Lignin peroxidase compound III. Mechanism of formation and decomposition

Lignin peroxidase compound III (LiPIII) was prepared via three procedures: (a) ferrous LiP + O2 (LiPIIIa), (b) ferric LiP + O2-. (LiPIIIb), and (c) LiP compound II + excess H2O2 followed by treatment with catalase (LiPIIIc). LiPIIIa, b, and c each have a Soret maximum at approximately 414 nm and visible bands at 543 and 578 nm. LiPIIIa, b, and c each slowly reverted to native ferric LiP, releasing stoichiometric amounts of O2-. in the process. Electronic absorption spectra of LiPIII reversion to the native enzyme displayed isosbestic points in the visible region at 470, 525, and 597 nm, suggesting a single-step reversion with no intermediates. The LiPIII reversion reactions obeyed first-order kinetics with rate constants of approximately 1.0 X 10(-3) s-1. In the presence of excess peroxide, at pH 3.0, native LiP, LiPII, and LiPIIIa, b, and c are all converted to a unique oxidized species (LiPIII*) with a spectrum displaying visible bands at 543 and 578 nm, but with a Soret maximum at 419 nm, red-shifted 5 nm from that of LiPIII. LiPIII* is bleached and inactivated in the presence of excess H2O2 via a biphasic process. The fast first phase of this bleaching reaction obeys second-order kinetics, with a rate constant of 1.7 X 10(1) M-1 s-1. Addition of veratryl alcohol to LiPIII* results in its rapid reversion to the native enzyme, via an apparent one-step reaction that obeys second-order kinetics with a rate constant of 3.5 X 10(1) M-1 s-1. Stoichiometric amounts of O2-. are released during this reaction. When this reaction was run under conditions that prevented further reactions, HPLC analysis of the products demonstrated that veratryl alcohol was not oxidized. These results suggest that the binding of veratryl alcohol to LiPIII* displaces O2-., thus returning the enzyme to its native state. In contrast, the addition of veratryl alcohol to LiPIII did not affect the rate of spontaneous reversion of LiPIII to the native enzyme.     

Oxidation of monomethoxylated aromatic compounds by lignin peroxidase: role of veratryl alcohol in lignin biodegradation

Lignin peroxidase (LiP), an extracellular heme enzyme from the lignin-degrading fungus Phanerochaete chrysosporium, catalyzes the H2O2-dependent oxidation of a variety of nonphenolic lignin model compounds. The oxidation of monomethoxylated lignin model compounds, such as anisyl alcohol (AA), and the role of veratryl alcohol (VA) in LiP reactions were studied. AA oxidation reached a maximum at relatively low H2O2 concentrations, beyond which the extent of the reactions decreased. The presence of VA did not affect AA oxidation at low molar ratios of H2O2 to enzyme; however, at ratios above 100, the presence of VA abolished the decrease in AA oxidation. Addition of stoichiometric amounts of AA to LiP compound II (LiPII) resulted in its reduction to the native enzyme at rates that were significantly faster than the spontaneous rate of reduction, indicating that AA and other monomethoxylated aromatics are directly oxidized by LiP, albeit slowly. Under steady-state conditions in the presence of excess H2O2 and VA, a visible spectrum for LiPII was obtained. In contrast, under steady-state conditions in the presence of AA a visible spectrum was obtained for LiPIII*, a noncovalent complex of LiPIII and H2O2. AA competitively inhibited the oxidation of VA by LiP; the Ki for AA inhibition was 32 microM. Addition of VA to LiPIII* resulted in its conversion to the native enzyme. In contrast, AA did not convert LiPIII* to the native enzyme; instead, LiPIII* was bleached in the presence of AA. Thus, AA does not protect LiP from inactivation by H2O2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Spectral characterization of diarylpropane oxygenase, a novel peroxide-dependent, lignin-degrading heme enzyme

