Oxidizing intermediates in cytochrome P450 model reactions

Oxoiron(IV) porphyrin -cation radicals have been considered as the sole reactive species in the catalytic oxidation of organic substrates by cytochromes P450 and their iron porphyrin models over the past two decades. Recent studies from several laboratories, however, have provided experimental evidence that multiple oxidizing species are involved in the oxygen transfer reactions and that the mechanism of oxygen transfer is much more complex than initially believed. In this Commentary, reactive intermediates that have been shown or proposed to be involved in iron porphyrin complex-catalyzed oxidation reactions are reviewed. Particularly, the current controversy on the oxoiron(IV) porphyrin -cation radical as a sole reactive species versus the involvement of multiple oxidizing species in oxygen transfer reactions is discussed.Abbreviations F₅PhIO pentafluoroiodosylbenzene - m-CPBA m-chloroperbenzoic acid - OEP dianion of octaethylporphyrin - PhIO iodosylbenzene - PPAA peroxyphenylacetic acid - TDCPP dianion of meso-tetrakis(2,6-dichlorophenyl)porphyrin - TMP dianion of meso-tetramesitylporphyrin - TPFPP dianion of meso-tetrakis(pentafluorophenyl)porphyrin - TPP dianion of meso-tetraphenylporphyrin - TTPPP dianion of meso-tetrakis(2,4,6-triphenylphenyl)porphyrin     

Prediction of binding modes for ligands in the cytochromes P450 and other heme-containing proteins

The cytochromes P450 (P450s) are a family of heme-containing monooxygenase enzymes involved in a variety of functions, including the metabolism of endogenous and exogenous substances in the human body. During lead optimization, and in drug development, many potential drug candidates are rejected because of the affinity they display for drug-metabolising P450s. Recently, crystal structures of human enzymes involved in drug metabolism have been determined, significantly augmenting the prospect of using structure-based design to modulate the binding and metabolizing properties of compounds against P450 proteins. An important step in the application of structure-based metabolic optimization is the accurate prediction of docking modes in heme binding proteins. In this paper we assess the performance of the docking program GOLD at predicting the binding mode of 45 heme-containing complexes. We achieved success rates of 64% and 57% for Chemscore and Goldscore respectively; these success rates are significantly lower than the value of 79% observed with both scoring functions for the full GOLD validation set. Re-parameterization of metal-acceptor interactions and lipophilicity of planar nitrogen atoms in the scoring functions resulted in a significant increase in the percentage of successful dockings against the heme binding proteins (Chemscore 73%, Goldscore 65%). The modified scoring functions will be useful in docking applications on P450 enzymes and other heme binding proteins.     

Spectroscopic investigations of intermediates in the reaction of cytochrome P450(BM3)-F87G with surrogate oxygen atom donors

Rapid mixing of substrate-free ferric cytochrome P450_BM3–F87G with m-chloroperoxybenzoic acid (mCPBA) resulted in the sequential formation of two high-valent intermediates. The first was spectrally similar to compound I species reported previously for P450_CAM and CYP 119 using mCPBA as an oxidant, and it featured a low intensity Soret absorption band characterized by shoulder at 370 nm. This is the first direct observation of a P450 compound I intermediate in a type II P450 enzyme. The second intermediate, which was much more stable at pH values below 7.0, was characterized by an intense Soret absorption peak at 406 nm, similar to that seen with P450_CAM [T. Spolitak, J.H. Dawson, D.P. Ballou, J. Biol. Chem. 280 (2005) 20300–20309]. Double mixing experiments in which NADPH was added to the transient 406 nm-absorbing intermediate resulted in rapid regeneration of the resting ferric state, with the flavins of the flavoprotein domain in their reduced state. EPR results were consistent with this stable intermediate species being a cytochrome c peroxidase compound ES-like species containing a protein-based radical, likely localized on a nearby Trp or Tyr residue in the active site. Iodosobenzene, peracetic acid, and sodium m-periodate also generated the intermediate at 406 nm, but not the 370 nm intermediate, indicating a probable kinetic barrier to accumulating compound I in reactions with these oxidants. The P450 ES intermediate has not been previously reported using iodosobenzene or m-periodate as the oxygen donor.     

