Spectroscopic characterization of the iron-oxo intermediate in cytochrome P450

From analogy to chloroperoxidase from Caldariomyces fumago, it is believed that the electronic structure of the intermediate iron-oxo species in the catalytic cycle of cytochrome P450 corresponds to an iron(IV) porphyrin-pi-cation radical (compound I). However, our recent studies on P450cam revealed that after 8 ms a tyrosine radical and iron(IV) were formed in the reaction of ferric P450 with external oxidants in the shunt pathway. The present study on the heme domain of P450BM3 (P450BMP) shows a similar result. In addition to a tyrosine radical, a contribution from a tryptophan radical was found in the electron paramagnetic resonance (EPR) spectra of P450BMP. Here we present comparative multi-frequency EPR (9.6, 94 and 285 GHz) and M?ssbauer spectroscopic studies on freeze-quenched intermediates produced using peroxy acetic acid as oxidant for both P450 cytochromes. After 8 ms in both systems, amino acid radicals occurred instead of the proposed iron(IV) porphyrin-pi-cation radical, which may be transiently formed on a much faster time scale. These findings are discussed with respect to other heme thiolate proteins. Our studies demonstrate that intramolecular electron transfer from aromatic amino acids is a common feature in these enzymes. The electron transfer quenches the presumably transiently formed porphyrin-pi-cation radical, which makes it extremely difficult to trap compound I.     

Spectroscopic features of cytochrome P450 reaction intermediates

Cytochromes P450 constitute a broad class of heme monooxygenase enzymes with more than 11,500 isozymes which have been identified in organisms from all biological kingdoms [1]. These enzymes are responsible for catalyzing dozens chemical oxidative transformations such as hydroxylation, epoxidation, N-demethylation, etc., with very broad range of substrates [2] and [3]. Historically these enzymes received their name from ‘pigment 450’ due to the unusual position of the Soret band in UV–vis absorption spectra of the reduced CO-saturated state [4] and [5]. Despite detailed biochemical characterization of many isozymes, as well as later discoveries of other ‘P450-like heme enzymes’ such as nitric oxide synthase and chloroperoxidase, the phenomenological term ‘cytochrome P450’ is still commonly used as indicating an essential spectroscopic feature of the functionally active protein which is now known to be due to the presence of a thiolate ligand to the heme iron [6]. Heme proteins with an imidazole ligand such as myoglobin and hemoglobin as well as an inactive form of P450 are characterized by Soret maxima at 420 nm [7]. This historical perspective highlights the importance of spectroscopic methods for biochemical studies in general, and especially for heme enzymes, where the presence of the heme iron and porphyrin macrocycle provides rich variety of specific spectroscopic markers available for monitoring chemical transformations and transitions between active intermediates of catalytic cycle.     

Electron paramagnetic resonance investigations of exogenous ligand complexes of low-spin ferric chloroperoxidase: further support for endogenous thiolate ligation to the heme iron.

Previous spectroscopic studies of chloroperoxidase have provided evidence for endogenous thiolate sulfur donor ligation to the central heme iron of the enzyme. This conclusion is further supported by recent DNA sequence data which revealed the existence of a third cysteine residue (in addition to a disulfide pair detected earlier) in the protein available for coordination to the heme iron. Thus, chloroperoxidase shares many spectroscopic properties with cytochrome P-450, the only other known thiolate-ligated heme protein. Surprisingly, a previous electron paramagnetic resonance (EPR) study of low-spin ferric chloroperoxidase-ligand complexes (Hollenberg, P.F., Hager, L.P., Blumberg, W.E. and Peisach, J. (1980) J. Biol. Chem. 255, 4801-4807) was unable to provide clear support for the presence of a thiolate ligand, although sulfur coordination was implicated. This was, in part, because an insufficient number of complexes was examined. In this work, we have significantly expanded upon the previous EPR study by using an extensive variety of over twenty exogenous ligands including carbon, nitrogen, oxygen, phosphorus and sulfur donors. Crystal field analysis, using the procedure of Blumberg and Peisach, of the present data in comparison with data for analogous complexes of cytochrome P-450-CAM, thiolate-ligated heme model systems, and myoglobin, is clearly indicative of endogenous thiolate ligation for chloroperoxidase. In addition, the UV-visible absorption and EPR spectral data suggest that a carboxylate ligand is a possible candidate for the endogenous sixth ligand to the heme iron that is responsible for the reversible conversion of ferric chloroperoxidase from high-spin to low-spin at low temperatures (less than 200 K).     