Diarylpropane oxygenase, an H2O2-dependent lignin-degrading enzyme from the basidiomycete fungus Phanerochaete chrysosporium, catalyzes the oxygenation of various lignin model compounds with incorporation of a single atom of dioxygen (O2). Diarylpropane oxygenase is also capable of oxidizing some alcohols to aldehydes and/or ketones. This enzyme (Mr = 41,000) contains a single iron protoporphyrin IX prosthetic group. Previous studies revealed that the Soret maximum of the ferrous-CO complex of diarylpropane oxygenase is at approximately 420 nm, as in ferrous-CO myoglobin (Mb), and not like the approximately 450 nm absorption of the CO complex of the ubiquitous heme monooxygenase, cytochrome P-450. This spectral difference between two functionally similar heme enzymes is of interest. To elucidate the structural requirements for heme iron-based oxygenase reactions, we have compared the electronic absorption, EPR, and resonance Raman (RR) spectral properties of diarylpropane oxygenase with those of other heme proteins and enzymes of known axial ligation. The absorption spectra of native (ferric), cyano, and ferrous diarylpropane oxygenase closely resemble those of the analogous myoglobin complexes. The EPR g values of native diarylpropane oxygenase, 5.83 and 1.99, also agree well with those of aquometMb. The RR spectra of ferric diarylpropane oxygenase have their spin- and oxidation-state marker bands at frequencies analogous to those of aquometMb and indicate a high-spin, hexacoordinate ferric iron. The RR spectra of ferrous diarylpropane oxygenase have frequencies analogous to those of deoxy-Mb that suggest a high-spin, pentacoordinate Fe(II) in the reduced form. The RR spectra of both ferric and ferrous diarylpropane oxygenase are less similar to those of horseradish peroxidase, catalase, or cytochrome c peroxidase and are clearly distinct from those of P-450. These observations suggest that the fifth ligand to the heme iron of diarylpropane oxygenase is a neutral histidine and that the iron environment must resemble that of the oxygen transport protein, myoglobin, rather than that of the peroxidases, catalase, or P-450. Given the functional similarity between diarylpropane oxygenase and P-450, this work implies that the mechanism of oxygen insertion for the two systems is different.     

Kinetic studies on the formation and decomposition of compounds II and III. Reactions of lignin peroxidase with H2O2.

The present study characterizes the serial reactions of H2O2 with compounds I and II of lignin peroxidase isozyme H1. These two reactions constitute part of the pathway leading to formation of the oxy complex (compound III) from the ferric enzyme. Compounds II and III are the only complexes observed; no compound III* is observed. Compound III* is proposed to be an adduct of compound III with H2O2, formed from the complexation of compound III with H2O2 (Wariishi, H., and Gold, M. H. (1990) J. Biol. Chem. 265, 2070-2077). We provide evidence that demonstrates that the spectral data, on which the formation of compound III* is based, are merely an artifact caused by enzyme instability and, therefore, rule out the existence of compound III*. The reactions of compounds II and III with H2O2 are pH-dependent, similar to that observed for reactions of compounds I and II with the reducing substrate veratryl alcohol. The spontaneous decay of the compound III of lignin peroxidase results in the reduction of ferric cytochrome c. The reduction is inhibited by superoxide dismutase, indicating that superoxide is released during the decay. Therefore, the lignin peroxidase compound III decays to the ferric enzyme through the dissociation of superoxide. This mechanism is identical with that observed with oxymyoglobin and oxyhemoglobin but different from that for horseradish peroxidase. Compound III is capable of reacting with small molecules, such as tetranitromethane (a superoxide scavenger) and fluoride (a ligand for the ferric enzyme), resulting in ferric enzyme and fluoride complex formation, respectively.     

Magnetic circular dichroism studies on horseradish peroxidase.

Magnetic circular dichroism (MCD) spectra were observed for native (Fe(III)) horseradish peroxidase (peroxidase, EC 1.11.1.7), its alkaline form and fluoro- and cyano-derivatives, and also for reduced (Fe(II)) horseradish peroxidase and its carbonmonoxy-- and cyano- derivatives. MCD spectra were obtained for the cyano derivative of Fe(III) horseradish peroxidase, and reduced horseradish peroxidase and its carbonmonoxy- derivative nearly identical with those for the respective myoglobin derivatives. The alkaline form of horseradish peroxidase exhibits a completely different MCD spectrum from that of myoglobin hydroxide. Thus it shows an MCD spectrum which falls into the ferric low-spin heme grouping. Native horseradish peroxidase and its fluoro derivatives show almost identical MCD spectra with those for the respective myoglobin derivatives in the visible region, though some changes were detected in the Soret region. Therefore it is concluded that the MCD spectra on the whole are sensitive to the spin state of the heme iron rather than to the porphyrin structures. The cyanide derivative of reduced horseradish peroxidase exhibited a characteristic MCD spectrum of the low-spin ferrous derivative like oxy-myoglobin.     