Highly reactive electrophilic oxidants in cytochrome P450 catalysis

The cytochrome P450 enzymes effect a wide range of oxidations in nature including difficult hydroxylation reactions of unactivated C-H. Most of the high energy reactions of these catalysts appear to involve highly electrophilic active species. Attempts to detect the reactive transients in the enzymes have met with limited success, but evidence has accumulated that two distinct electrophilic oxidants are produced in the P450 enzymes. The consensus electrophilic oxidant termed "iron-oxo" is usually thought to be an analogue of Compound I, an iron(IV)-oxo porphyrin radical cation species, but it is possible that a higher energy electronic isomer of Compound I is required to account for the facility of the C-H oxidation reactions. The second electrophilic oxidant of P450 is speculative; circumstantial evidence suggests that this species is iron-complexed hydrogen peroxide, but this oxidant might be a second spin state of iron-oxo. This overview discusses recent studies directed at detection of the electrophilic oxidants in P450 enzymes and the accumulated evidence for two distinct species.     

Mechanistic insight into the initiation step of the reaction of Burkholderia pseudomallei catalase-peroxidase with peroxyacetic acid

The reaction of the catalase-peroxidase of Burkholderia pseudomallei with peroxyacetic acid has been analyzed using stopped-flow spectrophotometry. Two well-defined species were observed, the first defined by an increase in intensity and narrowing of the Soret band at 407 nm and a 10-nm shift of the charge transfer band from 635 to 625 nm. These features are consistent with a ferric spectrum with a greater proportion of sixth-coordination character and are assigned to an Fe^III–peroxyacetic acid complex. Complementary 9-GHz EPR characterization of the changes in the ferric signal of the resting enzyme induced by the binding of acetate in the heme pocket substantiates the proposal. Kinetic analysis of the spectral changes as a function of peroxyacetic acid concentration revealed two independent peroxyacetic acid binding events, one coincident with formation of the Fe^III–peroxyacetic acid complex and the other coincident with the heme oxidation to the subsequent ferryl intermediate. A model to explain the need for two peroxyacetic acid binding events is proposed. The reaction of the W330F variant followed similar kinetics, although the characteristic spectral features of the Fe^IV=O Por^•+ species were detected. The variant D141A lacking an aspartate at the entrance to the heme cavity as well as the R108A and D141A/R108A variants showed no evidence for the Fe^III–peroxyacetic acid complex, only the formation of ferryl species with absorbance maxima at 414, 545, and 585 nm. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Reactive Intermediates in Cytochrome P450 Catalysis

Recently, we reported the spectroscopic and kinetic characterizations of cytochrome P450 compound I in CYP119A1, effectively closing the catalytic cycle of cytochrome P450-mediated hydroxylations. In this minireview, we focus on the developments that made this breakthrough possible. We examine the importance of enzyme purification in the quest for reactive intermediates and report the preparation of compound I in a second P450 (P450_ST). In an effort to bring clarity to the field, we also examine the validity of controversial reports claiming the production of P450 compound I through the use of peroxynitrite and laser flash photolysis.     

The enantiomer of progesterone (ent-progesterone) is a competitive inhibitor of human cytochromes P450c17 and P450c21

Human cytochrome P450c17 (17alpha-hydroxylase, 17,20-lyase) (CYP17) and cytochrome P450c21 (21-hydroxylase) (CYP21) differ by only 14 amino acids in length and share 29% amino acid identity. Both enzymes hydroxylate progesterone at carbon atoms that lie only 2.6A apart, but CYP17 also metabolizes other steroids and demonstrates additional catalytic activities. To probe the active site topologies of these related enzymes, we synthesized the enantiomer of progesterone and determined if ent-progesterone is a substrate or inhibitor of CYP17 and CYP21. Neither enzyme metabolizes ent-progesterone; however, ent-progesterone is a potent competitive inhibitor of CYP17 (K(I)=0.2 microM). The ent-progesterone forms a type I difference spectrum with CYP17, but molecular dynamics simulations suggest different binding orientations for progesterone and its enantiomer. The ent-progesterone also inhibits CYP21, with weaker affinity than for CYP17. We conclude that CYP17 accommodates the stereochemically unnatural ent-progesterone better than CYP21. Enantiomeric steroids can be used to probe steroid binding sites, and these compounds may be effective inhibitors of steroid biosynthesis.     

