Oxygen activation and electron transfer in flavocytochrome P450 BM3

In flavocytochrome P450 BM3, there is a conserved phenylalanine residue at position 393 (Phe393), close to Cys400, the thiolate ligand to the heme. Substitution of Phe393 by Ala, His, Tyr, and Trp has allowed us to modulate the reduction potential of the heme, while retaining the structural integrity of the enzyme's active site. Substrate binding triggers electron transfer in P450 BM3 by inducing a shift from a low- to high-spin ferric heme and a 140 mV increase in the heme reduction potential. Kinetic analysis of the mutants indicated that the spin-state shift alone accelerates the rate of heme reduction (the rate determining step for overall catalysis) by 200-fold and that the concomitant shift in reduction potential is only responsible for a modest 2-fold rate enhancement. The second step in the P450 catalytic cycle involves binding of dioxygen to the ferrous heme. The stabilities of the oxy-ferrous complexes in the mutant enzymes were also analyzed using stopped-flow kinetics. These were found to be surprisingly stable, decaying to superoxide and ferric heme at rates of 0.01-0.5 s(-)(1). The stability of the oxy-ferrous complexes was greater for mutants with higher reduction potentials, which had lower catalytic turnover rates but faster heme reduction rates. The catalytic rate-determining step of these enzymes can no longer be the initial heme reduction event but is likely to be either reduction of the stabilized oxy-ferrous complex, i.e., the second flavin to heme electron transfer or a subsequent protonation event. Modulating the reduction potential of P450 BM3 appears to tune the two steps in opposite directions; the potential of the wild-type enzyme appears to be optimized to maximize the overall rate of turnover. The dependence of the visible absorption spectrum of the oxy-ferrous complex on the heme reduction potential is also discussed.     

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.     

Spectroscopy and electrochemistry of cytochrome P450 BM3-surfactant film assemblies

We report analyses of electrochemical and spectroscopic measurements on cytochrome P450 BM3 (BM3) in didodecyldimethylammonium bromide (DDAB) surfactant films. Electronic absorption spectra of BM3-DDAB films on silica slides reveal the characteristic low-spin FeIII heme absorption maximum at 418 nm. A prominent peak in the absorption spectrum of BM3 FeII-CO in a DDAB dispersion is at 448 nm; in spectra of aged samples, a shoulder at approximately 420 nm is present. Infrared absorption spectra of the BM3 FeII-CO complex in DDAB dispersions feature a time-dependent shift of the carbonyl stretching frequency from 1950 to 2080 cm(-1). Voltammetry of BM3-DDAB films on graphite electrodes gave the following results: FeIII/II E(1/2) at -260 mV (vs SCE), approximately 300 mV positive of the value measured in solution; DeltaS degrees (rc), DeltaS degrees , and DeltaH degrees values for water-ligated BM3 in DDAB are -98 J mol(-1) K(-1), -163 J mol(-1) K(-1), and -47 kJ mol(-1), respectively; values for the imidazole-ligated enzyme are -8 J mol(-1) K(-1), -73 J mol(-1) K(-1), and -21 kJ mol(-1). Taken together, the data suggest that BM3 adopts a compact conformation within DDAB that in turn strengthens hydrogen bonding interactions with the heme axial cysteine, producing a P420-like species with decreased electron density around the metal center.     

Hydrogen-bonding interactions in the active sites of cytochrome P450cam and its site-directed mutants.

Resonance Raman spectroscopy is applied to the cyanide adducts of cytochrome P450cam and its T252A and D251N site-directed mutants, both in their substrate-free and camphor-bound forms, to probe active-site heme structure and, in particular, interactions of the FeCN fragment with potential active-site H-bond donors. In contrast to the ferrous CO and ferric NO adducts, which form only essentially linear (slightly distorted) FeXY fragments, the spectra of the ferric CN(-) adducts provide clear evidence the for the existence of an additional, rather highly bent, conformer; that is, the cyanide complexes form both linear and bent conformers in both the substrate-free and substrate-bound forms. Formation of this bent conformer is most reasonably attributed to the presence of off-axis H-bond donors, which induce distortion on the FeCN fragment but not the FeCO and FeNO fragments, which are poorer H-bond acceptors. For all three proteins, the substrate-free form exhibits a complex spectral pattern which arises because one of the modes associated with the FeCN fragment is coupled with two heme macrocycle deformation modes. Significantly, no evidence for such coupling is observed in the spectra of the camphor-bound forms. While various unknown factors may possibly give rise to selective activation of such coupling in the substrate-free derivative, given the known facts about the active-site architecture of this enzyme, a plausible explanation is that the bent conformer is oriented toward the water-filled substrate-binding site in the substrate-free form, but oppositely, toward the proposed proton delivery shuttle, in the substrate-bound form. Sensitivity of the FeCN modes to H(2)O/D(2)O exchange in the two camphor-bound mutants, which is apparently absent for the camphor-bound native protein, is most reasonably attributed to the known presence of extra water in the active sites of these mutants.     

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.     

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.     

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.     

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.     

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.     

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.     

Substrate binding favors enhanced NO binding to P450cam

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

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.     

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.     

Reaction of cytochrome P450BM3 and peroxynitrite yields nitrosyl complex

Peroxynitrite has come into the spotlight in recent years. Its effects on proteins have been implicated in several diseases such as acute lung injury, rheumatoid arthritis, implant rejection, artherosclerosis, Parkinson's disease, and Alzheimer's disease. Peroxynitrite is thought to inactivate a variety of proteins including thiolate-ligated heme proteins such as cytochrome P450 2B1 and PGI2 synthase, through the nitration of tyrosine residues. In previous studies it was reported that thiolate-ligated heme enzymes react with peroxynitrite to form a ferryl intermediate. In an effort to spectroscopically characterize this species in P450BM3, we discovered that the peroxynitrite-generated intermediate is not an FeIVoxo, but rather an iron-nitrosyl [FeNO]6 complex. We present density functional calculations as well as M?ssbauer and stopped-flow spectroscopic characterizations of the peroxynitrite-generated intermediate in P450BM3.     