Formation of a bis(histidyl) heme iron complex in manganese peroxidase at high pH and restoration of the native enzyme structure by calcium

Manganese peroxidase (MnP) from Phanerochaete chrysosporium undergoes a pH-dependent conformational change evidenced by changes in the electronic absorption spectrum. This high- to low-spin alkaline transition occurs at approximately 2 pH units lower in an F190I mutant MnP when compared to the wild-type enzyme. Herein, we provide evidence that these spectral changes are attributable to the formation of a bis(histidyl) heme iron complex in both proteins at high pH. The resonance Raman (RR) spectra of both ferric proteins at high pH are similar, indicating similar heme environments in both proteins, and resemble that of ferric cytochrome b(558), a protein that contains a bis-His iron complex. Upon reduction with dithionite at high pH, the visible spectra of both the wild-type and F190I MnP exhibit absorption maxima at 429, 529, and 558 nm, resembling the absorption spectrum of ferrous cytochrome b(558). RR spectra of the reduced wild-type and F190I mutant proteins at high pH are also similar to the RR spectrum of ferrous cytochrome b(558), further suggesting that the alkaline low-spin species is a bis(histidyl) heme derivative. No shift in the low-frequency RR bands was observed in 75% (18)O-labeled water, indicating that the low-spin species is most likely not a hydroxo-heme derivative. Electronic and RR spectra also indicate that addition of Ca(2+) to either the ferric or ferrous enzymes at high pH completely restores the high-spin pentacoordinate species. Other divalent metals, such as Mn(2+), Mg(2+), Zn(2+), or Cd(2+), do not restore the enzyme under the conditions studied.     

Binding of protoporphyrin IX and metal derivatives to the active site of wild-type mouse ferrochelatase at low porphyrin-to-protein ratios

Resonance Raman (RR) spectroscopy is used to examine porphyrin substrate, product, and inhibitor interactions with the active site of murine ferrochelatase (EC 4.99.1.1), the terminal enzyme in the biosynthesis of heme. The enzyme catalyzes in vivo Fe(2+) chelation into protoporphyrin IX to give heme. The RR spectra of native ferrochelatase show that the protein, as isolated, contains varying amounts of endogenously bound high- or low-spin ferric heme, always at much less than 1 equiv. RR data on the binding of free-base protoporphyrin IX and its metalated complexes (Fe(III), Fe(II), and Ni(II)) to active wild-type protein were obtained at varying ratios of porphyrin to protein. The binding of ferric heme, a known inhibitor of the enzyme, leads to the formation of a low-spin six-coordinate adduct. Ferrous heme, the enzyme's natural product, binds in the ferrous high-spin five-coordinate state. Ni(II) protoporphyrin, a metalloporphyrin that has a low tendency toward axial ligation, becomes distorted when bound to ferrochelatase. Similarly for free-base protoporphyrin, the natural substrate of ferrochelatase, the RR spectra of porphyrin-protein complexes reveal a saddling distortion of the porphyrin. These results corroborate and extend our previous findings that porphyrin distortion, a crucial step of the catalytic mechanism, occurs even in the absence of bound metal substrate. Moreover, RR data reveal the presence of an amino acid residue in the active site of ferrochelatase which is capable of specific axial ligation to metals.     

Spectral characterization of manganese peroxidase, an extracellular heme enzyme from the lignin-degrading basidiomycete, Phanerochaete chrysosporium