Hydroperoxoferric heme intermediate as a second electrophilic oxidant in cytochrome P450-catalyzed reactions

Experimental evidence supporting the catalytic activity of the peroxoferric and hydroperoxoferric cytochrome P450 intermediates as alternative oxidants to the compound I (ferryl) state in the oxygenation of organic substrates is reviewed. The peroxoferric P450 state is proposed to function as a nucleophile in the lyase step of the P450-aromatase reaction. Several systems are reviewed in which the hydroperoxoferric P450 intermediate likely functions as a second electrophilic oxidant, the two-oxidants model. These include alkene epoxidation, sulfoxidation, and hydroxylation of methyl groups on cyclopropane rings. The key use of the P450 mutants from different sources in which the conserved threonine in the distal substrate binding pocket is replaced with alanine, in order to minimize the formation of the compound I intermediate and unmask the reactivity of the hydroperoxoferric state, is emphasized. These data are discussed in the context of the two-states model, which proposes that the compound I P450 intermediate has both high- and low-spin states with different reactivities. A complicated reaction profile emerges for the wide range of P450 reactions involving up to three reactive intermediates, of which the most reactive, the compound I P450 state, has two spin states with different reactivities.     

Spectroscopic investigations of intermediates in the reaction of cytochrome P450BM3–F87G with surrogate oxygen atom donors

Rapid mixing of substrate-free ferric cytochrome P450_BM3–F87G with m-chloroperoxybenzoic acid (mCPBA) resulted in the sequential formation of two high-valent intermediates. The first was spectrally similar to compound I species reported previously for P450_CAM and CYP 119 using mCPBA as an oxidant, and it featured a low intensity Soret absorption band characterized by shoulder at 370 nm. This is the first direct observation of a P450 compound I intermediate in a type II P450 enzyme. The second intermediate, which was much more stable at pH values below 7.0, was characterized by an intense Soret absorption peak at 406 nm, similar to that seen with P450_CAM [T. Spolitak, J.H. Dawson, D.P. Ballou, J. Biol. Chem. 280 (2005) 20300–20309]. Double mixing experiments in which NADPH was added to the transient 406 nm-absorbing intermediate resulted in rapid regeneration of the resting ferric state, with the flavins of the flavoprotein domain in their reduced state. EPR results were consistent with this stable intermediate species being a cytochrome c peroxidase compound ES-like species containing a protein-based radical, likely localized on a nearby Trp or Tyr residue in the active site. Iodosobenzene, peracetic acid, and sodium m-periodate also generated the intermediate at 406 nm, but not the 370 nm intermediate, indicating a probable kinetic barrier to accumulating compound I in reactions with these oxidants. The P450 ES intermediate has not been previously reported using iodosobenzene or m-periodate as the oxygen donor.     

On the identity and reactivity patterns of the “second oxidant” of the T252A mutant of cytochrome P450cam in the oxidation of 5-methylenenylcamphor