Design of a five-coordinate heme protein maquette: a spectroscopic model of deoxymyoglobin

The substitution of 1-methyl-l-histidine for the histidine heme ligands in a de novo designed four-alpha-helix bundle scaffold results in conversion of a six-coordinate cytochrome maquette into a self-assembled five-coordinate mono-(1-methyl-histidine)-ligated heme as an initial maquette for the dioxygen carrier protein myoglobin. UV-vis, magnetic circular dichroism, and resonance Raman spectroscopies demonstrate the presence of five-coordinate mono-(1-methyl-histidine) ligated ferrous heme spectroscopically similar to deoxymyoglobin. Thermodynamic analysis of the ferric and ferrous heme dissociation constants indicates greater destabilization of the ferric state than the ferrous state. The ferrous heme protein reacts with carbon monoxide to form a (1-methyl-histidine)-Fe(II)(heme)-CO complex; however, reaction with dioxygen leads to autoxidation and ferric heme dissociation. These results indicate that negative protein design can be used to generate a five-coordinate heme within a maquette scaffold.     

Novel Conformational Transitions of Human Cytochrome P450 2C8 during Thermal and Acid-induced Unfolding

Transitions among various heme coordination/spin states, heme environments and protein conformations of human cytochrome P450 2C8 were investigated under different denaturing conditions by means of electronic absorption and circular dichroism spectroscopies. It is the first report of it's kind. Our results indicated that the thermal and acid‐induced denaturation could convert P450 2C8 to various P420 forms. In the thermal unfolding process, the ferric P420 thermal form emerged with weakened Fe‐S (thiolate) bond. An absorption band at ca. 425 nm of the ferrous P420 2C8 thermal form was observed, suggesting that the axial Cys435 was protonated or displaced by other ligand. Moreover, the new coordination bond was stabilized when the temperature was cooled down. When binding with CO, the ferrous P420 2C8 thermal form had the protonated thiol of Cys435 as the axial ligand. X‐ray structure of P450 2C8 suggested that the specific structure of the β‐bulge where the axial cysteine ligand located might be the reason of the formation of these P420 2C8 thermal forms. In the acid‐induced unfolding studies, we found that at pH 3.0 the heme could be irreversibly released from the heme pocket of ferric and ferrous P450 2C8. Interestingly, the released heme could form a new coordination bond with an unidentified ligand at the surface of partially unfolded protein when binding with CO at reduced state.     

Crystals in Minutes: Instant On‐Site Microcrystallisation of Various Flavours of the CYP102A1 (P450BM3) Haem Domain

Despite CYP102A1 (P450BM3) representing one of the most extensively researched metalloenzymes, crystallisation of its haem domain upon modification can be a challenge. Crystal structures are indispensable for the efficient structure‐based design of P450BM3 as a biocatalyst. The abietane diterpenoid derivative N‐abietoyl‐l ‐tryptophan (AbiATrp) is an outstanding crystallisation accelerator for the wild‐type P450BM3 haem domain, with visible crystals forming within 2 hours and diffracting to a near‐atomic resolution of 1.22 Å. Using these crystals as seeds in a cross‐microseeding approach, an assortment of P450BM3 haem domain crystal structures, containing previously uncrystallisable decoy molecules and diverse artificial metalloporphyrins binding various ligand molecules, as well as heavily tagged haem‐domain variants, could be determined. Some of the structures reported herein could be used as models of different stages of the P450BM3 catalytic cycle.     

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.     

The H93G myoglobin cavity mutant as a versatile template for modeling heme proteins: ferrous, ferric, and ferryl mixed-ligand complexes with imidazole in the cavity

One of the difficulties in preparing accurate ambient-temperature model complexes for heme proteins, particularly in the ferric state, has been the generation of mixed-ligand adducts: complexes with different ligands on either side of the heme. The difference in the accessibility of the two sides of the heme in the H93G cavity mutant of myoglobin (Mb) provides a potential general solution to this problem. To demonstrate the versatility of H93G Mb for the preparation of heme protein models, numerous mixed-ligand adducts of ferrous, ferric, and ferryl imidazole-ligated H93G (H93G(Im) Mb) have been prepared. The complexes have been characterized by electronic absorption and magnetic circular dichroism (MCD) spectroscopy in comparison to analogous derivatives of wild type Mb. The starting ferric H93G(Im) Mb state spectroscopically resembles wild-type ferric Mb as expected for a complex containing a single imidazole in the proximal cavity and water bound on the distal side. Addition of a sixth ligand to ferric H93G(Im) Mb, whether charge neutral (imidazole) or anionic (cyanide and azide), results in formation of six-coordinate low-spin complexes with MCD characteristics similar to those of parallel derivatives of wild-type ferric Mb. Reduction of ferric H93G(Im) Mb and subsequent exposure to either CO, NO, or O2 produces ferrous complexes (deoxy, CO, NO, and O2) that consistently exhibit MCD spectra similar to the analogous ferrous species of wild-type ferrous Mb. Most interestingly, reaction of ferric H93G(Im) Mb with H2O2 results in the formation of a stable high-valent oxoferryl complex with MCD characteristics that are essentially identical to those of oxoferryl wild-type Mb. The generation of such a wide array of mixed-ligand heme complexes demonstrates the efficacy of the H93G Mb cavity mutant as a template for the preparation of heme protein model complexes.