Manganese peroxidase (MnP) is a component of the lignin degradation system of the basidiomycetous fungus, Phanerochaete chrysosporium. This novel MnII-dependent extracellular enzyme (Mr = 46,000) contains a single protoporphyrin IX prosthetic group and oxidizes phenolic lignin model compounds as well as a variety of other substrates. To elucidate the heme environment of this enzyme, we have studied its electron paramagnetic resonance and resonance Raman spectroscopic properties. These studies indicate that the native enzyme is predominantly in the high-spin ferric form and has a histidine as fifth ligand. The reduced enzyme has a high-spin, pentacoordinate ferrous heme. Fluoride and cyanide readily bind to the sixth coordination position of the heme iron in the native form, thereby changing MnP into a typical high-spin, hexacoordinate fluoro adduct or a low-spin, hexacoordinate cyano adduct, respectively. EPR spectra of 14NO- and 15NO-adducts of ferrous MnP were compared with those of horseradish peroxidase (HRP); the presence of a proximal histidine ligand was confirmed from the pattern of superhyperfine splittings of the NO signals centered at g approximately equal to 2.005. The appearance of the FeII-His stretch at approximately 240 cm-1 and its apparent lack of deuterium sensitivity suggest that the N delta proton of the proximal histidine of the enzyme is more strongly hydrogen bonded than that of oxygen carrier globins and that this imidazole ligand may be described as having a comparatively strong anionic character. Although resonance Raman frequencies for the spin- and coordination-state marker bands of native MnP, nu 3 (1487), nu 19 (1565), and nu 10 (1622 cm-1), do not fall into frequency regions expected for typical penta- or hexacoordinate high-spin ferric heme complexes, ligation of fluoride produces frequency shifts of these bands very similar to those observed for cytochrome c peroxidase and HRP. Hence, these data strongly suggest that the iron in native MnP is predominantly high-spin pentacoordinate. Analysis of the Raman frequencies indicates that the dx2-y2 orbital of the native enzyme is at higher energy than that of metmyoglobin. These features of the heme in MnP must be favorable for the peroxidase catalytic mechanism involving oxidation of the heme iron to FeIV. Consequently, it is most likely that the heme environment of MnP resembles those of HRP, cytochrome c peroxidase, and lignin peroxidase.     

Cationic ascorbate peroxidase isoenzyme II from tea: structural insights into the heme pocket of a unique hybrid peroxidase

The novel class III ascorbate peroxidase isoenzyme II from tea leaves (TcAPXII), with an unusually high specific ascorbate peroxidase activity associated with stress response, has been characterized by resonance Raman (RR), electronic absorption, and Fourier transform infrared (FT-IR) spectroscopies. Ferric and ferrous forms and the complexes with fluoride, cyanide, and CO have been studied at various pH values. The overall blue shift of the electronic absorption spectrum, the high RR frequencies of the core size marker bands, similar to those of 6-coordinate low-spin heme, and the complex RR spectrum in the low-frequency region of ferric TcAPXII indicate that this protein contains an unusual 5-coordinate quantum mechanically mixed-spin heme. The spectra of both the fluoride and the CO adducts suggest that these exogenous ligands are strongly hydrogen-bonded with a residue that appears to be unique to this peroxidase. Electronic absorption spectra also emphasize structural differences between the benzhydroxamic acid binding sites of TcAPXII and horseradish peroxidases (HRPC). It is concluded that TcAPXII is a paradigm peroxidase since it is the first example of a hybrid enzyme that combines spectroscopic signatures, structural elements, and substrate specificities previously reported only for distinct class I and class III peroxidases.     

Protein conformational perturbations affect the photoreduction of native cytochrome c peroxidase (III) at alkaline pH.

Ferric cytochrome c peroxidase (CCP) undergoes a ligation-state transition from a pentacoordinate, high-spin (5c/hs) heme to a hexacoordinate, low-spin (6c/1s) heme when titrated over a pH range of 7.30-9.70. This behavior is similar to that exhibited by the ferrous form of the enzyme. However, the photodissociation of the low-spin, axial ligand, exhibited by ferrous CCP at alkaline pH, is not observed for ferric CCP. Instead, a photoinduced reduction of the ferric heme is apparent in the pH range 7.90-9.70. In the absence of O2 and redox mediators such as methyl viologen (MV2+), the reoxidation of the photoreduced enzyme is very slow (tau 1/2 approximately 3 min). F(-)-bound CCP(III) (6c/hs) displays similar pH-dependent photoreduction. Horseradish peroxidase, however, does not. The formation of 6c/1s heme coincides with the onset of appreciable photoreduction (between laser pulses, > 60 ms) of CCP (III) at alkaline pH, suggesting a global protein conformational rearrangement within or around its heme pocket. Photoreduction of alkaline CCP(III) most likely involves intramolecular electron transfer (ET) from the aromatic residue in the proximal heme pocket to the photoexcited heme. We speculate that the kinetics of electron transfer are affected by changes in the orientation of Trp-191.     