Density functional calculations show that in the absence of Compound I, the primary oxidant species of P450, the precursor species, Compound 0 (FeOOH), can effect double bond activation of 5-methylenylcamphor (1). The mechanism is initiated by homolytic cleavage of the O–O bond and formation of an OH radical bound to the Compound II species by hydrogen bonding interactions. Subsequently, the so-formed OH radical can either activate the double bond of 1 or attack the meso position of the heme en route to heme degradation. The calculations show that double bond activation is preferred over attack on the heme. Past the double bond activation, the intermediate can either lead to epoxidation or to a glycol formation. The glycol formation is predicted to be preferred, although in the P450_cam pocket the competition may be closer. Therefore, in the absence of Compound I, Compound 0 will be capable of epoxidizing double bonds. Previous studies [E. Derat, D. Kumar, H. Hirao, S. Shaik, J. Am. Chem. Soc. 128 (2006) 473–484] showed that in the case of a substrate that can undergo only C–H activation, the bound OH prefers heme degradation over hydrogen abstraction. Since the epoxidation barrier for Compound I is much smaller than that of Compound 0 (12.8 vs. 18.9 kcal/mol), when Compound I is present in the cycle, Compound 0 will be silent. As such, our mechanism explains lucidly why T252A P450_cam can epoxidize olefins like 5-methylenylcamphor but is ineffective in camphor hydroxylation [S. Jin, T.M. Makris, T. A. Bryson, S.G. Sligar, J.H. Dawson, J. Am. Chem. Soc. 125 (2003) 3406–3407]. Our calculations show that the glycol formation is a marker reaction of Compound 0 with 5-methylenylcamphor. If this product can be found in T252A P450_cam or in similar mutants of other P450 isozymes, this will constitute a more definitive proof for the action of Cpd 0 in P450 enzymes.     

Substrate modulates compound I formation in peroxide shunt pathway of Pseudomonas putida cytochrome P450(cam)

The active oxygenating intermediate, a ferryl-oxo-(II) porphyrin cation radical (compound I), in substrate-bound cytochrome P450(cam) (P450(cam)) has eluded detection and kinetic analysis for several decades. Upon rapid mixing of peroxides-H(2)O(2) and m-CPBA with substrate-bound forms of P450(cam), we observed an intermediate with spectral features characteristic of compound I. Unlike in H(2)O(2), kinetic investigation on the reaction of m-CPBA with various substrate (camphor, adamantone, and norcamphor)-bound P450(cam) and its Y96A mutant shows a preferential binding of the aromatic end group of m-CPBA to the active-site of the enzyme and modulation of compound I formation by the local environment of heme active-site. The results presented in this paper describe the importance of heme environment in modulating formation of compound I, and form the first kinetic analysis of this intermediate in the peroxide shunt pathway of substrate-bound P450(cam).     

Cytochrome P450-type hydroxylation and epoxidation in a tyrosine-liganded hemoprotein, catalase-related allene oxide synthase

The ability of hemoproteins to catalyze epoxidation or hydroxylation reactions is usually associated with a cysteine as the proximal ligand to the heme, as in cytochrome P450 or nitric oxide synthase. Catalase-related allene oxide synthase (cAOS) from the coral Plexaura homomalla, like catalase itself, has tyrosine as the proximal heme ligand. Its natural reaction is to convert 8R-hydroperoxy-eicosatetraenoic acid (8R-HPETE) to an allene epoxide, a reaction activated by the ferric heme, forming product via the Fe(IV)-OH intermediate, Compound II. Here we oxidized cAOS to Compound I (Fe(V)=O) using the oxygen donor iodosylbenzene and investigated the catalytic competence of the enzyme. 8R-hydroxyeicosatetraenoic acid (8R-HETE), the hydroxy analog of the natural substrate, normally unreactive with cAOS, was thereby epoxidized stereospecifically on the 9,10 double bond to form 8R-hydroxy-9R,10R-trans-epoxy-eicosa-5Z,11Z,14Z-trienoic acid as the predominant product; the turnover was 1/s using 100 μm iodosylbenzene. The enantiomer, 8S-HETE, was epoxidized stereospecifically, although with less regiospecificity, and was hydroxylated on the 13- and 16-carbons. Arachidonic acid was converted to two major products, 8R-HETE and 8R,9S-eicosatrienoic acid (8R,9S-EET), plus other chiral monoepoxides and bis-allylic 10S-HETE. Linoleic acid was epoxidized, whereas stearic acid was not metabolized. We conclude that when cAOS is charged with an oxygen donor, it can act as a stereospecific monooxygenase. Our results indicate that in the tyrosine-liganded cAOS, a catalase-related hemoprotein in which a polyunsaturated fatty acid can enter the active site, the enzyme has the potential to mimic the activities of typical P450 epoxygenases and some capabilities of P450 hydroxylases.     