Axial ligation of the high-potential heme center in an Arabidopsis cytochrome b561

Arabidopsis has four putative, di-heme cytochrome b561 proteins, including one localized in the tonoplast (TCytb). From a comparative electron paramagnetic resonance (EPR), UV–Vis absorption and resonance Raman study, on wild type, H83A/H156A-TCytb and H83L/H156L-TCytb double mutants, it follows that the H83 and H156 residues are binding one of the two hemes. These measurements show that the high-potential heme site is situated at the cytoplasmic side of the membrane and allow the unambiguous differentiation between two models on the heme localization in cytochrome b561 proteins.     

The ferric-hydroperoxo complex of chloroperoxidase

The hydroperoxo-ferric complex, or Compound 0 (Cpd 0), is an unstable transient intermediate common for oxygen activating heme enzymes such as the cytochromes P450, nitric oxide synthases, and heme oxygenases, as well as the peroxidases and catalases which utilize hydrogen peroxide as a source of oxygen and reducing equivalents. Detailed understanding of the mechanism of oxygen activation and formation of the higher valent catalytically active intermediates in heme enzyme catalysis requires the structural and spectroscopic characterization of this immediate precursor, Cpd 0. Using the method of cryoradiolytic reduction of the oxy-ferrous heme complex, we have prepared and characterized hydroperoxo-ferric complex in chloroperoxidase (CPO) and compared this to the same intermediate generated in cytochrome P450 CYP101. Optical absorption spectrum of Cpd 0 in CPO has a Soret band at 449 nm and poorly resolved α, β bands at 576 and 546 nm.     

Isolation and spectroscopic characterization of a recombinant bell pepper hydroperoxide lyase

Fatty acid hydroperoxide (HPO) lyase is a component of the oxylipin pathway and holds a central role in elicited plant defense. HPO lyase from bell pepper has been identified as a heme protein which shares 40% homology with allene oxide synthase, a cytochrome P450 (CYP74A). HPO lyase of immature bell pepper fruits was expressed in Escherichia coli and the enzyme was purified and characterized by spectroscopic techniques. The electronic structure and ligand coordination properties of the heme were investigated by using a series of exogenous ligands. The various complexes were characterized by using UV-visible absorption and electron paramagnetic resonance spectroscopy. The spectroscopic data demonstrated that the isolated recombinant HPO lyase has a pentacoordinate, high-spin heme with thiolate ligation. Addition of the neutral ligand imidazole or the anionic ligand cyanide results in the formation of hexacoordinate adducts that retain thiolate ligation. The striking similarities between both the ferric and ferrous HPO lyase-NO complexes with the analogous P450 complexes, suggest that the active sites of HPO lyase and P450 share common structural features.     

Electronic and stereochemical characterizations of intermediates in the photolysis of ferric cytochrome P450scc nitrosyl complexes. Effects of cholesterol and its analogues on ligand binding structures.

