret maximum (by 0.5 nm) despite giving type I difference spectra, implying that the heteroatoms of these substituents may interact with a heme-bound water (Supporting information, Fig. S2, Table S2). The spin state experiments also suggest that NADH oxidation by CYP199A4 in the presence of these substrates will be slow because electron transfer is gated by displacement of the water and the related changes in spin state and redox potential [17,19,[30][31][32][33]. If these substrates were to bind exclusively with the meta-substituent on the side of the benzene ring which is further away from the heme, we would predict little or no product to be formed. ...
The cytochrome P450 metalloenzyme (CYP) CYP199A4 from Rhodopseudomonas palustris HaA2 catalyzes the highly efficient oxidation of para-substituted benzoic acids. Here we determined crystal structures of CYP199A4, and the binding and turnover parameters, with different meta-substituted benzoic acids in order to establish which criteria are important for efficient catalysis. When compared to the para isomers, the meta-substituted benzoic acids were less efficiently oxidized. For example, 3-formylbenzoic acid was oxidized with lower activity than the equivalent para isomer and 3-methoxybenzoic acid did not undergo O-demethylation by CYP199A4. The structural data highlighted that the meta-substituted benzoic acids bound in the enzyme active site in a modified position with incomplete loss of the distal water ligand of the heme moiety. However, for both sets of isomers the meta- or para-substituent pointed towards, and was in close proximity, to the heme iron. The absence of oxidation activity with 3-methoxybenzoic acid was assigned to the observation that the CH bonds of this molecule point away from the heme iron. In contrast, in the para isomer they are in an ideal location for abstraction. These findings were confirmed by using the bulkier 3-ethoxybenzoic acid as a substrate which removed the water ligand and reoriented the meta-substituent so that the methylene hydrogens pointed towards the heme, enabling more efficient oxidation. Overall we show relatively small changes in substrate structure and position in the active site can have a dramatic effect on the activity.
... P450 cam (CYP101A1) is a bacterial P450 that catalyses the stereospecific hydroxylation of (1R)-camphor to 5-exo-hydroxycamphor [9,12]. It uses a Class I electron transfer system, comprising of the FAD dependent, putidaredoxin reductase (PdR) and the [2Fe-2S] ferredoxin, putidaredoxin (Pdx) to obtain electrons from NADH [13,14]. The development of active site mutants which catalyse the oxidation of alternative substrates highlighted the biocatalytic potential of CYP101A1 and this family of enzymes in general [9,[15][16][17][18][19]. ...
... [16][17][18][19][20] As a result of the extensive body of biochemical and structural data, the P450cam system is considered the model of the bacterial class I CYPs, despite its idiosyncrasies. 21 This class contains a multitude of other CYPs with potential for biotechnological applications but their kinetic properties inhibit widespread development. 22 Numerous CYPs are not genetically associated with their physiological electron transfer partners, including those from metagenomic libraries. ...
Cytochrome P450 (CYP) enzymes catalyze the insertion of oxygen into carbon–hydrogen bonds and have great potential for enzymatic synthesis. Application development of class I CYPs is hampered by their dependence on two redox partners (a ferredoxin and ferredoxin reductase), slowing catalysis compared to self-sufficient CYPs such as CYP102A1 (P450BM3). Previous attempts to address this have fused all three components in several permutations and geometries, with much reduced activity compared to the native system. We report here the new approach of fusing putidaredoxin reductase (PdR) to the carboxy-terminus of CYP101A1 (P450cam) via a linker peptide and reconstituting camphor hydroxylase activity with free putidaredoxin (Pdx). Initial purification of a P450cam–PdR fusion yielded 2.0% heme incorporation. Co-expression of E. coli ferrochelatase, lengthening the linker from 5 to 20 residues, and altering culture conditions for enzyme production furnished 85% heme content. Fusion co-expression with Pdx gave a functional system with comparable in vivo camphor oxidation activity as the native system. In vitro, the fused system's steady state NADH oxidation rate was two-fold faster than that of the native system. In contrast to the native system, NADH oxidation rates for the fusion enzyme showed non-hyperbolic dependence on Pdx concentration, suggesting a role for the PdR domain; these data were consistent with a kinetic model based on two-site binding of Pdx by P450cam–PdR and inactive dimer formation of the fusion. P450cam–PdR is the first example of a class I P450 fusion that exhibits significantly more favorable behavior than that of the native system.
... In both cases, a single redox couple corresponding to heme Fe III/II transition was detected. Reduction potentials measured from SWV were −284 mV and −247 mV versus Ag/AgCl for substratefree and -bound rArom, respectively (Table 1), consistent with published data on direct electrochemistry of CYP enzymes [16,[20][21][22][23]. CV applied to protein previously denatured by 6 M guanidinium hydrochloride resulted in a midpoint potential of −42 mV (versus Ag/AgCl). ...
... In both cases, a single redox couple corresponding to heme Fe III/II transition was detected. Reduction potentials measured from SWV were −284 mV and −247 mV versus Ag/AgCl for substratefree and -bound rArom, respectively (Table 1), consistent with published data on direct electrochemistry of CYP enzymes [16,[20][21][22][23]. CV applied to protein previously denatured by 6 M guanidinium hydrochloride resulted in a midpoint potential of −42 mV (versus Ag/AgCl). ...
... The electron transfer rate constant (k 0 ) of S. oneidensis MR-1 MtrABC complex on a graphite electrode was estimated to be 195 s −1 [29], which is at the same order of magnitude with the constant of each individual protein in the complex [34] (Table 1). Those values are also comparable to the k 0 of S. loihica PV-4 OMCs (150 ± 10 s −1 ) [34], as well as the enzyme-electrode optimized for biosensors [35,36]. In comparison with other anaerobic respiration processes, the electron transfer rate of electrode reduction by S. oneidensis MR-1 in several orders of magnitude higher than haematite reduction but is significantly lower than the reductions of soluble electron acceptors [37]. ...
... As a result of these changes, cytochromes P450 saturated with substrates are usually reduced much faster than in the substrate-free state, due to a positive shift of the redox potential by 80 -130 mV67686970. In addition, faster electron transfer in the presence of substrate may be partly due to the lower reorganization energy7172. One-electron reduction of the heme iron to the ferrous state [3] is necessary for oxygen binding and formation of and oxyferrous complex [4] , which has predominantly ferricsuperoxo character. ...
Cooperative functional properties and allosteric regulation in cytochromes P450 play an important role in xenobiotic metabolism and define one of the main mechanisms of drug-drug interactions. Recent experimental results suggest that ability to bind simultaneously two or more small organic molecules can be the essential feature of cytochrome P450 fold, and often results in rich and complex pattern of allosteric behavior. Manifestations of non-Michaelis kinetics include homotropic and heterotropic activation and inhibition effects depending on the stoichiometric ratios of substrate and effector, changes in the regio- and stereospecificity of catalytic transformations, and often give rise to the clinically important drug-drug interactions. In addition, functional response of P450 systems is modulated by the presence of specific and non-specific effector molecules, metal ions, membrane incorporation, formation of homo- and hetero-oligomers, and interactions with the protein redox partners. In this article we briefly overview the main factors contributing to the allosteric effects in cytochromes P450 with the main focus on the sources of cooperative behavior in xenobiotic metabolizing monomeric heme enzymes with their conformational flexibility and extremely broad substrate specificity. The novel mechanism of functional cooperativity in P450 enzymes does not require substantial binding cooperativity, rather it implies the presence of one or more binding sites with higher affinity than the single catalytically active site in the vicinity of the heme iron.
