Electrochemistry of cytochrome P450 BM3 in sodium dodecyl sulfate films

Direct electrochemistry of the cytochrome P450 BM3 heme domain (BM3) was achieved by confining the protein within sodium dodecyl sulfate (SDS) films on the surface of basal-plane graphite (BPG) electrodes. Cyclic voltammetry revealed the heme FeIII/II redox couple at -330 mV (vs Ag/AgCl, pH 7.4). Up to 10 V/s, the peak current was linear with the scan rate, allowing us to treat the system as surface-confined within this regime. The standard heterogeneous rate constant determined at 10 V/s was estimated to be 10 s-1. Voltammograms obtained for the BM3-SDS-BPG system in the presence of dioxygen exhibited catalytic waves at the onset of FeIII reduction. The altered heme reduction potential of the BM3-SDS-graphite system indicates that SDS is likely bound in the enzyme active-site region. Compared to other P450-surfactant systems, we find redox potentials and electron-transfer rates that differ by approximately 100 mV and >10-fold, respectively, indicating that the nature of the surfactant environment has a significant effect on the observed heme redox properties.     

Laser photoexcitation of NAD(P)H induces reduction of P450 BM3 heme domain on the microsecond time scale

We demonstrate that photoexcitation of NAD(P)H at 355 nm using a Nd:YAG laser leads to rapid reduction of the heme domain of the Bacillus megaterium fatty acid hydroxylase flavocytochrome P450 BM3. An aqueous electron derived from photoexcited NAD(P)H is rapidly transferred to the heme domain, enabling the formation of a carbon monoxy complex of the ferrous P450 (FeII-CO) on the microsecond time scale. Using this approach we have determined the limiting rate constant (1770 s-1 for substrate-free heme domain) for formation of the FeII-CO complex. We find no dependence of the observed rate of FeII-CO complex formation on NAD(P)H concentration but demonstrate a hyperbolic dependence on carbon monoxide concentration. The apparent dissociation constant for the complex of carbon monoxide bound noncovalently to the ferric form of the BM3 heme domain (and with NADH as reductant) is 323 microM. Binding of a P450 substrate (N-palmitoylglycine) weakened the complex between carbon monoxide and the ferric BM3 heme domain (Kd increased to 1404 microM) but enhanced the rate of formation of the FeII-CO complex (3036 s-1 for substrate-free heme domain). This study demonstrates the applicability of NAD(P)H photoexcitation as a method for rapid electron delivery to P450 enzymes and provides a new route to probing the P450 catalytic cycle and its transient intermediates.     

Direct Hydroxylation of Benzene to Phenol by Cytochrome P450BM3 Triggered by Amino Acid Derivatives

The selective hydroxylation of benzene to phenol, without the formation of side products resulting from overoxidation, is catalyzed by cytochrome P450BM3 with the assistance of amino acid derivatives as decoy molecules. The catalytic turnover rate and the total turnover number reached 259 min⁻¹ P450BM3⁻¹ and 40 200 P450BM3⁻¹ when N‐heptyl‐l ‐proline modified with l ‐phenylalanine (C7‐l ‐Pro‐l ‐Phe) was used as the decoy molecule. This work shows that amino acid derivatives with a totally different structure from fatty acids can be used as decoy molecules for aromatic hydroxylation by wild‐type P450BM3. This method for non‐native substrate hydroxylation by wild‐type P450BM3 has the potential to expand the utility of P450BM3 for biotransformations.     

Light-initiated hydroxylation of lauric acid using hybrid P450 BM3 enzymes

We have developed hybrid P450 BM3 enzymes consisting of a Ru(II)-diimine photosensitizer covalently attached to non-native single cysteine residues of P450 BM3 heme domain mutants. These enzymes are capable, upon light activation, of selectively hydroxylating lauric acid with 40 times higher total turnover numbers compared to the peroxide shunt.     