Low temperature photolysis of nitric oxide from the nitrosyl complexes of ferric cytochrome P450scc was examined by EPR spectroscopy to elucidate the stereochemical interaction between heme-bound ligand and side-chain of cholesterol or its hydroxylated analogues at the substrate-binding site. The photoproducts of the NO complexes trapped at 5 K exhibited new EPR absorptions providing information on the steric crowding of the distal heme moiety. Without substrate, the photoproduct exhibited a broad EPR absorption at g-8 due to magnetic dipole-dipole interaction between the photo-dissociated NO (S = 1/2) and the ferric iron (S = 5/2). This indicates that the photo-dissociated NO can move far away from the heme iron in the less restricted distal heme moiety of the substrate-free cytochrome P450scc. In the presence of substrates, such as cholesterol, 20(S)-hydroxycholesterol, 22(S)-hydroxycholesterol, 22(R)-hydroxycholesterol, and 25-hydroxycholesterol, the EPR spectra of the photoproducts exhibited many variations having broad g-8 absorptions and/or the widespread signals together with zero-field absorption. Among the steroid complexes used, 20(S)-hydroxycholesterol complex exhibited a conspicuously widespread EPR signal with a distinct zero-field absorption due to a spin-coupled interaction between the ferric iron (S = 5/2) and the photolyzed NO (S = 1/2). These results indicate that the 20(S)-hydroxycholesterol complex has restricted substrate-binding structure and that the hydroxylation of the cholesterol side-chain at the 22R position is necessary to proceed the side-chain cleavage reaction properly in cytochrome P450scc.     

Structures of the high-valent metal-ion haem-oxygen intermediates in peroxidases, oxygenases and catalases

Peroxidases, oxygenases and catalases have similar high-valent metal-ion intermediates in their respective reaction cycles. In this review, haem-based examples will be discussed. The intermediates of the haem-containing enzymes have been extensively studied for many years by different spectroscopic methods like UV-Vis, EPR (electron paramagnetic resonance), resonance Raman, M?ssbauer and MCD (magnetic circular dichroism). The first crystal structure of one of these high-valent intermediates was on cytochrome c peroxidase in 1987. Since then, structures have appeared for catalases in 1996, 2002, 2003, putatively for cytochrome P450 in 2000, for myoglobin in 2002, for horseradish peroxidase in 2002 and for cytochrome c peroxidase again in 1994 and 2003. This review will focus on the most recent structural investigations for the different intermediates of these proteins. The structures of these intermediates will also be viewed in light of quantum mechanical (QM) calculations on haem models. In particular quantum refinement, which is a combination of QM calculations and crystallography, will be discussed. Only small structural changes accompany the generation of these intermediates. The crystal structures show that the compound I state, with a so called pi-cation radical on the haem group, has a relatively short iron-oxygen bond (1.67-1.76A) in agreement with a double-bond character, while the compound II state or the compound I state with a radical on an amino acid residue have a relatively long iron-oxygen bond (1.86-1.92A) in agreement with a single-bond character where the oxygen-atom is protonated.     

Adrenodoxin-cytochrome P450scc interaction as revealed by EPR spectroscopy: comparison with the putidaredoxin-cytochrome P450cam system.

The cholesterol side-chain cleavage reaction catalyzed by cytochrome P450scc comprises three consecutive monooxygenase reactions (22R-hydroxylation, 20S-hydroxylation, and C(20)-C(22) bond scission) that produces pregnenolone. The electron equivalents necessary for the oxygen activation are supplied from a 2Fe-2S type ferredoxin, adrenodoxin. We found that 1:1 stoichiometric binding of oxidized adrenodoxin to oxidized cytochrome P450scc complexed with cholesterol or 25-hydroxycholesterol caused shifts of the high-spin EPR signals of the heme moiety at 5 K. Such shifts were not observed for the low-spin EPR signals. Ligation of CO or NO to the reduced heme of cytochrome P450scc complexed with reduced adrenodoxin and various steroid substrates did not cause any change in the axial EPR spectrum of the reduced iron-sulfur center at 77 K. These results are in remarkable contrast to those obtained for the cytochrome P450cam-d-camphor-putidaredoxin ternary complex, suggesting that the mode of cross talk between adrenodoxin and cytochrome P450scc is very different from that in the Pseudomonas system. The difference may be primarily due to the location of the charged amino acid residues of the ferredoxins important for the interaction with the partner cytochrome P450.     