... Binding of a substrate sufficiently close to the haem could displace the haem iron sixth ligand, water, leading to an increase in the haem reduction potential and a decrease in the reorganisation energy of electron transfer because the haem in both the ferric and ferrous forms would be five-coordinate. 48 However, these criteria do not restrict the motion of the bound substrate-as long as it is sufficiently close to the haem to cause dissociation of the water ligand, fast electron transfer should ensue. We have suggested previously that the Y96F mutation promotes the binding and oxidation of hydrophobic organic molecules by increasing the active site hydrophobicity and facilitating the displacement of water molecules from the active site. ...
The haem monooxygenase cytochrome P450cam from Pseudomonas putida has been engineered into an alkane hydroxylase. Active site amino acid residues were substituted with residues that have bulkier and more hydrophobic side-chains. The residues F87, Y96, V247 and V396, which are further away from the haem, were targeted first for substitution in order to constrain the small alkanes n-butane and propane to bind closer to the haem. We found that just two mutations could increase the alkane oxidation activity of P450cam by two orders of magnitude. The F87W/ Y96F/V247L triple mutant was then used as a basis for introducing further substitutions, at the residues T101, L244, V395 and D297 which are closer to the haem, to improve the enzyme/alkane fit and hence the alkane hydroxylase activity. The F87W/Y96F/T101L/V247L mutant oxidised n-butane with a catalytic turnover rate of 755 nmol(nmol P450cam)(-1) (min)(-1), which is comparable to the camphor oxidation activity of the wild-type (1000 min(-1)). The F87W/ Y96F/T101L/L244M/V247L mutant had lower n-butane oxidation activity but the highest propane oxidation rate (176 min(-1)) of the P450cam enzymes studied. All P450cam enzymes gave 2-butanol and 2-propanol as the only products. Determination of the extent of uncoupling showed that hydrogen peroxide generation was the dominant uncoupling mechanism. The data indicate that further mutations at residues higher up in the active site are required to localise the substrates close to the haem and to reduce substrate mobility. These next-generation mutants will have higher activity, and may be able to catalyse the oxidation of ethane and methane.
... Those results reveal a strict correlation between the rate of substrate N-demethylation and its coupling to NADPH oxidation with the amplitude of the substrate-induced spin shift. Furthermore, the low-to-high spin transition is known to increase the rate of electron transfer to cytochromes P450 (Tamburini et al., 1984; Backes et al., 1985; Backes and Eyer, 1989) due to a considerable decrease in the reorganization energy associated with the reduction process (Honeychurch et al., 1999; Denisov et al., 2005) along with a positive displacement of the P450 redox potential (Fisher and Sligar, 1985). This has been demonstrated with CYP3A4 (Das et al., 2007) in particular. ...
The basis of decreased cooperativity in substrate binding in the cytochrome P450 3A4 mutants F213W, F304W, and L211F/D214E was studied with fluorescence resonance energy transfer and absorbance spectroscopy. Although in the wild type enzyme, the absorbance changes reflecting the interactions with 1-pyrenebutanol exhibit a Hill coefficient (n(H)) around 1.7 (S(50) = 11.7 µM), the mutants showed no cooperativity (n(H) ≤ 1.1) with unchanged S(50) values. Contrary to the premise that the mutants lack one of the two binding sites, the mutants exhibited at least two substrate binding events. The high-affinity interaction is characterized by a dissociation constant (K(D)) ≤ 1.0 µM, whereas the K(D) of the second binding has the same magnitude as the S(50). Theoretical analysis of a two-step binding model suggests that n(H) values may vary from 1.1 to 2.2 depending on the amplitude of the spin shift caused by the first binding event. Alteration of cooperativity in the mutants is caused by a partial displacement of the "spin-shifting" step. Although in the wild type the spin shift occurs in the ternary complex only, the mutants exhibit some spin shift on binding of the first substrate molecule.
... [38] In the majority of P450 enzymes, the first electron transfer is slow in the absence of substrate, but this is not the case with I401P (k f = 254 s À1 ), meaning the catalytic cycle for the mutant is not "gated" by the requirement for substrate binding to induce the structural changes required for fast first-electron transfer. [39,40] The leak rate was nonetheless significantly lower than the k f value. The k cat value with palmitic acid was also substantially lower than the palmitate-bound k f value measured at a lower temperature. ...
The crystal structures of the haem domains of Ala330Pro and Ile401Pro, two single-site proline variants of CYP102A1 (P450(BM3)) from Bacillus megaterium, have been solved. In the A330P structure, the active site is constricted by the relocation of the Pro329 side chain into the substrate access channel, providing a basis for the distinctive C-H bond oxidation profiles given by the variant and the enhanced activity with small molecules. I401P, which is exceptionally active towards non-natural substrates, displays a number of structural similarities to substrate-bound forms of the wild-type enzyme, notably an off-axial water ligand, a drop in the proximal loop, and the positioning of two I-helix residues, Gly265 and His266, the reorientation of which prevents the formation of several intrahelical hydrogen bonds. Second-generation I401P variants gave high in vitro oxidation rates with non-natural substrates as varied as fluorene and propane, towards which the wild-type enzyme is essentially inactive. The substrate-free I401P haem domain had a reduction potential slightly more oxidising than the palmitate-bound wild-type haem domain, and a first electron transfer rate that was about 10 % faster. The electronic properties of A330P were, by contrast, similar to those of the substrate-free wild-type enzyme.
Histamine dehydrogenase from the gram-negative bacterium Rhizobium sp. 4-9 (HaDHR) is a member of a small family of dehydrogenases containing a covalently attached FMN, and the only member so far identified to date that does not exhibit substrate inhibition. In this study, we present the 2.1 Å resolution crystal structure of HaDHR. This new structure allowed for the identification of the internal electron transfer pathway to abiological ferrocene-based mediators. Alanine 437 was identified as the exit point of electrons from the Fe4S4 cluster. The enzyme was modified with a Ser436Cys mutation to facilitate covalent attachment of a ferrocene moiety. When modified with Fc-maleimide, this new construct demonstrated direct electron transfer from the enzyme to a gold electrode in a histamine concentration-dependent manner without the need for any additional electron mediators.