Crossover of a high-spin (S=7/2, 3/2) to a low-spin (S=1/2) iron-selenium cluster in FA and FB of photosystem I on rebinding of PsaC onto P700-FX cores

 PsaC is a tightly bound ferredoxin in the Photosystem I (PS I) reaction center which contains two [4Fe-4S] clusters named F_A and F_B. We recently proposed that the mixed-ligand F_B cluster in C14D_PsaC and the mixed-ligand F_A cluster in C51D_PsaC exist in a spin state of S=3/2, and that a spin state crossover to S=1/2 occurs when the PsaC mutants are rebound onto P700-F_X cores. Since EPR signals from a highly rhombic S=3/2 spin state can be difficult to study, wild-type PsaC was reconstituted with iron and selenium to introduce an easily detected S=7/2 spin state similar to that shown for Clostridial ferredoxin. When the unbound [4Fe-4Se] PsaC was chemically reduced, a sharp derivative resonance was found at g=5.171 attributed to the excited ±3/2 doublet from an S=7/2 spin multiplet. An additional peak was found at g=5.616 attributed to the superimposed ±1/2 and ±3/2 doublets from a highly rhombic S=3/2 spin multiplet, and an axial set of resonances found around g=2.0 attributed, in part, to a classical S=1/2 spin state. When the [4Fe-4Se] PsaC was rebound onto P700-F_X cores, the spin population derived from the S=7/2 and 3/2 spin states was negligible. Illumination of the rebuilt PS I complex at 15 K resulted in two rhombic sets of resonances, one with g values of 2.043, 1.941 and 1.854, diagnostic of F_A, and the other with g values of 2.067, 1.941 and 1.878, diagnostic of F_B. Chemical reduction with sodium dithionite at pH 10.5 or photoaccumulation by freezing during illumination resulted in a set of resonances with g values of 2.046, 1.938, 1.920 and 1.883, characteristic of a spin-coupled F_A ^–/F_B ^– pair. The spin state crossover in this iron chalcogenide cluster is the first known to be induced by protein-protein association and reinforces the hypothesis that an S=3/2 to 1/2 crossover occurs in the PS I-rebound mutants C14D_PsaC and C51D_PsaC. Received: 6 August 1996 / Accepted: 28 December 1996     

Molecular basis for the inability of an oxygen atom donor ligand to replace the natural sulfur donor heme axial ligand in cytochrome P450 catalysis. Spectroscopic characterization of the Cys436Ser CYP2B4 mutant

All cytochrome P450s (CYPs) contain a cysteinate heme iron proximal ligand that plays a crucial role in their mechanism of action. Conversion of the proximal Cys436 to Ser in NH₂-truncated microsomal CYP2B4 (ΔCYP2B4) transforms the enzyme into a two-electron NADPH oxidase producing H₂O₂ without monooxygenase activity [K.P. Vatsis, H.M. Peng, M.J. Coon, J. Inorg. Biochem. 91 (2002) 542–553]. To examine the effects of this ligation change on the heme iron spin-state and coordination structure of ΔC436S CYP2B4, the magnetic circular dichroism and electronic absorption spectra of several oxidation/ligation states of the variant have been measured and compared with those of structurally defined heme complexes. The spectra of the substrate-free ferric mutant are indicative of a high-spin five-coordinate structure ligated by anionic serinate. The spectroscopic properties of the dithionite-reduced (deoxyferrous) protein are those of a five-coordinate (high-spin) state, and it is concluded that the proximal ligand has been protonated to yield neutral serine (ROH-donor). Low-spin six-coordinate ferrous complexes of the mutant with neutral sixth ligands (NO, CO, and O₂) examined are also likely ligated by neutral serine, as would be expected for ferric complexes with anionic sixth ligands such as the hydroperoxo-ferric catalytic intermediate. Ligation of the heme iron by neutral serine vs. deprotonated cysteine is likely the result of the large difference in their acidity. Thus, without the necessary proximal ligand push of the cysteinate, although the ΔC436S mutant can accept two electrons and two protons, it is unable to heterolytically cleave the O–O bond of the hydroperoxo-ferric species to generate Compound I and hydroxylate the substrate.     