Chloroperoxidase: P-450 type absorption in the absence of sulfhydryl groups.

The oxidation state of the two half-cystine residues in the native ferric form of chloroperoxidase and in the reduced ferrous chloroperoxidase has been examined in order to evaluate the role of sulfhydryl groups as determinants of P-450 type spectra. M?ssbauer and optical spectroscopy studies indicate that the ferrous forms of P-450cam and chloroperoxidase have very similar or identical heme environments. Model studies have suggested that sulfhydryl groups may function as axial ligands for developing P-450 character. However, chemical studies involving both sulfhydryl reagents and amperometric titrations show that neither the ferric nor the chemically produced ferrous forms of chloroperoxidase contain a sulfhydryl group. These results rule out the hypothesis that sulfhydryl groups are unique components for P-450 absorption characteristics. The optical and electron paramagnetic resonance (EPR) spectra of the nitric oxide complex of chloroperoxidase have been obtained and compared to those of myoglobin, hemoglobin, and cytochrome c and horseradish peroxidase. The EPR spectrum of the NO-ferrous chloroperoxidase complex, which is similar to that of cytochrome P-450cam, does not show the extra nitrogen hyperfine structure which appears to be characteristic of those hemoproteins which have a nitrogen atom as an axial heme ligand.     

Chromaffin granule membranes contain at least three heme centers: direct evidence from EPR and absorption spectroscopy

Low-temperature electron paramagnetic resonance (EPR) spectroscopy, circular dichroism and two-component redox titration have previously provided evidence for two different ascorbate-reducible heme centers in cytochrome b(561) present in chromaffin granule membranes. These species have now been observed by room and liquid nitrogen temperature absorption spectroscopy. The visualization of these heme centers becomes possible as a consequence of utilizing chromaffin granule membranes prepared by a mild procedure. Additionally, a new redox center, not reducible by ascorbate, was discovered by both EPR and absorption spectroscopy. It constitutes about 15% of the heme absorbance of chromaffin membranes at 561 nm and has EPR characteristics of a well-organized highly axial low-spin heme center (thus making it unlikely that it is a denatured species). This species is either an alternative form of one of the hemes of cytochrome b(561) that has a very low redox potential or a b-type cytochrome distinct from b(561).     

Renal cytochrome P-450's-electrophoretic and electron paramagnetic resonance studies.

The cytochrome P-450's of the microsomal mixed function oxidase systems from the rabbit renal cortex, outer medulla, inner medulla, and the liver were compared. Sodium dodecyl sulfate-(SDS) gel electrophoresis and electron paramagnetic resonance (EPR) studies detected cytochrome P-450 proteins in the liver, renal cortex, and outer medulla but not the inner medulla of normal animals. Two cytochrome P-450 peptides, which had molecular weights of 54,500 and 58,900 and which comigrated with known hepatic cytochrome P-450's on SDS gels, were identified in the cortex and outer medulla. Treatment of animals with 3-methylcholanthrene (MC) enhanced the 54,500 and 58,900 peptides in the liver and cortex but produced little change in outer medulla. MC treatment induced faint cytochrome P-450 bands in the inner medulla. The EPR studies detected low spin heme iron absorption lines at g = 2.42, 2.26, and 1.92 in liver, cortex, and outer medulla from untreated animals. The amplitude of the low spin absorption lines was increased by ethanol, a reverse type I compound, and reduced by chloroform, a type I compound, in these tissues. MC treatment increased the amplitude of the heme absorption lines in these tissues, and it induced a barely detectable heme spectrum in the inner medulla. Differences in exogenous substrate binding between hepatic and renal microsomes from MC-treated animals were detected by EPR and optical difference spectroscopy. Acetone, 1-butanol, and 2-propanol gave evidence of binding to the hepatic cytochrome P-450's but no evidence of binding to renal cortical microsomes. These results, along with previous enzymatic studies, suggest that the liver and each area of the kidney contain different substrate specificities and pathways for the metabolism of organic compounds.     