Polymorphism is an important aspect in drug metabolism responsible for different individual response to drug dosage, often leading to adverse drug reactions. Here human CYP2C9 as well as its polymorphic variants CYP2C9*2 and CYP2C9*3 present in approximately 35% of the Caucasian population have been engineered by linking their gene to the one of D. vulgaris flavodoxin (FLD) that acts as regulator of the electron flow from the electrode surface to the haem. The redox properties of the immobilised proteins were investigated by cyclic voltammetry and electrocatalysis was measured in presence of the largely used anticoagulant drug S-warfarin, marker substrate for CYP2C9. Immobilisation of the CYP2C9-FLD, CYP2C9*2-FLD and CYP2C9*3-FLD on DDAB modified glassy carbon electrodes showed well defined redox couples on the oxygen-free cyclic voltammograms and mid-point potentials of all enzymes were calculated. Electrocatalysis in presence of substrate and quantification of the product formed showed lower catalytic activities for the CYP2C9*3-FLD (2.73 ± 1.07 min⁻¹) and CYP2C9*2-FLD (12.42 ± 2.17 min⁻¹) compared to the wild type CYP2C9-FLD (18.23 ± 1.29 min⁻¹). These differences in activity among the CYP2C9 variants are in line with the reported literature data, and this set the basis for the use of the bio-electrode for the measurement of the different catalytic responses towards drugs very relevant in therapy.
The interprotein electron‐transfer (ET) mechanism between lytic polysaccharide monooxygenases (LPMOs) and cellobiose dehydrogenase (CDH) was deciphered by multiscale simulations. Our simulations show that I) the specific ET pathway involves water molecules; II) the long‐range ET is enhanced by exothermic oxygen binding; III) enhanced ET is spin‐regulated and favors the quartet state over the doublet state.
Abstract
Long‐range electron transfer (ET) in metalloenzymes is a general and fundamental process governing O2 activation and reduction. Lytic polysaccharide monooxygenases (LPMOs) are key enzymes for the oxidative cleavage of insoluble polysaccharides, but their reduction mechanism by cellobiose dehydrogenase (CDH), one of the most commonly used enzymatic electron donors, via long‐range ET is still an enigma. Using multiscale simulations, we reveal that interprotein ET between CDH and LPMO is mediated by the heme propionates of CDH and solvent waters. We also show that oxygen binding to the copper center of LPMO is coupled with the long‐range interprotein ET. This process, which is spin‐regulated and enhanced by the presence of O2, directly leads to LPMO−CuII−O2⁻, bypassing the formation of the generally assumed LPMO−CuI species. The uncovered ET mechanism rationalizes experimental observations and might have far‐reaching implications for LPMO catalysis as well as the O2‐ or CO‐binding‐enhanced long‐range ET processes in other metalloenzymes.
The cytochrome P450 superfamily of heme monooxygenases catalyze important chemical reactions across nature. The changes in the optical spectra of these enzymes, induced by the addition of substrates or inhibitors, are critical for assessing how these molecules bind to the P450, enhancing or inhibiting the catalytic cycle. Here we use the bacterial CYP199A4 en-zyme (Uniprot ID; Q2IUO2), from Rhodopseudomonas palustris HaA2, and a range of substituted benzoic acids to investi-gate different binding modes. 4-Methoxybenzoic acid elicits an archetypal type I spectral response due to a 95% switch from the low- to high-spin state with concomitant dissociation of the sixth aqua ligand. 4-(Pyridin-3-yl)- and 4-(pyridin-2-yl)-benzoic acid induced different type II UV-vis spectral responses in CYP199A4. The former induced a greater red shift in the Soret wavelength (424 versus 422 nm) along with a larger overall absorbance change and other differences in the α-, β- and δ-bands. There were also variations in the ferrous UV-vis spectra of these two substrate-bound forms with a spectrum indicative of Fe-N bond formation with 4-(pyridin-3-yl)benzoic acid. The crystal structures of CYP199A4, with the pyridinyl com-pounds bound, revealed that while the nitrogen of 4-(pyridin-3-yl)benzoic acid is coordinated to the heme, with 4-(pyridin-2-yl)benzoic acid an aqua ligand remains. Continuous wave and pulse EPR data in frozen solution revealed that the sub-strates are bound in the active site in a form consistent with the crystal structures. The redox potential of each CYP199A4-substrate combination was measured, allowing correlation between binding modes, spectroscopic properties and the ob-served biochemical activity.
This study has evaluated the use of the P450 metalloenzymes CYP176A1, CYP101A1 and CYP102A1, together with engineered protein variants of CYP101A1 and CYP102A1, to alter the regioselectivity of 1,8- and 1,4-cineole hydroxylation. CYP176A1 was less selective for 1,4-cineole oxidation when compared to its preferred substrate, 1,8-cineole. The CYP102A1 variants significantly improved the activity over the WT enzyme for oxidation of 1,4- and 1,8-cineole. The CYP102A1 R47L/Y51F/A74G/F87V/L188Q mutant generated predominantly (1S)-6α-hydroxy-1,8-cineole (78% e.e.) from 1,8-cineole. Oxidation of 1,4-cineole by the CYP102A1 R47L/Y51F/F87A/I401P variant generated the 3α product in >90% yield. WT CYP101A1 formed a mixture metabolites with 1,8-cineole and very little product was generated with 1,4-cineole. In contrast the F87W/Y96F/L244A/V247L and F87W/Y96F/L244A variants of CYP101A1 favoured formation of 5α-hydroxy-1,8-cineole (>88%, 1S 86% e.e.) while the F87V/Y96F/L244A variant generated (1S)-6α-hydroxy-1,8-cineole in excess (90% regioselective, >99% e.e.). The CYP101A1 F87W/Y96F/L244A/V247L and F87W/Y96F/L244A mutants improved the oxidation of 1,4-cineole generating an excess of the 3α metabolite (1S > 99% e.e. with the latter). The CYP101A1 F87L/Y96F variant also improved the oxidation of this substrate but shifted the site of oxidation to the isopropyl group, (8-hydroxy-1,4-cineole). When this 8-hydroxy metabolite was generated in significant quantities desaturation of C8–C9 to the corresponding alkene was also detected.
The cytochrome P450 enzymes execute a range of selective oxidative biotransformations across many biological systems. The bacterial enzyme CYP199A4 catalyses the oxidative demethylation of 4-methoxybenzoic acid. The benzoic acid moiety of the molecule binds in the active site of the enzyme such that the functional group at the para-position is held close to the heme iron. Therefore, CYP199A4 has the potential to catalyse alternative monooxygenase reactions with different para-substituted benzoic acid substrates such as thioethers and alkylamines. The oxidation of 4-methyl- and 4-ethyl-thiobenzoic acids by CYP199A4 resulted in sulfur oxidation. 4-Ethylthiobenzoic acid sulfoxidation and 4-ethylbenzoic acid hydroxylation by CYP199A4 occurred with high enantioselectivity (>74% enantiomeric excess). By way of contrast, CYP199A4 catalysed exclusive oxidative N-demethylation over N-oxide formation with 4-methyl- and 4-dimethyl-aminobenzoic acids. Unexpectedly acetamide formation by CYP199A4 competes with dealkylation in the turnover of 4-ethyl- and diethyl-aminobenzoic acids. No oxidative dealkylation was observed with 3,4-ethylenedioxybenzoic with only hydroxylation to form a cyclic hemiacetal being detected. The X-ray crystal structures of four substrate-bound forms of the enzyme were solved and revealed subtle changes in the location of the para substituent which, when combined with the reactivity of the substituents, provided a basis for understanding the changes in selectivity. Furthermore, in the 4-ethylthiobenzoic acid bound structure the active site residue Phe298 moves to accommodate the substituent which points away from the heme iron. As such the CYP199A4 enzyme provides ready access to a combination of structural, binding and activity data with which to study a variety of reactions which are catalyzed by the P450 superfamily of enzymes.