Metal-substituted protein MRI contrast agents engineered for enhanced relaxivity and ligand sensitivity

Engineered metalloproteins constitute a flexible new class of analyte-sensitive molecular imaging agents detectable by magnetic resonance imaging (MRI), but their contrast effects are generally weaker than synthetic agents. To augment the proton relaxivity of agents derived from the heme domain of cytochrome P450 BM3 (BM3h), we formed manganese(III)-containing proteins that have higher electron spin than their native ferric iron counterparts. Metal substitution was achieved by coexpressing BM3h variants with the bacterial heme transporter ChuA in Escherichia coli and supplementing the growth medium with Mn3+-protoporphyrin IX. Manganic BM3h variants exhibited up to 2.6-fold higher T1 relaxivities relative to native BM3h at 4.7 T. Application of ChuA-mediated porphyrin substitution to a collection of thermostable chimeric P450 domains resulted in a stable, high-relaxivity BM3h derivative displaying a 63% relaxivity change upon binding of arachidonic acid, a natural ligand for the P450 enzyme and an important component of biological signaling pathways. This work demonstrates that protein-based MRI sensors with robust ligand sensitivity may be created with ease by including metal substitution among the toolkit of methods available to the protein engineer.     

Cofactor‐Free Light‐Driven Whole‐Cell Cytochrome P450 Catalysis

Cytochromes P450 can catalyze various regioselective and stereospecific oxidation reactions of non‐functionalized hydrocarbons. Here, we have designed a novel light‐driven platform for cofactor‐free, whole‐cell P450 photo‐biocatalysis using eosin Y (EY) as a photosensitizer. EY can easily enter into the cytoplasm of Escherichia coli and bind specifically to the heme domain of P450. The catalytic turnover of P450 was mediated through the direct transfer of photoinduced electrons from the photosensitized EY to the P450 heme domain under visible light illumination. The photoactivation of the P450 catalytic cycle in the absence of cofactors and redox partners is successfully conducted using many bacterial P450s (variants of P450 BM3) and human P450s (CYPs 1A1, 1A2, 1B1, 2A6, 2E1, and 3A4) for the bioconversion of different substrates, including marketed drugs (simvastatin, lovastatin, and omeprazole) and a steroid (17β‐estradiol), to demonstrate the general applicability of the light‐driven, cofactor‐free system.     

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

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

Identification of mutant Asp251Gly/Gln307His of cytochrome P450 BM3 for the generation of metabolites of diclofenac, ibuprofen and tolbutamide

The soluble, catalytically self-sufficient cytochrome P450 BM3 from Bacillus megaterium is a good candidate as biocatalyst for the synthesis of drug metabolites. To this end, error-prone polymerase chain reaction (PCR) was used to generate a library of P450 BM3 mutants with novel activities toward drugs. The double mutant Asp251Gly/Gln307His (A2) with activities towards diclofenac, ibuprofen and tolbutamide was identified by screening with the alkali method. This is based on the detection of NADPH oxidation during enzymatic turnover on whole Escherichia coli cells heterologously expressing the P450 BM3 mutants in the presence of the target substrates. The three drugs screened are marker substrates of human liver cytochromes P450 belonging to the 2C subfamily. Interestingly the mutations Asp251Gly/Gln307His are located on the protein surface and they are not directly involved in substrate binding and turnover. Dissociation constants and K(M) values of mutant A2 for diclofenac, ibuprofen and tolbutamide are in the micromolar range. Catalysis leads to hydroxylations in specific positions, producing 4'-hydroxydiclofenac, 2-hydroxyibuprofen and 4-hydroxytolbutamide, respectively.     

Structure, electronic properties and catalytic behaviour of an activity-enhancing CYP102A1 (P450(BM3)) variant

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-? 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.     