Spectroscopic characterization of secondary amine mono-oxygenase. Comparison to cytochrome P-450 and myoglobin

Secondary amine mono-oxygenase from Pseudomonas aminovorans catalyzes the NAD(P)H- and dioxygen-dependent N-dealkylation of secondary amines to yield a primary amine and an aldehyde. Heme iron, flavin, and non-heme iron prosthetic groups are known to be present in the oligomeric enzyme. The N-dealkylation reaction is also catalyzed by the only other heme-containing mono-oxygenase, cytochrome P-450. In order to identify the heme iron axial ligands of secondary amine mono-oxygenase so as to better define the structural requirements for oxygen activation by heme enzymes, we have investigated the spectroscopic properties of the enzyme. The application of three different spectroscopic techniques, UV-visible absorption, magnetic circular dichroism and electron paramagnetic resonance, to study eight separate enzyme derivatives has provided extensive and convincing evidence for the presence of a proximal histidine ligand. This conclusion is based primarily on comparisons of the spectral properties of the enzyme with those of parallel derivatives of myoglobin (histidine proximal ligand) and P-450 (cysteinate proximal ligand). Spectral studies of ferric secondary amine mono-oxygenase as a function of pH have led to the proposal that the distal ligand is water. Deprotonation of the distal water ligand occurs upon either raising the pH to 9.0 or substrate (dimethylamine) binding. In contrast, the deoxyferrous enzyme appears to have a weakly bound nitrogen donor distal ligand. Initial spectroscopic studies of the iron-sulfur units in the enzyme are interpreted in terms of a pair of Fe2S2 clusters. Secondary amine mono-oxygenase is unique in its ability to function as cytochrome P-450 in activating molecular oxygen but to do so with a myoglobin-like active site. As such, it provides an important system with which to probe structure-function relations in heme-containing oxygenases.     

Modifications in heme iron of free and vesicle bound cytochrome c by tert-butyl hydroperoxide: a magnetic circular dichroism and electron paramagnetic resonance investigation

To characterize changes to the heme and the influence of membrane lipids in the reaction of cytochrome c with peroxides, we studied the reaction of cytochrome c with tert-butyl hydroperoxide (tert-BuOOH) by magnetic circular dichroism (MCD) and direct electron paramagnetic resonance (EPR) in the presence and absence of different liposomes. Direct low-temperature (11 degrees K) EPR analysis of the cytochrome c heme iron on exposure to tert-BuOOH shows a gradual (180 s) conversion of the low-spin form to a high-spin Fe(III) species of rhombic symmetry (g = 4.3), with disappearance of a prior peroxyl radical signal (g(o) = 2.014). The conversion to high spin precedes Soret band bleaching, observable by UV/Vis spectroscopy and by magnetic circular dichroism (MCD) at room temperature, that indicates loss of iron coordination by the porphyrin ring. The presence of cardiolipin-containing liposomes delayed formation of the peroxyl radical and conversion to high-spin iron, while dicetylphosphate (DCP) liposomes accelerated these changes. Correspondingly, bleaching of cytochrome c by tert-BuOOH at room temperature was accelerated by several negatively charged liposome preparations, and inhibited by mitochondrial-mimetic phosphatidylcholinephosphatidylethanolaminecardiolipin (PCPECL) liposomes. Concomitant with bleaching, spin-trapping measurements with 5,5-dimethyl-1-pyroline-N-oxide showed that while the relative production of peroxyl, alkoxyl, and alkyl radicals was unaffected by DCP liposomes, PCPECL liposomes decreased the spin-trapped alkoxyl radical signal by 50%. The EPR results show that the primary initial change on exposure of cytochrome c to tert-BuOOH is a change to a high-spin Fe(III) species, and together with MCD measurements show that unsaturated cardiolipin-containing lipid membranes influence the interaction of tert-BuOOH with cytochrome c heme iron, to alter radical production and decrease damage to the cytochrome.     