CYP109E1 is a cytochrome P450 monooxygenase from Bacillus megaterium with a hydroxylation activity for testosterone and vitamin D3. This study reports the screening of a focused library of statins, terpene-derived and steroidal compounds to explore the substrate spectrum of this enzyme. Catalytic activity of CYP109E1 towards the statin drug-precursor compactin and the prodrugs lovastatin and simvastatin as well as biotechnologically relevant terpene compounds including ionones, nootkatone, isolongifolen-9-one, damascones, and β-damascenone was found in vitro. The novel substrates induced a type I spin-shift upon binding to P450 and thus permitted to determine dissociation constants. For the identification of conversion products by NMR spectroscopy, a B. megaterium whole-cell system was applied. NMR analysis revealed for the first time the ability of CYP109E1 to catalyze an industrially highly important reaction, the production of pravastatin from compactin, as well as regioselective oxidations generating drug metabolites (6′β-hydroxy-lovastatin, 3′α-hydroxy-simvastatin, and 4″-hydroxy-simvastatin) and valuable terpene derivatives (3-hydroxy-α-ionone, 4-hydroxy-β-ionone, 11,12-epoxy-nootkatone, 4(R)-hydroxy-isolongifolen-9-one, 3-hydroxy-α-damascone, 4-hydroxy-β-damascone, and 3,4-epoxy-β-damascone). Besides that, a novel compound, 2-hydroxy-β-damascenone, produced by CYP109E1 was identified. Docking calculations using the crystal structure of CYP109E1 rationalized the experimentally observed regioselective hydroxylation and identified important amino acid residues for statin and terpene binding.
The cytochrome P450 monooxygenase CYP101B1, from a Novosphingobium bacterium is able to bind and oxidise aromatic substrates but at a lower activity and efficiency compared to norisoprenoids and monoterpenoid esters. Histidine 85 of CYP101B1 aligns with tyrosine 96 of CYP101A1, which in this enzyme forms the only hydrophilic interaction with its substrate, camphor. The histidine residue of CYP101B1 was modified to a phenylalanine with the aim of improving the activity of the enzyme for hydrophobic substrates. The H85F mutant lowered the binding affinity and activity of the enzyme for β-ionone and altered the oxidation selectivity. This variant also showed enhanced affinity and activity towards alkylbenzenes, styrenes and methylnaphthalenes. For example the product formation rate of acenaphthene oxidation was improved 6-fold to 245 nmol.nmol-CYP-1.min-1. Certain disubstituted naphthalenes and substrates such as phenylcyclohexane, and biphenyls, were oxidised with lower activity by the H85F variant. Variants at H85 (A and G) designed to introduce additional space in the active site to accommodate these larger substrates did not engender improvements in the oxidation activity. As the H85F mutant of CYP101B1 improved the oxidation of hydrophobic substrates this residue is likely to be in the substrate binding pocket or the access channel of the enzyme. The side chain of the histidine may interact with the carbonyl groups of the favoured norisoprenoid substrates of CYP101B1.
Heme (iron protoporphyrin IX) proteins and enzymes play crucial roles in all living organisms. Iron is very tightly bound to the porphyrin and does not dissociate under physiological conditions. Indeed the heme group is almost like a separate element altogether. It is redox-active, and heme proteins such as cytochrome c have important electron-transfer functions. The strong π donor nature of heme iron(II) also means that heme proteins are involved in the binding and activation of small molecules such as oxygen. The P450 heme monooxygenases use two electrons and two protons to activate O2, giving one molecule of water and a high-valent iron-oxo intermediate that is sufficiently reactive to attack aliphatic CH bonds in a diverse range of organic molecules. The heme dioxygenases, as the name implies, insert both oxygen atoms of the O2 molecule into the substrate. The known heme dioxygenases are indoleamine ring cleavage enzymes but have not been studied in detail, and no crystal structure of the enzymes is available. It is known that the active form of the enzyme has Fe(II), and that both oxygen and superoxide could be the source of the two oxygen atoms inserted into substrates. The main focus of this article is on the cytochrome P450 monooxygenases.
Stone artifacts from the Bose basin, South China, are associated with tektites dated to 803,000 +/- 3000 years ago and represent the oldest known Large cutting tools (LCTs) in East Asia. Bose toolmaking is compatible with Mode 2 (Acheulean) technologies in Africa in its targeted manufacture and biased spatial distribution of LCTs, Large-scale flaking, and high flake scar counts. Acheulean-Like tools in the mid-Pleistocene of South China imply that Mode 2 technical advances were manifested in East Asia contemporaneously with handaxe technology in Africa and western Eurasia. Bose Lithic technology is associated with a tektite airfall and forest burning.
The complex multistep mechanism of oxygen activation in P450 is reviewed as a sequence of the following reactions: Substrate binding, reduction of the heme iron from ferric to the ferrous state, binding of dioxygen, second electron transfer and formation of peroxo-ferric intermediate, two sequential protonation events to give hydroperoxo-ferric intermediate, and finally, after O–O bond scission, the ferryl-oxo intermediate, termed compound I. Details of these processes and the role of interactions with redox partners, as well as substrate variability in the overall efficiency of P450 catalysis, are discussed. In addition, common points and variations between the soluble prokaryotic and membrane-bound eukaryotic cytochromes P450 with respect to the oxygen activation mechanisms are briefly compared.
Background:
Metastasis is the primary cause of prostate cancer (PCa) lethality and poses a huge clinical obstacle. Lipocalin 2 (LCN2), a member of the lipocalin family, is aberrantly expressed in some human cancers and has been implicated in the progression of some tumors. However, the role of LCN2 in the metastatic capacity of prostate cancer (PCa) is poorly understood.
Methods:
LCN2 expression was examined by RT-qPCR and/or immunoblotting in human prostate tissue specimens and prostate cancer cell lines LNCaP, C4-2, 22RV1, PC3, DU-145, and PC3MM2. LCN2 protein level in human serum samples was determined by ELISA. Lentiviruses-mediated over-expression of LCN2 and knockdown of LCN2 was conducted to evaluate the role of LCN2 in cell migratory and invasive capacities of prostate cancer cells. Cell migration and invasion was examined by transwell chamber assay. Knockdown of SLUG by lentivirus was performed to investigate its role in LCN2-promoted cell migration and invasion in vitro (22RV1 cell line) and metastasis in vivo (tail vein metastasis assay in nude mice). Role of ERK signaling in LCN2-mediated up-regulation of SLUG was assayed by using ERK inhibitor U0126.