Spectral studies with lactoperoxidase and thyroid peroxidase: interconversions between native enzyme, compound II, and compound III

Spectral scans in both the visible (650-450 nm) and the Soret (450-380 nm) regions were recorded for the native enzyme, Compound II, and Compound III of lactoperoxidase and thyroid peroxidase. Compound II for each enzyme (1.7 microM) was prepared by adding a slight excess of H2O2 (6 microM), whereas Compound III was prepared by adding a large excess of H2O2 (200 microM). After these compounds had been formed it was observed that they were slowly reconverted to the native enzyme in the absence of exogenous donors. The pathway of Compound III back to the native enzyme involved Compound II as an intermediate. Reconversion of Compound III to native enzyme was accompanied by the disappearance of H2O2 and generation of O2, with approximately 1 mol of O2 formed for each 2 mol of H2O2 that disappeared. A scheme is proposed to explain these observations, involving intermediate formation of the ferrous enzyme. According to the scheme, Compound III participates in a reaction cycle that effectively converts H2O2 to O2. Iodide markedly affected the interconversions between native enzyme, Compound II, and Compound III for lactoperoxidase and thyroid peroxidase. A low concentration of iodide (4 microM) completely blocked the formation of Compound II when lactoperoxidase or thyroid peroxidase was treated with 6 microM H2O2. When the enzymes were treated with 200 microM H2O2, the same low concentration of iodide completely blocked the formation of Compound III and largely prevented the enzyme degradation that otherwise occurred in the absence of iodide. These effects of iodide are readily explained by (i) the two-electron oxidation of iodide to hypoiodite by Compound I, which bypasses Compound II as an intermediate, and (ii) the rapid oxidation of H2O2 to O2 by the hypoiodite formed in the reaction between Compound I and iodide.     

Preparation and properties of ferrous chloroperoxidase complexes with dioxygen, nitric oxide, and an alkyl isocyanide. Spectroscopic dissimilarities between the oxygenated forms of chloroperoxidase and cytochrome P-450

Extensive spectroscopic investigations of chloroperoxidase and cytochrome P-450 have consistently revealed close similarities between these two functionally distinct enzymes. Although the CO-bound ferrous states were the first to display such resemblance, additional comparisons have focused on the native ferric and ferrous and the ligand-bound ferric derivatives of the enzymes. In order to test the extent to which the spectral properties of the two enzymes match each other, we have prepared the NO, alkyl isocyanide, and O2 adducts of ferrous chloroperoxidase, the latter two for the first time. As expected, the NO adducts of the two proteins have similar UV-visible absorption and magnetic circular dichroism spectra; the same behavior is observed for the alkyl isocyanide complexes. Unexpectedly, the dioxygen adduct of ferrous chloroperoxidase (i.e. Compound III), generated in cryogenic solvents at -30 degrees C by bubbling with O2, is spectrally distinct from oxy-P-450-CAM. Identification of this derivative as oxygenated chloroperoxidase is based on the following criteria: It is EPR-silent at 77 K. The bound O2 is dissociable as judged by the uniform conversion to the CO-bound form. Oxy-chloroperoxidase autoxidizes to form the native ferric enzyme without detectable intermediates at a rate comparable to that determined for oxy-P-450-CAM. Oxy-chloroperoxidase exhibits optical absorption (lambda nm (epsilon mM) = 354 (41), 430 (94), 554 (16.5), 587 (12.5)) and magnetic circular dichroism spectra that are clearly distinct from those of histidine-ligated heme proteins such as oxy-myoglobin or oxy-horseradish peroxidase. Surprisingly, several of its spectral properties, namely the red-shifted Soret peak and discrete alpha peak, are also unlike those of oxy-P-450-CAM. Since considerable evidence has accumulated supporting the ligation of an endogenous thiolate to the heme iron of chloroperoxidase, as has been established for the P-450 enzyme, the observed dissimilarities suggest that the electronic properties of the two dioxygen adducts are quite sensitive to differences in their active site heme environment. This, in turn may be related to the functional differences between the two enzymes.     

The role of superoxide radicals in lactoperoxidase-catalysed H2O2-metabolism and in irreversible enzyme inactivation

Irreversible inactivation of lactoperoxidase in the presence of excess H2O2 has been investigated. Serial overlay absorption spectra of the Soret region show that the rate and total amount of enzyme inactivation depend on the proton concentration. Perhydroxyl or superoxide radicals (HO.2 or O-2) cannot be established as the inactivating species in this mechanism, but they influence the rate of reconversion of the intermediate lactoperoxidase-compound III back to the resting ferric form of the enzyme.     