Reduction of dioxygen catalyzed by pyrene-wired heme domain cytochrome P450 BM3 electrodes

We have electronically wired the cytochrome P450 BM3 heme domain to a graphite electrode with the use of a pyrene-terminated tether. AFM images clearly reveal that pyrene-wired enzyme molecules are adsorbed to the electrode surface. The enzyme-electrode system undergoes rapid and reversible electron transfer, displaying a standard rate constant higher than that of any other P450-electrode system. We also show that the graphite-pyrene-BM3 system catalyzes the four-electron reduction of dioxygen to water.     

Conformational dynamics of the cytochrome P450 BM3/N-palmitoylglycine complex: the proposed "proximal-distal" transition probed by temperature-jump spectroscopy

The ferric spin state equilibrium of the heme iron was analyzed in wild-type cytochrome P450 BM3 and its F87G mutant by using temperature (T)-jump relaxation spectroscopy in combination with static equilibrium experiments. No relaxation process was measurable in the substrate-free enzyme indicating a relaxation process with a rate constant>10,000 s(-1). In contrast, a slow spin state transition process was observed in the N-palmitoylglycine (NPG)-bound enzyme species. This transition occurred with an observed rate constant (298 K) of approximately 800 s(-1) in the wild-type, and approximately 2500 s(-1) in the F87G mutant, suggesting a significant contribution of the phenylalanine side chain to a reaction step rate limiting the actual spin state transition. These findings are discussed in terms of an equilibrium between different binding modes of the substrate, including a position 7.5 A away from the heme iron ("distal") and the catalytically relevant "proximal" binding site, and are in accordance with results from X-ray crystallography, NMR studies, and molecular dynamics simulations.     

Fluorescence detection of ligand binding to labeled cytochrome P450 BM3

The cytochrome P450 superfamily of monoxygenases are highly relevant for pharmaceutical, environmental and biocatalytical applications. The binding of a substrate to their catalytic site is usually detectable by UV-vis spectroscopy as a low-to-high spin state transition of the heme iron. However, the discovery of potential new substrates is limited by the fact that some compounds do not cause the typical spin-shift even if they are oxidised by P450 enzymes. Here we report a fluorescence-based method able to detect the binding of such substrates to the heme domain of cytochrome P450 BM3 from Bacillus megaterium. The protein was labeled with the fluorescent probe N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-Yl)-ethylenediamine (IANBD). Arachidonic and lauric acids are substrates of P450 BM3 and were used to validate the method, as their binding can be detected both by a spin-shift of the Soret peak from 419 to 397 nm and by the fluorescence change of the labelled protein. The fluorescence emission of the probe linked to the protein increased by a value corresponding to 121 ± 9% and 52 ± 5% with respect to the initial one, upon titration with arachidonic or lauric acids respectively. The dissociation constants were calculated by both UV-vis and fluorescence spectroscopy. Three drugs, propranolol, chlorzoxazone and nifedipine, known to be oxidized by P450 BM3 and that bind without causing spin-shift, were also tested and the fluorescence emission of IANBD was found to decrease by 29 ± 5%, 21 ± 2% and 23 ± 3%, respectively, allowing the measurement of their dissociation constants.     

Whole‐Cell Biotransformation of Benzene to Phenol Catalysed by Intracellular Cytochrome P450BM3 Activated by External Additives

An Escherichia coli whole‐cell biocatalyst for the direct hydroxylation of benzene to phenol has been developed. By adding amino acid derivatives as decoy molecules to the culture medium, wild‐type cytochrome P450BM3 (P450BM3) expressed in E.coli can be activated and non‐native substrates hydroxylated, without supplementing with NADPH. The yield of phenol reached 59 % when N‐heptyl‐l ‐prolyl‐l ‐phenylalanine (C7‐Pro‐Phe) was employed as the decoy molecule. It was shown that decoy molecules, especially those lacking fluorination, reached the cytosol of E. coli, thus imparting in vivo catalytic activity for the oxyfunctionalisation of non‐native substrates to intracellular P450BM3.     