Results:
We confirmed that LCN2 levels were correlated positively with invasive prostate cancer in human tissue and serum samples, and were also consistently associated with the invasive capacity of prostate cancer cell lines. The over-expression of LCN2 in 22RV1 cells (not highly invasive) promoted the epithelial-mesenchymal transition (EMT), increasing cell motility and invasiveness, while the knockdown of LCN2 in PC3 cells (highly invasive) inhibited EMT, decreasing cell motility and invasiveness. Among the multiple EMT transcription factors, LCN2 specifically induces the expression of SLUG, which was shown here to be required for the LCN2-induced increase in the invasive capacity of prostate cancer cells both in vitro and in vivo. Mechanistically, LCN2 promoted SLUG expression via activating ERK signaling pathway.
Conclusion:
LCN2 plays an important role in promoting cell migration and invasion of prostate cancer by inducing EMT through the ERK/SLUG axis. Therefore, targeted inhibition of LCN2 may represent a therapeutic strategy to prevent the metastasis of prostate cancer.
Using a combination of self-assembly and synthesis, bioinspired electrodes having dilute iron porphyrin active sites bound to axial thiolate and imidazole axial ligands are created atop self-assembled monolayers (SAMs). Resonance Raman data indicate that a picket fence architecture results in a high-spin (HS) ground state (GS) in these complexes and a hydrogen-bonding triazole architecture results in a low-spin (LS) ground state. The reorganization energies (λ) of these thiolate- and imidazole-bound iron porphyrin sites for both HS and LS states are experimentally determined. The λ of 5C HS imidazole and thiolate-bound iron porphyrin active sites are 10-16 kJ/mol, which are lower than their 6C LS counterparts. Density functional theory (DFT) calculations reproduce these data and indicate that the presence of significant electronic relaxation from the ligand system lowers the geometric relaxation and results in very low λ in these 5C HS active sites. These calculations indicate that loss of one-half a π bond during redox in a LS thiolate bound active site is responsible for its higher λ relative to a σ-donor ligand-like imidazole. Hydrogen bonding to the axial ligand leads to a significant increase in λ irrespective of the spin state of the iron center. The results suggest that while the hydrogen bonding to the thiolate in the 5C HS thiolate bound active site of cytochrome P450 (cyp450) shifts the potential up, resulting in a negative ΔG, it also increases λ resulting in an overall low barrier for the electron transfer process.
Constructing renormalizable models on non-commutative spaces constitutes a
big challenge. Only few examples of renormalizable theories are known, such as
the scalar Grosse-Wulkenhaar model. Gauge fields are even more difficult, since
new renormalization techniques are required which are compatible with the
inherently non-local setting on the one hand, and also allow to properly treat
the gauge symmetry on the other hand. In this proceeding (which is based on my
talk given at the ``Workshop on Noncommutative Field Theory and Gravity'' in
Corfu/Greece, September 8 -- 15, 2013), I focus on this last point and present
new extensions to existing renormalization schemes (which are known to work for
gauge field theories in commutative space) adapted to non-commutative Moyal
space.
We analyze the polynomial part of the Iwasawa realization of the coset representative of non compact symmetric Riemannian spaces. We start by studying the role of Kostant's principal SU(2)P subalgebra of simple Lie algebras, and how it determines the structure of the nilpotent subalgebras. This allows us to compute the maximal degree of the polynomials for all faithful representations of Lie algebras. In particular the metric coefficients are related to the scalar kinetic terms while the representation of electric and magnetic charges is related to the coupling of scalars to vector field strengths as they appear in the Lagrangian. We consider symmetric scalar manifolds in 𝒩-extended supergravity in various space-time dimensions, elucidating various relations with the underlying Jordan algebras and normed Hurwitz algebras. For magic supergravity theories, our results are consistent with the Tits-Satake projection of symmetric spaces and the nilpotency degree turns out to depend only on the space-time dimension of the theory. These results should be helpful within a deeper investigation of the corresponding supergravity theory, e.g. in studying ultraviolet properties of maximal supergravity in various dimensions.
Protected cyclohexanol and cyclohex-2-enol substrates, containing benzyl ether and benzoate ester moieties, were designed to fit into the active site of the Tyr96Ala mutant of cytochrome P450cam. The protected cyclohexanol substrates were efficiently and selectively hydroxylated by the mutant enzyme at the trans C-H bond of C-4 on the cyclohexyl ring. The selectivity of oxidation of the benzoate ester protected cyclohexanol could be altered by making alternative amino acid substitutions in the P450cam active site. The addition of the double bond in the cyclohexyl ring of the benzoate ester protected cyclohex-2-enol has a debilitative effect on the activity of the Tyr96Ala mutant with this substrate. However, the Phe87Ala/Tyr96Phe double mutant, which introduces space at a different location in the active site than the Tyr96Ala mutant, was able to efficiently hydroxylate the C-H bonds of 1-cyclohex-2-enyl benzoate at the allylic C-4 position. Mutations at Phe87 improved the selectivity of the oxidation of 1-phenyl-1-cyclohexylethylene to trans-4-phenyl-ethenylcyclohexanol (92%) when compared to single mutants at Tyr96 of P450cam.
The energetics of protein-DNA interactions are often modeled using so-called statistical potentials, that is, energy models derived from the atomic structures of protein-DNA complexes.Many statistical protein-DNA potentials based on differing theoretical assumptions have been investigated, but little attention has been paid to the types of data and the parameter estimation process used in deriving the statistical potentials.We describe three enhancements to statistical potential inferencethat significantly improve the accuracy of predicted protein-DNA interactions: (i) incorporation of binding energy data of protein-DNA complexes, in conjunction with their X-ray crystal structures, (ii) use of spatially-aware parameter fitting, and (iii) use of ensemble-based parameter fitting.We apply these enhancements to three widely-used statistical potentials and use the resulting enhanced potentials in a structure-based prediction of the DNA binding sites of proteins.These enhancements are directly applicable to all statistical potentials used in protein-DNAmodeling, and we show that they can improve the accuracy of predicted DNA binding sites by up to 21%. Proteins 2012. © 2012 Wiley Periodicals, Inc.
Cytochrome P450cam (CYP101) catalyzes the oxidation of D(+)-camphor at the 5 position. The enzyme couples the reduction of dioxygen to the oxidation of the substrate. To transfer electrons from the reductant (NADH) to the cytochrome, two additional proteins are required. These are putidaredoxin (PdX) and putidaredoxin reductase (PdR). We have chemically linked a form of PdX with a histidine tag at the C-terminus to the P450. To accomplish this, we have modified cysteine 334 on P450 with a bipyridinyl group, and co-ordinated the C-terminal histidine tag of PdX by the addition of Ni2+ or Ru3+. The Ru3+ complex was the most stable. The non-linked system gave mostly 5-ketocamphor, a product of two consecutive hydroxylations, and H2O2, a product of 2-electron uncoupling. The Ni2+ complex gave both 5-exo-hydroxycamphor and 5-ketocamphor, but it also uncoupled. The Ru3+ complex gave a single product (5-exo-hydroxycamphor) and did not uncouple at the optimal PdR concentration. Our results are consistent with other studies of this system, in that strong binding of PdX to P450 is crucial for good coupling and for release of 5-exo-hydroxycamphor.