Reduction of Photosystem I Reaction Center in DrgA Mutant of the Cyanobacterium Synechocystis sp. PCC 6803 Lacking Soluble NAD(P)H:Quinone Oxidoreductase

Photoautotrophically grown cells of the cyanobacterium Synechocystis sp. PCC 6803 wild type and the Ins2 mutant carrying an insertion in the drgA gene encoding soluble NAD(P)H:quinone oxidoreductase (NQR) did not differ in the rate of light-induced oxygen evolution and Photosystem I reaction center (P700+) reduction after its oxidation with a white light pulse. In the presence of DCMU, the rate of P700+ reduction was lower in mutant cells than in wild type cells. Depletion of respiratory substrates after 24 h dark-starvation caused more potent decrease in the rate of P700+ reduction in DrgA mutant cells than in wild type cells. The reduction of P700+ by electrons derived from exogenous glucose was slower in photoautotrophically grown DrgA mutant than in wild type cells. The mutation in the drgA gene did not impair the ability of Synechocystis sp. PCC 6803 cells to oxidize glucose under heterotrophic conditions and did not impair the NDH-1-dependent, rotenone-inhibited electron transfer from NADPH to P700+ in thylakoid membranes of the cyanobacterium. Under photoautotrophic growth conditions, NADPH-dehydrogenase activity in DrgA mutant cells was less than 30% from the level observed in wild type cells. The results suggest that NQR, encoded by the drgA gene, might participate in the regulation of cytoplasmic NADPH oxidation, supplying NADP+ for glucose oxidation in the pentose phosphate cycle of cyanobacteria.     

Modulation of photosynthetic electron transport in the absence of terminal electron acceptors: Characterization of the rbcL deletion mutant of tobacco

Tobacco rbcL deletion mutant, which lacks the key enzyme Rubisco for photosynthetic carbon assimilation, was characterized with respect to thylakoid functional properties and protein composition. The ΔrbcL plants showed an enhanced capacity for dissipation of light energy by non-photochemical quenching which was accompanied by low photochemical quenching and low overall photosynthetic electron transport rate. Flash-induced fluorescence relaxation and thermoluminescence measurements revealed a slow electron transfer and decreased redox gap between Q_A and Q_B, whereas the donor side function of the Photosystem II (PSII) complex was not affected. The 77 K fluorescence emission spectrum of ΔrbcL plant thylakoids implied a presence of free light harvesting complexes. Mutant plants also had a low amount of photooxidisible P700 and an increased ratio of PSII to Photosystem I (PSI). On the other hand, an elevated level of plastid terminal oxidase and the lack of F₀ ‘dark rise’ in fluorescence measurements suggest an enhanced plastid terminal oxidase-mediated electron flow to O₂ in ΔrbcL thylakoids. Modified electron transfer routes together with flexible dissipation of excitation energy through PSII probably have a crucial role in protection of PSI from irreversible protein damage in the ΔrbcL mutant under growth conditions. This protective capacity was rapidly exceeded in ΔrbcL mutant when the light level was elevated resulting in severe degradation of PSI complexes.     

Crystal structure of the 16 heme cytochrome from Desulfovibrio gigas: a glycosylated protein in a sulphate-reducing bacterium

Sulphate-reducing bacteria have a wide variety of periplasmic cytochromes involved in electron transfer from the periplasm to the cytoplasm. HmcA is a high molecular mass cytochrome of 550 amino acid residues that harbours 16 c-type heme groups. We report the crystal structure of HmcA isolated from the periplasm of Desulfovibrio gigas. Crystals were grown using polyethylene glycol 8K and zinc acetate, and diffracted beyond 2.1 A resolution. A multiple-wavelength anomalous dispersion experiment at the iron absorption edge enabled us to obtain good-quality phases for structure solution and model building. DgHmcA has a V-shape architecture, already observed in HmcA isolated from Desulfovibrio vulgaris Hildenborough. The presence of an oligosaccharide molecule covalently bound to an Asn residue was observed in the electron density maps of DgHmcA and confirmed by mass spectrometry. Three modified monosaccharides appear at the highly hydrophobic vertex, possibly acting as an anchor of the protein to the cytoplasmic membrane.