Electrochemical Activation of the Natural Catalytic Cycle of Cytochrome P450s in Human Liver Microsomes

The natural catalytic cycle of cytochrome (cyt) P450 enzymes in human liver microsome (HLM) films was activated electrochemically via the electron transfer sequence electrode→cyt P450 reductase (CPR)→cyt P450. Cyclic voltammograms for HLM films had midpoint potentials of ?0.50 V vs. SCE at pH 7.4 characteristic of CPR, not cyt P450s. HLM and CPR microsomes without cyt P450s did not electrocatalytically reduce H₂O₂, and did not shift midpoint potential when CO was added, also indicating that the peaks do not correspond to iron heme cyt P450 enzymes. Electrochemical activation of the natural cyt P450 cycle for substrate conversion via CPR in HLM films was confirmed by catalytic electrolysis in an electrochemical microfluidic array designed to generate and detect reactive metabolites by measuring their reactivity with DNA.     

Spectroscopic Observation of Calcium‐Induced Reorientation of Cellobiose Dehydrogenase Immobilized on Electrodes and its Effect on Electrocatalytic Activity

Cellobiose dehydrogenase catalyzes the oxidation of various carbohydrates and is considered as a possible anode catalyst in biofuel cells. It has been shown that the catalytic performance of this enzyme immobilized on electrodes can be increased by presence of calcium ions. To get insight into the Ca²⁺‐induced changes in the immobilized enzyme we employ surface‐enhanced vibrational (SERR and SEIRA) spectroscopy together with electrochemistry. Upon addition of Ca²⁺ ions electrochemical measurements show a shift of the catalytic turnover signal to more negative potentials while SERR measurements reveal an offset between the potential of heme reduction and catalytic current. Comparing SERR and SEIRA data we propose that binding of Ca²⁺ to the heme induces protein reorientation in a way that the electron transfer pathway of the catalytic FAD center to the electrode can bypass the heme cofactor, resulting in catalytic activity at more negative potentials.     

Improving the Activity of Cytochrome P450 BM-3 Catalyzing Indole Hydroxylation by Directed Evolution

Cytochrome P450 BM-3 (A74G/F87V/L188Q) could catalyze indole to produce indigo. To further improve this capability, random mutagenesis was performed on the heme domain of P450 BM-3 (A74G/F87V/L188Q) with error-prone PCR. A single mutant V445A was selected out from the error-prone library and exhibited the highest specific activity toward indole among the mutants obtained. The kinetic parameters of V445A were also highly improved. Compared with the parent enzyme, the turnover rate (k _cat) of V445A was increased by 7.5 times, while its K _m value decreased by 9.2 %. Consequently, the catalytic efficiency (k _cat/K _m) of V445A was raised to 8.2 times than that of the parent enzyme. Moreover, alanine was confirmed as the best amino acid substitution by saturated mutagenesis in Val445 position. Three-dimensional structure analysis was also used to rationalize the effect on the enzyme properties of the mutation. This study showed that random mutagenesis was efficient to identify mutants with potential values in industry and increased our insight into P450 BM-3.     

P450 versus P420: correlation between cyclic voltammetry and visible absorption spectroscopy of the immobilized heme domain of cytochrome P450 BM3

Cyclic voltabsorptometry is used for the first time to distinguish and characterize electrochemically the active (P450) and inactive (P420) forms of cytochromes P450 immobilized on an electrode during voltammetry experiments. This was achieved by using the heme domain (BMP) of the bacterial cytochrome P450 BM3 from Bacillus megaterium (CYP102A1) immobilized on mesopouros tin-oxide (SnO2) electrodes. We demonstrate that the formation of either the P450 form or the P420 one can be obtained by modifying the mesoporous electrode surface with polycations with different properties such as polyethylenimmine (PEI) and polydiallyldimethylammonium chloride (PDDA). Potential step spectroelectrochemistry allowed measurement of reduction potentials of the active P450 form. Values of -0.39+/-0.01 V and -0.58+/-0.01 V (both versus Ag/AgCl) were calculated for the active P450 form immobilized on the BMP/PDDA-SnO2 and BMP/PEI-SnO2 electrodes, respectively. The cyclic voltabsorptometric experiments showed how, when both the active and inactive forms are present on the PEI film, the inactive P420 species tends to dominate the cyclic voltammetric signal.     