The Phe-193 residue on the surface of cytochrome P450cam is part of a cluster of residues proposed to undergo dynamic fluctuations to permit the entry of substrates into the active site pocket. The role of this residue in the activity of P450cam has been investigated. The F193A, F193V, F193I, and F193L mutations were introduced into the Y96F mutant, which had been shown to oxidize a wider range of molecules at faster rates than the wild-type enzyme. The F193L mutation had very little effect, while the F193A and F193I mutations reduced the camphor oxidation rate and almost abolished the styrene and naphthalene oxidation activity of the Y96F mutant. In contrast, the high activity of the Y96F mutant for the oxidation of adamantane, hexane, and 3-methylpentane was largely retained, although the product distributions were significantly altered. This dramatic difference between the F193L and F193I mutations warrants further investigation. The turnover rates of the Y96F–F193I with all the substrates showed the same dependence on the Pd:P450cam concentration ratio as for the Y96F mutant, clearly indicating that if the F193 mutations had affected substrate access, substrate entry was still fast compared to the first electron transfer, which remained the rate-limiting step for the overall reaction. We concluded that the F193A and F193I mutations shifted the substrate specificity of P450cam by causing structural changes that were relayed from their surface position down to the vicinity of the heme. The altered substrate binding resulted in differential electron transfer kinetics between classes of compounds.
BACKGROUND
In bone metastatic sites, prostate cancer cells proliferate on interacting with osteoclasts. Tranilast, which is used for an antiallergic drug, has been shown to inhibit growth of several cancers and stromal cells. The present study was conducted to assess suppressive effects of Tranilast on prostate cancer growth and osteoclast differentiation in vivo and in vitro.METHODS
In vivo, rat prostate cancer tissue was transplanted onto cranial bones of F344 rats and Tranilast was given for 9 days at doses of 0, 200, or 400 mg/kg/day. In vitro, human prostate cancer cell lines, LNCaP, PC3, and DU145, the rat prostate cancer cell line, PLS-10, and rat bone marrow cells were similarly treated with the agent.RESULTSIn vivo, tumor volumes were significantly decreased in the high dose group. While cell proliferation did not appear to be affected, apoptosis was induced and tumor necrosis was apparent. Cranial bone defects were decreased in the high dose group. In vitro, cell proliferation rates of all four cell lines were reduced by Tranilast and increased apoptosis was observed in LNCaP and PLS-10. In addition, Tranilast significantly reduced osteoclast differentiation of rat bone marrow cells. Western blot analysis of PLS-10 and LNCaP revealed that phospho-GSK3β was up-regulated and phospho-Akt was down-regulated.CONCLUSIONS
Tranilast here suppressed rat prostate cancer growth and osteoclast differentiation. Growth of human prostate cancer cells was also inhibited. Thus, this agent deserves consideration as a candidate for conventional therapy of bone metastatic prostate cancer. Prostate 70: 229–238, 2010. © 2009 Wiley-Liss, Inc.
Heme (iron protoporphyrin IX) proteins and enzymes play crucial roles in all living organisms. Iron is very tightly bound to the porphyrin and does not dissociate under physiological conditions. Indeed the heme group is almost like a separate element altogether. It is redox-active, and heme proteins such as cytochrome c have important electron-transfer functions. The strong π donor nature of heme iron(II) also means that heme proteins are involved in the binding and activation of small molecules such as oxygen. The P450 heme monooxygenases use two electrons and two protons to activate O2, giving one molecule of water and a high-valent iron-oxo intermediate that is sufficiently reactive to attack aliphatic CH bonds in a diverse range of organic molecules. The heme dioxygenases, as the name implies, insert both oxygen atoms of the O2 molecule into the substrate. The known heme dioxygenases are indoleamine ring cleavage enzymes but have not been studied in detail, and no crystal structure of the enzymes is available. It is known that the active form of the enzyme has Fe(II), and that both oxygen and superoxide could be the source of the two oxygen atoms inserted into substrates. The main focus of this article is on the cytochrome P450 monooxygenases.
The human cytochromes P450 are responsible for the clearance of ∼90% of xenobiotics yet comparatively little is known about their electrochemistry. Here we report the first direct electrochemistry of P450s from the 2C subfamily; one of the major groups of enzymes from this family. Specifically, the proteins that we have examined are recombinant human P450s 2C9, 2C18 and 2C19 and reversible FeIII/II couples are seen in the absence of dioxygen. Even in the presence of trace amounts of dioxygen, a pronounced cathodic response is seen which is assigned to catalytic reduction of the bound dioxygen ligand by the ferrous P450.
CYP108D1 from Novosphingobium aromaticivorans DSM12444 binds a range of aromatic hydrocarbons such as phenanthrene, biphenyl and phenylcyclohexane. Its structure, which is reported here at 2.2 Å resolution, is closely related to that of CYP108A1 (P450terp), an α-terpineol-oxidizing enzyme. The compositions and structures of the active sites of these two enzymes are very similar; the most significant changes are the replacement of Glu77 and Thr103 in CYP108A1 by Thr79 and Val105 in CYP108D1. Other residue differences lead to a larger and more hydrophobic access channel in CYP108D1. These structural features are likely to account for the weaker α-terpineol binding by CYP108D1 and, when combined with the presence of three hydrophobic phenylalanine residues in the active site, promote the binding of aromatic hydrocarbons. The haem-proximal surface of CYP108D1 shows a different charge distribution and topology to those of CYP101D1, CYP101A1 and CYP108A1, including a pronounced kink in the proximal loop of CYP108D1, which may result in poor complementarity with the [2Fe-2S] ferredoxins Arx, putidaredoxin and terpredoxin that are the respective redox partners of these three P450 enzymes. The unexpectedly low reduction potential of phenylcyclohexane-bound CYP108D1 (-401 mV) may also contribute to the low activity observed with these ferredoxins. CYP108D1 appears to function as an aromatic hydrocarbon hydroxylase that requires a different electron-transfer cofactor protein.
Cytochrome P450 enzymes (P450s) are exceptionally versatile monooxygenases, mediating hydroxylations of unactivated C–H bonds,
epoxidations, dealkylations, and N- and S-oxidations as well as other less common reactions. In the conventional view of the catalytic cycle, based upon studies of
P450s in vitro, substrate binding to the Fe(III) resting state facilitates the first 1-electron reduction of the heme. However, the resting
state of P450s in vivo has not been examined. In the present study, whole cell difference spectroscopy of bacterial (CYP101A1 and CYP176A1, i.e. P450cam and P450cin) and mammalian (CYP1A2, CYP2C9, CYP2A6, CYP2C19, and CYP3A4) P450s expressed within intact Escherichia coli revealed that both Fe(III) and Fe(II) forms of the enzyme are present in the absence of substrates. The relevance of this
finding was supported by similar observations of Fe(II) P450 heme in intact rat hepatocytes. Electron paramagnetic resonance
(EPR) spectroscopy of the bacterial forms in intact cells showed that a proportion of the P450 in cells was in an EPR-silent
form in the native state consistent with the presence of Fe(II) P450. Coexpression of suitable cognate electron donors increased
the degree of endogenous reduction to over 80%. A significant proportion of intracellular P450 remained in the Fe(II) form
after vigorous aeration of cells. The addition of substrates increased the proportion of Fe(II) heme, suggesting a kinetic
gate to heme reduction in the absence of substrate. In summary, these observations suggest that the resting state of P450s
should be regarded as a mixture of Fe(III) and Fe(II) forms in both aerobic and oxygen-limited conditions.