DiGeorge critical region 8 (DGCR8) is a double-cysteine-ligated heme protein

All known heme-thiolate proteins ligate the heme iron using one cysteine side chain. We previously found that DiGeorge Critical Region 8 (DGCR8), an essential microRNA processing factor, associates with heme of unknown redox state when overexpressed in Escherichia coli. On the basis of the similarity of the 450-nm Soret absorption peak of the DGCR8-heme complex to that of cytochrome P450 containing ferrous heme with CO bound, we identified cysteine 352 as a probable axial ligand in DGCR8. Here we further characterize the DGCR8-heme interaction using biochemical and spectroscopic methods. The DGCR8-heme complex is highly stable, with a half-life exceeding 4 days. Mutation of the conserved proline 351 to an alanine increases the rate of heme dissociation and allows the DGCR8-heme complex to be reconstituted biochemically. Surprisingly, DGCR8 binds ferric heme without CO to generate a hyperporphyrin spectrum. The electronic absorption, magnetic circular dichroism, and electron paramagnetic resonance spectra of the DGCR8-heme complex suggest a ferric heme bearing two cysteine ligands. This model was further confirmed using selenomethionine-substituted DGCR8 and mercury titration. DGCR8 is the first example of a heme-binding protein with two endogenous cysteine side chains serving as axial ligands. We further show that native DGCR8 binds heme when expressed in eukaryotic cells. This study provides a chemical basis for understanding the function of the DGCR8-heme interaction in microRNA maturation.     

Refolding processes of cytochrome P450cam from ferric and ferrous acid forms to the native conformation. Formations of folding intermediates with non-native heme coordination state

Changes in heme coordination state and protein conformation of cytochrome P450(cam) (P450(cam)), a b-type heme protein, were investigated by employing pH jump experiments coupled with time-resolved optical absorption, fluorescence, circular dichroism, and resonance Raman techniques. We found a partially unfolded form (acid form) of ferric P450(cam) at pH 2.5, in which a Cys(-)-heme coordination bond in the native conformation was ruptured. When the pH was raised to pH 7.5, the acid form refolded to the native conformation through a distinctive intermediate. Formations of similar acid and intermediate forms were also observed for ferrous P450(cam). Both the ferric and ferrous forms of the intermediate were found to have an unidentified axial ligand of the heme at the 6th coordination sphere, which is vacant in the high spin ferric and ferrous forms at the native conformation. For the ferrous form, it was also indicated that the 5th axial ligand is different from the native cysteinate. The folding intermediates identified in this study demonstrate occurrences of non-native coordination state of heme during the refolding processes of the large b-type heme protein, being akin to the well known folding intermediates of cytochromes c, in which c-type heme is covalently attached to a smaller protein.     

Interaction of nitric oxide with cytochrome P450 BM3

The interaction of nitric oxide with cytochrome P450 BM3 from Bacillus megaterium has been analyzed by spectroscopic techniques and enzyme assays. Nitric oxide ligates tightly to the ferric heme iron, inducing large changes in each of the main visible bands of the heme and inhibiting the fatty acid hydroxylase function of the protein. However, the ferrous adduct is unstable under aerobic conditions, and activity recovers rapidly after addition of NADPH to the flavocytochrome due to reduction of the heme via the reductase domain and displacement of the ligand. The visible spectral properties revert to that of the oxidized resting form. Aerobic reduction of the nitrosyl complex of the BM3 holoenzyme or heme domain by sodium dithionite also displaces the ligand. A single electron reduction destabilizes the ferric-nitrosyl complex such that nitric oxide is released directly, as shown by the trapping of released nitric oxide. Aerobically and in the absence of exogenous reductant, nitric oxide dissociates completely from the P450 over periods of several minutes. However, recovery of the nativelike visible spectrum is accompanied by alterations in the catalytic activity of the enzyme and changes in the resonance Raman spectrum. Specifically, resonance Raman spectroscopy identifies the presence of internally located nitrated tyrosine residue(s) following treatment with nitric oxide. Analysis of a Y51F mutant indicates that this is the major nitration target under these conditions. While wild-type P450 BM3 does not form an aerobically stable ferrous-nitrosyl complex, a site-directed mutant of P450 BM3 (F393H) does form an isolatable ferrous-nitrosyl complex, providing strong evidence for the role of this residue in controlling the electronic properties of the heme iron. We report here the spectroscopic characterization of the ferric- and ferrous-nitrosyl complexes of P450 BM3 and describe the use of resonance Raman spectroscopy to identify nitrated tyrosine residue(s) in the enzyme. Nitration of tyrosine in P450 BM3 may exemplify a typical mechanism by which the ubiquitous messenger molecule nitric oxide exerts a regulatory function over the cytochromes P450.     