Enhanced Activity and Substrate Specificity by Site-Directed Mutagenesis for the P450 119 Peroxygenase Catalyzed Sulfoxidation of Thioanisole

P450 119 peroxygenase was found to catalyze the sulfoxidation of thioanisole and the sulfonation of sulfoxide in the presence of tert-butyl hydroperoxide (TBHP) for the first time with turnover rates of 1549 min⁻¹ and 196 min⁻¹ respectively. Several mutants were designed to improve the peroxygenation activity and thioanisole specificity by site-directed mutagenesis. The F153G/T213G mutant gave an increase of sulfoxide yield and a decrease of sulfone yield. Moreover the S148P/I161T/K199E/T214V mutant and the K199E mutant with acidic Glu residue contributed to improving the product ratio of sulfoxide to sulfone. Addition of short-alkyl-chain organic acids to the P450 119 peroxygenase-catalyzed sulfur oxidation of thioanisole was investigated. Octanoic acid was found to induce a preferred sulfoxidation of thioanisole catalyzed by the F153G/T213G mutant to give approximately 2.4-fold increase in turnover rate with a k_cat value of 3687 min⁻¹ relative to that of the wild-type, and by the F153G mutant to give the R-sulfoxide up to 30 % ee. The experimental control and the proposed mechanism for the P450 119 peroxygenase-catalyzed sulfoxidation of thioanisole in the presence of octanoic acid suggested that octanoic acid could partially occupy the substrate pocket; meanwhile the F153G mutation could enhance the substrate specificity, which could lead to efficiently regulate the spatial orientation of thioanisole and facilitate the formation of Compound I. This is the most effective catalytic system for the P450 119 peroxygenase-catalyzed sulfoxidation of thioanisole.     

Enhanced electron transfer and lauric acid hydroxylation by site-directed mutagenesis of CYP119

CYP119, a cytochrome P450 from a thermophilic organism for which a crystal structure is available, is shown here to hydroxylate lauric acid in a reaction supported by putidaredoxin and putidaredoxin reductase. This fatty acid hydroxylation activity is increased 15-fold by T214V and D77R mutations. The T214V mutation increases the rate by facilitating substrate binding and enhancing the associated spin state change, whereas the D77R mutation improves binding of the heterologous redox partner putidaredoxin to CYP119 and the rate of electron transfer from it to the heme group. A sequence alignment with P450(cam) can, therefore, be used to identify a part of the binding site for putidaredoxin on an unrelated P450 enzyme. This information can be used to engineer by mutagenesis an improved complementarity of the protein-protein interface that results in improved electron transfer from putidaredoxin to the P450 enzyme. As a result, the catalytic activity of the thermo- and barostable CYP119 has been incorporated into a catalytic system that hydroxylates fatty acids.     

Selective C–H bond functionalization with light-driven P450 biocatalysts

The unique photochemical properties of Ru(II)-diimine photosensitizers have enabled light-induced electron transfers in hybrid P450 heme domain enzymes. Rapid quenching of the excited state by soluble molecules generates either a highly oxidative or reductive species depending on the nature of the quencher. Under flash quench oxidative conditions, the heme cofactor of the P450 BM3 enzyme is oxidized to a high-valent ferryl species. Meanwhile, a photogenerated reductive species is able to deliver the necessary electrons to P450 heme active sites and sustain photocatalytic activity in the selective hydroxylation of substrate C–H bonds.