Most prostate cancer-related deaths occur in patients with castration-resistant prostate cancer (CRPC). Recent preclinical and clinical studies have identified intracellular signaling pathways and changes in the tumor and bone microenvironment as potential key drivers of CRPC. This increased understanding of mechanisms associated with CRPC has driven the development of numerous new agents, many of which are poised to alter the current CRPC treatment landscape.
A review of literature was conducted to identify ongoing and planned phase III studies of novel agents to treat CRPC.
Multiple studies were identified, including novel androgen biosynthesis inhibitors (abiraterone, TAK-700), androgen-receptor inhibitors (MDV3100), angiogenesis inhibitors (aflibercept, tasquinimod), endothelin antagonists (zibotentan, atrasentan), a Src tyrosine kinase inhibitor (dasatinib), a novel radiotherapy (radium-223), and new immunotherapies (ipilimumab and ProstVac). In addition, both sipuleucel-T (an immunotherapy) and cabazitaxel (third-generation taxane) and the RANK-L inhibitor, denosumab, have recently been approved by the US Food and Drug Administration.
Various combinations of these agents could theoretically be used to treat future patients with CRPC by targeting multiple signaling pathways as well as aspects of the tumor and bone microenvironments. Additional research will be needed to understand how to best use these agents and individualize care to optimize CRPC patient outcomes.
SNARE proteins and fusogenic viral membrane proteins represent the major classes of integral membrane proteins that mediate fusion of eukaryotic lipid bilayers. Although both classes have different primary structures, they share a number of basic architectural features. There is ample evidence that the fusogenic function of representative fusion proteins is influenced by the primary structure of the single transmembrane domain (TMD) and the region linking it to the soluble assembly domains. Here, we used comprehensive non-redundant datasets to examine potential over- and underrepresentation of amino acid types in the TMDs and flanking regions relative to control proteins that share similar biosynthetic origins. Our results reveal conserved overall and/or site-specific enrichment of β-branched residues and Gly within the TMDs, underrepresentation of Gly and Pro in regions flanking the TMD N-terminus, and overrepresentation of the same residue types in C-terminal flanks of SNAREs and viral fusion proteins. Furthermore, the basic Lys and Arg are enriched within SNARE N-terminal flanking regions. These results suggest evolutionary conservation of key structural features of fusion proteins and are discussed in light of experimental findings that link these features to the fusogenic function of these proteins.
The substrate-free crystal structure of a five-mutation directed evolution variant of CYP102A1 (P450(BM3)) with generic activity-enhancing properties ("KT2") has been determined to 1.9-Å resolution. There is a close resemblance to substrate-bound structures of the wild-type enzyme (WT). The disruption of two salt bridges that link the G- and I-helices in WT causes conformational changes that break several hydrogen bonds and reduce the angle of the kink in the I-helix where dioxygen activation is thought to take place. The side-chain of a key active site residue, Phe87, is rotated in one molecule of the asymmetric unit, and the side-chains of Phe158 and Phe261 cascade into the orientations found in fatty-acid-bound forms of the enzyme. The iron is out of the porphyrin plane, towards the proximal cysteine. Unusually, the axial water ligand to the haem iron is not hydrogen-bonded to Ala264. The first electron transfer from the reductase domain to the haem domain of substrate-free KT2 is almost as fast as in palmitate-bound WT even though the reduction potential of the haem domain is only slightly more oxidising than that of substrate-free WT. However, NADPH is turned over slowly in the absence of substrate, so the catalytic cycle is gated by a step subsequent to the first electron transfer-a contrast to WT. Propylbenzene binding slightly raises the first electron transfer rate in WT but not in KT2. It is proposed that the generic rate accelerating properties of KT2 arise from the substrate-free form being in a catalytically ready conformation, such that substrate-induced changes to the structure play a less significant role in promoting the first electron transfer than in WT.
Introduction:
There is increasing evidence of physical interactions (association) among cytochromes P450 in the membranes of the endoplasmic reticulum. Functional consequences of these interactions are often underestimated.
Areas covered:
This article provides a comprehensive overview of available experimental material regarding P450-P450 interactions. Special emphasis is given to the interactions between different P450 species and to the functional consequences of homo- and heterooligomerization.
Expert opinion:
Recent advances provide conclusive evidence for a substantial degree of P450 oligomerization in membranes. Interactions between different P450 species resulting in the formation of mixed oligomers with altered activity and substrate specificity have been demonstrated clearly. There are important indications that oligomerization impedes electron flow to a fraction of the P450 population, which renders some P450 species nonfunctional. Functional consequences of P450-P450 interactions make the integrated properties of the microsomal monooxygenase remarkably different from a simple summation of the properties of the individual P450 species. This complexity compromises the predictive power of the current in vitro models of drug metabolism and warrants an urgent need for development of new model systems that consider the interactions of multiple P450 species.
A study of the single turnover kinetics of the reaction between oxycytochrome P-450cam and reduced putidaredoxin was performed using the inhibitor metyrapone to trap the cytochrome immediately after release of the product, 5-exo-hydroxycamphor. EPR determinations of the concentrations of reduced putidaredoxin and ferric metyrapone-bound cytochrome at the same time points showed that there is no time lag between the oxidation of reduced putidaredoxin and the appearance of metyrapone-bound cytochrome. This implies that the rate constant for electron transfer is smaller than the rate constant for the later processes involved in product formation and release, lumped into a single step. Taking this restriction into account and doing computer simulation of absorbance versus time curves, previously obtained at various putidaredoxin concentrations using stopped-flow spectrophotometry, allowed bounds to be determined for rate constants of the processes within the reaction. At 4 degrees C in buffer at pH 7.4 with 0.50 M KCl, the rate constant for the bimolecular association of the two enzymes is between 3 and 20/microM.s; the rate constant for dissociation is between 12 and 600/s; the rate constant for electron transfer is between 60 and 100/s; and the rate constant for the later processes is at least 200/s.
The oxidation-reduction behavior of the iron sulfide protein, putidaredoxin, isolated from camphor-grown Pseudomonas putida has been examined in the native sulfur and in the selenide-substituted forms. This change in the replaceable chalcogenide ligand shifts the potential at pH 7 from -235 mv (sulfur) to -245 mv (selenide). Analysis of the formal potentials of the sulfur protein over the range pH 6.5 to 10 indicates two proton-linked processes in the oxidized form (K′ o1 = 1.0 x 10⁻⁸; K′o2 = 1.0 x 10⁻¹⁰) and one in the reduced form (K′ r1 = 1.0 x 10⁻⁹). The potential versus pH curves for the sulfur and selenide proteins merge at high pH.