Effects of heme ligand mutations including a pathogenic variant, H65R, on the properties of human cystathionine beta-synthase

Human cystathionine beta-synthase is a hemeprotein that catalyzes a pyridoxal phosphate (PLP)-dependent condensation of serine and homocysteine into cystathionine. Biophysical characterization of this enzyme has led to the assignment of the heme ligands as histidine and cysteinate, respectively, which has recently been confirmed by crystal structure determination of the catalytic core of the protein. Using site-directed mutagenesis, we confirm that C52 and H65 represent the thiolate and histidine ligands to the heme. Conversion of C52 to alanine or serine results in spectral properties of the resulting hemeprotein that are consistent with the loss of a thiolate ligand. Thus, the Soret peak blue-shifts from 428 to 415 and 417 nm in the ferric forms of the C52S and C52A mutants, respectively, and from 450 to 423 nm in the ferrous states of both mutants. Addition of CO to the dithionite-reduced ferrous C52 mutants results in spectra with Soret peaks at 420 nm. EPR spectroscopy of the ferric C52 variants reveals the predominance of a high-spin species. The H65R mutant, a variant described in a homocystinuric patient, has Soret peaks at 424, 421, and 420 nm in the ferric, ferrous, and ferrous CO states, respectively. EPR spectroscopy reveals predominance of the low-spin species. Both C52A and C52S mutations lead to protein with substoichiometric heme (19% with respect to wild type); however, the PLP content is comparable to that of wild-type enzyme. The heme and PLP contents of the H65R mutant are 40% and 75% that of wild-type enzyme. These results indicate that heme saturation does not dictate PLP saturation in these mutant enzymes. Both H65 and C52 variants display low catalytic activity, revealing that changes in the heme binding domain modulate activity, consistent with a regulatory role for this cofactor.     

Allosteric modulation of Euphorbia peroxidase by nickel ions

A class III peroxidase, isolated and characterized from the latex of the perennial Mediterranean shrub Euphorbia characias, contains one ferric iron-protoporphyrin IX pentacoordinated with a histidine 'proximal' ligand as heme prosthetic group. In addition, the purified peroxidase contained 1 mole of endogenous Ca(2+) per mole of enzyme, and in the presence of excess Ca(2+), the catalytic efficiency was enhanced by three orders of magnitude. The incubation of the native enzyme with Ni(2+) causes reversible inhibition, whereas, in the presence of excess Ca(2+), Ni(2+) leads to an increase of the catalytic activity of Euphorbia peroxidase. UV/visible absorption spectra show that the heme iron remains in a quantum mechanically mixed-spin state as in the native enzyme after addition of Ni(2+), and only minor changes in the secondary or tertiary structure of the protein could be detected by fluorescence or CD measurements in the presence of Ni(2+). In the presence of H(2)O(2) and in the absence of a reducing agent, Ni(2+) decreases the catalase-like activity of Euphorbia peroxidase and accelerates another pathway in which the inactive stable species accumulates with a shoulder at 619 nm. Analysis of the kinetic measurements suggests that Ni(2+) affects the H(2)O(2)-binding site and inhibits the formation of compound I. In the presence of excess Ca(2+), Ni(2+) accelerates the reduction of compound I to the native enzyme. The reported results are compatible with the hypothesis that ELP has two Ni(2+)-binding sites with opposite functional effects.     

Magnetic resonance spectral characterization of the heme active site of Coprinus cinereus peroxidase

Examination of the peroxidase isolated from the inkcap Basidiomycete Coprinus cinereus shows that the 42,000-dalton enzyme contains a protoheme IX prosthetic group. Reactivity assays and the electronic absorption spectra of native Coprinus peroxidase and several of its ligand complexes indicate that this enzyme has characteristics similar to those reported for horseradish peroxidase. In this paper, we characterize the H2O2-oxidized forms of Coprinus peroxidase compounds I, II, and III by electronic absorption and magnetic resonance spectroscopies. Electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) studies of this Coprinus peroxidase indicate the presence of high-spin Fe(III) in the native protein and a number of differences between the heme site of Coprinus peroxidase and horseradish peroxidase. Carbon-13 (of the ferrous CO adduct) and nitrogen-15 (of the cyanide complex) NMR studies together with proton NMR studies of the native and cyanide-complexed Coprinus peroxidase are consistent with coordination of a proximal histidine ligand. The EPR spectrum of the ferrous NO complex is also reported. Protein reconstitution with deuterated hemin has facilitated the assignment of the heme methyl resonances in the proton NMR spectrum.     