Flavocytochrome P450 BM3 mutant A264E undergoes substrate-dependent formation of a novel heme iron ligand set

A conserved glutamate covalently attaches the heme to the protein backbone of eukaryotic CYP4 P450 enzymes. In the related Bacillus megaterium P450 BM3, the corresponding residue is Ala264. The A264E mutant was generated and characterized by kinetic and spectroscopic methods. A264E has an altered absorption spectrum compared with the wild-type enzyme (Soret maximum at approximately 420.5 nm). Fatty acid substrates produced an inhibitor-like spectral change, with the Soret band shifting to 426 nm. Optical titrations with long-chain fatty acids indicated higher affinity for A264E over the wild-type enzyme. The heme iron midpoint reduction potential in substrate-free A264E is more positive than that in wild-type P450 BM3 and was not changed upon substrate binding. EPR, resonance Raman, and magnetic CD spectroscopies indicated that A264E remains in the low-spin state upon substrate binding, unlike wild-type P450 BM3. EPR spectroscopy showed two major species in substrate-free A264E. The first has normal Cys-aqua iron ligation. The second resembles formate-ligated P450cam. Saturation with fatty acid increased the population of the latter species, suggesting that substrate forces on the glutamate to promote a Cys-Glu ligand set, present in lower amounts in the substrate-free enzyme. A novel charge-transfer transition in the near-infrared magnetic CD spectrum provides a spectroscopic signature characteristic of the new A264E heme iron ligation state. A264E retains oxygenase activity, despite glutamate coordination of the iron, indicating that structural rearrangements occur following heme iron reduction to allow dioxygen binding. Glutamate coordination of the heme iron is confirmed by structural studies of the A264E mutant (Joyce, M. G., Girvan, H. M., Munro, A. W., and Leys, D. (2004) J. Biol. Chem. 279, 23287-23293).     

Freeze-quenched iron-oxo intermediates in cytochromes P450

Since the discovery of cytochromes P450 and their assignment to heme proteins a reactive iron-oxo intermediate as the hydroxylating species has been discussed. It is believed that the electronic structure of this intermediate corresponds to an iron(IV)-porphyrin-pi-cation radical system (Compound I). To trap this intermediate the reaction of P450 with oxidants (shunt pathway) has been used. The common approaches are stopped-flow experiments with UV-visible spectroscopic detection or rapid-mixing/freeze-quench studies with EPR and M?ssbauer spectroscopic characterization of the trapped intermediate. Surprisingly, the two approaches seem to give conflicting results. While the stopped-flow data indicate the formation of a porphyrin-pi-cation radical, no such species is seen by EPR spectroscopy, although the M?ssbauer data indicate iron(IV) for P450cam (CYP101) and P450BMP (CYP102). Instead, radicals on tyrosine and tryptophan residues are observed. These findings are reviewed and discussed with respect to intramolecular electron transfer from aromatic amino acids to a presumably transiently formed porphyrin-pi-cation radical.