In the ten years that have elapsed since the first edition of this book went to press, the cytochrome P450 field has completed the transition to a discipline in which structure and mechanism, even regulation and biological function, are dealt with in molecular terms. The twin forces that have propelled this remarkable progress have been the widespread adoption of molecular biological approaches and the successful application of modem structural techniques. Only a few P450 primary sequences were available in 1985, whereas hundreds of P450 sequences are now available. Site-specific mutagenesis was then mostly a proverbial gleam in the eye of the P450 community, but it is now a standard technique in the research repertoire. The first crystal structure of a cytochrome P450 enzyme had just been solved in 1985 and appeared on the cover of the first edition. Today, the high-reso lution crystal structures of four soluble bacterial P450 enzymes are available and the race is on to develop approaches that will permit us to determine the structures of the membrane-bound forms of the enzyme. The past ten years has seen phenomenal progress let us hope that the next ten will prove equally exciting. The book is informally divided into four sections. In order to hold the book close to the advancing front of research, some of the chapter topics from the first edition have been dropped to make room for new or expanded topics.
Major advances have been made in recent years in clarifying the molecular properties of the cytochrome P-450 system. These advances stem, in practical terms, from the generally recognized importance of cytochrome P-450 in the metabolism of drugs and in the bioactivation of xenobiotics to toxic products. The fascinating multiplicity and differential regulation of cytochrome P-450 isozymes, and their ability to catalyze extraordinarily difficult chemical transformations, have independently drawn many chemists and biochemists into the P-450 circle. Progress in the field, from a technical point of view, has been propelled by the de velopment of reliable procedures for the purification of membrane-bound enzymes, by the growing repertoire of molecular biological techniques, and by the development of chemical models that mimic the catalytic action of P-450. As a result, our understanding of the P-450 system is moving from the descriptive, pharmacological level into the tangible realm of atomic detail. The rapid progress and multidisciplinary character of the cytochrome P-450 field, which cuts across the lines that traditionally divide disciplines as diverse as inorganic chemistry and genetics, have created a need for an up-to-date evaluation of the advances that have been made. It is hoped that this book, with its molecular focus on the cytochrome P-450 system, will alleviate this need. The authors of the individual chapters have strived to emphasize recent results without sacrificing the background required to make their chapters comprehensible to informed nonspecialists.
The purpose of this chapter is twofold. First, we will attempt to summarize the existing knowledge on the wide variety of bacterial cytochrome P-450 hemeproteins that have been discovered. As such, this represents the first comprehensive review of the bacterial cytochromes P-450. It should become evident to the reader that these cytochromes comprise a class just as diverse, if not more so, than their eukaryotic counterparts. Perhaps the best known and most extensively characterized of these bacterial enzymes is P-450cam isolated from Pseudomonas putida. From the initial isolation in Gunsalus’s laboratory in 1967 and subsequent investigations in many laboratories, the numerous studies employing this cytochrome have shed much light on the mechanisms associated with the family of P-450-dependent monooxygenases as a whole. A second goal of this chapter is to review the detailed biophysical, biochemical, and molecular biological studies conducted with this system. It should be noted that “P-450cam” and “bacterial P-450” are not synonymous. The various P-450 molecules described herein are in all probability only a very small fraction of those present in the myriad of bacterial cells in the biosphere. In view of the variety of substrate transformations that this enzyme family is able to catalyze, some of which are energetically very difficult, their distribution in nature is no doubt ubiquitous. One need only look through the American Type Culture Collection Catalogue1 under such diverse genera as Pseudomonas, Rhodococcus, Bacillus, and Nocardia among others to see the potential roles of yet undiscovered P-450 monooxygenases in the degradation and transformation of a plethora of compounds. Even if only a small portion of the enzymes involved in these various biotransformations are P-450 isozymes, there would correspond many hundreds of P-450 hemeproteins with definable specificities.
Electron-transfer reactions between ions and
molecules in solution have been the subject of
considerable experimental study during the past
three decades. Experimental results have also been
obtained on related phenomena, such as reactions
between ions or molecules and electrodes, charge-transfer
spectra, photoelectric emission spectra of
ionic solutions, chemiluminescent electron transfers,
electron transfer through frozen media, and
electron transfer through thin hydrocarbon-like
films on electrodes.
This chapter discusses the determination of midpoint potentials of oxidation–reduction components of biological electron-transfer systems using redox potentiometry. The experimental goal is to measure the redox potential, Eh, and a corresponding state of oxidation or reduction of a redox couple: Eh is measured by electrodes, while the state of oxidation-reduction of a redox component is measured by some physical technique, usually some form of spectrometry. The character of the biological redox component usually dictates the chosen technique; the character also will dictate whether the oxidized or reduced form is measured. Several such correlations of Eh and state of oxidation or reduction should be taken at Eh values that extend over the range that encompasses the central part of the expected Nernst curve. To make the analysis straightforward it is preferable to obtain readings for the state of oxidation-reduction as nearly fully oxidized and reduced as possible. The information in the chapter describes the Nernst curve of the redox couple and provides the standard half reduction or midpoint potential value and the number of electrons.
Cytochrome P450cam was subjected to high pressures of 2.2 kbar, converting the enzyme to its inactive form P420cam. The resultant protein was characterized by electron paramagnetic resonance, magnetic circular dichroism, circular dichroism, and electronic absorption spectroscopy. A range of exogenous ligands has been employed to probe the coordination structure of P420cam. The results suggest that conversion to P420cam involves a conformational change which restricts the substrate binding site and/or alters the ligand access channel. The reduction potential of P420cam is essentially the same in the presence or absence of camphor (-211 +/- 10 and -210 +/- 15 mV, respectively). Thus, the well-documented thermodynamic regulation of enzymatic activity for P450cam in which the reduction potential is coupled to camphor binding is not found with P420cam. Further, cyanide binds more tightly to P420cam (Kd = 1.1 +/- 0.1 mM) than to P450cam (Kd = 4.6 +/- 0.2 mM), reflecting a weakened iron-sulfur ligation. Spectral evidence reported herein for P420cam as well as results from a parallel investigation of the spectroscopically related inactive form of chloroperoxidase lead to the conclusion that a sulfur-derived proximal ligand is coordinated to the heme of ferric cytochrome P420cam.
Flavocytochrome P-450 BM3 from Bacillus megaterium is a 119 kDa polypeptide whose heme and diflavin domains are fused to produce a catalytically self-sufficient fatty acid monooxygenase. Redox potentiometry studies have been performed with intact flavocytochrome P-450 BM3 and with its component heme, diflavin, FAD, and FMN domains. Results indicate that electron flow occurs from the NADPH donor through FAD, then FMN and on to the heme center where fatty acid substrate is bound and monooxygenation occurs. Prevention of futile cycling of electrons is avoided through an increase in redox potential of more than 100 mV caused by binding of fatty acids to the active site of P-450. Redox potentials are little altered for the component domains with respect to their values in the larger constructs, providing further evidence for the discrete domain organization of this flavocytochrome. The reduction potentials of the 4-electron reduced diflavin domain and 2-electron reduced FAD domain are considerably lower than those for the blue FAD semiquinone species observed during reductive titrations of these enzymes and that of the physiological electron donor (NADPH), indicating that the FAD hydroquinone is thermodynamically unfavorable and does not accumulate under turnover conditions. In contrast, the FMN hydroquinone is thermodynamically more favorable than the semiquinone.
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