Resonance Raman investigations of chloroperoxidase, horseradish peroxidase, and cytochrome c using Soret band laser excitation.

Resonance Raman spectra of the heme protein chloroperoxidase in its native and reduced forms and complexed with various small ions are obtained by using laser excitation in the Soret region (350-450 nm). Additionally, Raman spectra of horseradish peroxidase, cytochrome P-450cam, and cytochrome c, taken with Soret excitation, are presented and discussed. The data support previous findings that indicate a strong analogy between the active site environments of chloroperoxidase and cytochrome P-450cam. The Raman spectra of native chloroperoxidase are found to be sensitive to temperature and imply that a high leads to low spin transition of the heme iron atom takes place as the temperature is lowered. Unusual peak positions are also found for native and reduced chloroperoxidase and indicate a weakening of porphyrin ring bond strengths due to the presence of a strongly electron-donating axial ligand. Enormous selective enhancements of vibrational modes at 1360 and 674 cm-1 are also observed in some low-spin ferrous forms of the enzyme. These vibrational frequencies are assigned to primary normal modes of expansion of the prophyrin macrocycle upon electronic excitation.     

Identification of heme axial ligands of cytochrome c' from Alcaligenes sp. N.C.I.B. 11015

The spectral properties of both ferric and ferrous cytochromes c' from Alcaligenes sp. N.C.I.B. 11015 are reported. The EPR spectra at 77 K and the electronic, resonance Raman, CD and MCD spectra at room temperature have been compared with those of the other cytochromes c' and various hemoproteins. In the ferrous form, all the spectral results at physiological pH strongly indicated that the heme iron(II) is in a high-spin state. In the ferric form, the EPR and electronic absorption spectra were markedly dependent upon pH. EPR and electronic spectral results suggested that the ground state of heme iron(III) at physiological pH consists of a quantum mechanical admixture of an intermediate-spin and a high-spin state. Under highly alkaline conditions, identification of the axial ligands of heme iron(III) was attempted by crystal field analysis of the low-spin EPR g values. Upon the addition of sodium dodecyl sulfate to ferric and ferrous cytochrome c', the low-spin type spectra were induced. The heme environment of this low-spin species is also discussed.     

Extensive studies of the heme coordination structure of indoleamine 2,3-dioxygenase and of tryptophan binding with magnetic and natural circular dichroism and electron paramagnetic resonance spectroscopy

In order to probe the active site of the heme protein indoleamine 2,3-dioxygenase, magnetic and natural circular dichroism (MCD and CD) and electron paramagnetic resonance (EPR) studies of the substrate (L-tryptophan)-free and substrate-bound enzyme with and without various exogenous ligands have been carried out. The MCD spectra of the ferric and ferrous derivatives are similar to those of the analogous myoglobin and horseradish peroxidase species. This provides strong support for histidine imidazole as the fifth ligand to the heme iron of indoleamine 2,3-dioxygenase. The substrate-free native ferric enzyme exhibits predominantly high-spin EPR signals (g perpendicular = 6, g parallel = 2) along with weak low-spin signals (g perpendicular = 2.86, 2.28, 1.60); similar EPR, spin-state and MCD features are found for the benzimidazole adduct of ferric myoglobin. This suggests that the substrate-free ferric enzyme has a sterically hindered histidine imidazole nitrogen donor sixth ligand. Upon substrate binding, noticeable MCD and EPR spectral changes are detected that are indicative of an increased low spin content (from 30 to over 70% at ambient temperature). Concomitantly, new low spin EPR signals (g = 2.53, 2.18, 1.86) and MCD features characteristic of hydroxide complexes of histidine-ligated heme proteins appear. For almost all of the other ferric and ferrous derivatives, only small substrate effects are observed with MCD spectroscopy, while substantial substrate effects are seen with CD spectroscopy. Thus, changes in the heme coordination structure of the ferric enzyme and in the protein conformation at the active site of the ferric and ferrous enzyme are induced by substrate binding. The observed substrate effects on the ferric enzyme may correlate with the previously observed kinetic substrate inhibition of indoleamine 2,3-dioxygenase activity, while such effects on the ferrous enzyme suggest the possibility that the substrate is activated during turnover.