Heme-linked ionization of horseradish peroxidase compound II monitored by the resonance Raman Fe(IV)=O stretching vibration

Fe(IV)=O resonance Raman stretching vibrations were recently identified by this laboratory for horseradish peroxidase compound II and ferryl myoglobin. In the present report it is shown that Fe(IV)=O stretching frequency for horseradish peroxidase compound II will switch between two values depending on pH, with pKa values corresponding to the previously reported compound II heme-linked ionizations of pKa = 6.9 for isoenzyme A-2 and pKa = 8.5 for isoenzyme C. Similar pH-dependent shifts of the Fe(IV)=O frequency of ferryl myoglobin were not detected above pH 6. The Fe(IV)=O stretching frequencies of compound II of the horseradish peroxidase isoenzymes at pH values above the transition points were at a high value approaching the Fe(IV)=O stretching frequency of ferryl myoglobin. Below the transition points the horseradish peroxidase frequencies were found to be 10 cm-1 lower. Frequencies of the Fe(IV)=O stretching vibrations of horseradish peroxidase compound II for one set of isoenzymes were found to be sensitive to deuterium exchange below the transition point but not above. These results were interpreted to be indicative of an alkaline deprotonation of a distal amino acid group, probably histidine, which is hydrogen bonded to the oxyferryl group below the transition point. Deprotonation of this group at pH values above the pKa disrupts hydrogen bonding, raising the Fe(IV)=O stretching frequency, and is proposed to account for the lowering of compound II reactivity at alkaline pH. The high value of the Fe(IV)=O vibration of compound II above the transition point appears to be identical in frequency to what is believed to be the Fe(IV)=O vibration of compound X.     

Comparison of the heme structures of horseradish peroxidase compounds X and II by resonance Raman spectroscopy

Horseradish peroxidase will catalyze the chlorination of certain substrates by sodium chlorite through an intermediate known as compound X. A chlorite-derived chlorine atom is known to be retained by compound X and has been proposed to be located at the heme active site. Although several heme structures have been proposed for compound X, including an Fe(IV)-OCl group, preliminary data previously reported by our laboratory suggested that compound X contained a heme Fe(IV) = O group, based on the similarity of a compound X resonance Raman band at 788 cm-1 to resonance Raman Fe(IV) = O stretching vibrations recently identified for horseradish peroxidase compound II and ferryl myoglobin. Isotopic studies now confirm that the 788 cm-1 resonance Raman band of compound X is, in fact, due to a heme Fe(IV) = O group, with the oxygen atom derived from chlorite. The Fe(IV) = O frequency of compound X, of horseradish peroxidase isoenzymes B and C, undergoes a pH-induced frequency shift, with behavior which appears to be the same as that previously reported for compound II, formed from the same isoenzymes. These observations strongly suggest that compounds II and X have very similar, if not identical, heme structures. The chlorine atom thus appears not to be heme-bound and may rather be located on an amino acid residue. The studies on compound X reported here were done in a pH region above pH 8, where compound X is moderately stable. The present results do not necessarily apply to compound X below pH 8.     

Resonance Raman spectra of bovine liver catalase compound II. Similarity of the heme environment to horseradish peroxidase compound II

Resonance Raman spectroscopy has been used to investigate the structure and environment of the heme group in bovine liver catalase compound II. Both Soret- and Q-band excitation have been employed to observe and assign the skeletal stretching frequencies of the porphyrin ring. The oxidation state marker band v4 increases in frequency from 1373 cm-1 in ferricatalase to 1375 cm-1 in compound II, consistent with oxidation of the iron atom to the Fe(IV) state. Oxidation of five-coordinate, high-spin ferricatalase to compound II is accompanied by a marked increase of the porphyrin core marker frequencies that is consistent with a six-coordinate low-spin state with a contracted core. An Fe(IV) = O stretching band is observed at 775 cm-1 for compound II at neutral pH, indicating that there is an oxo ligand at the sixth site. At alkaline pH, the Fe(IV) = O stretching band shifts to 786 cm-1 in response to a heme-linked ionization that is attributed to the distal His-74 residue. Experiments carried out in H218O show that the oxo ligand of compound II exchanges with bulk water at neutral pH, but not at alkaline pH. This is essentially the same behavior exhibited by horseradish peroxidase compound II and the exchange reaction at neutral pH for both enzymes is attributed to acid/base catalysis by a distal His residue that is believed to be hydrogen-bonded to the oxo ligand. Thus, the structure and environment of the heme group of the compound II species of catalase and horseradish peroxidase are very similar. This indicates that the marked differences in their reactivities as oxidants are probably due to the manner in which the protein controls access of substrates to the heme group.     

Oxygen exchange between the Fe(IV) = O heme and bulk water for the A2 isozyme of horseradish peroxidase

Resonance Raman spectra were observed for compound II of horseradish peroxidase A2, and the Fe(IV) = O stretching Raman line was identified at 775 cm-1. This Raman line shifted to 741 cm-1 upon a change of solvent from H2(16)O to H2(18)O, indicating occurrence of the oxygen exchange between the Fe(IV) = O heme and bulk water. The oxygen exchange took place only at the acidic side of the heme-linked ionization with pKa = 6.9.     

Resonance Raman study on cytochrome c peroxidase and its intermediate. Presence of the Fe(IV) = O bond in compound ES and heme-linked ionization

Resonance Raman spectra of ferrous and ferric cytochrome c peroxidase and Compound ES and their pH dependences were investigated in resonance with Soret band. The Fe(IV) = O stretching Raman line of Compound ES was assigned to a broad band around 767 cm-1, which was shifted to 727 cm-1 upon 18O substitution. The 18O-isotopic frequency shift was recognized for Compound ES derived in H218O, but not in H216O. This clearly indicated occurrence of an oxygen exchange between the Fe(IV) = O heme and bulk water. The Fe(IV) = O stretching Raman band was definitely more intense and of higher frequency in D2O than in H2O as in Compound II of horseradish peroxidase, but in contrast with this its frequency was unaltered between pH 4 and 11. The Fe(II)-histidine stretching Raman line was assigned on the basis of the frequency shift observed for 54Fe isotopic substitution. From the intensity analysis of this band, the pKa of the heme-linked ionization of ferrocytochrome c peroxidase was determined to be 7.3. The Raman spectrum of ferricytochrome c peroxidase strongly suggested that the heme is placed under an equilibrium between the 5- and 6-coordinate high-spin structures. At neutral pH it is biased to the 5-coordinate structure, but at the acidic side of the transition of pKa = 5.5 the 6-coordinate heme becomes dominant. F- was bound to the heme iron at pH 6, but Cl- was bound only at acidic pH. Acidification by HNO3, H2SO4, CH3COOH, HBr, or HI resulted in somewhat different populations of the 5- and 6-coordinate forms when they were compared at pH 4.3. Accordingly, it is inferred that a water molecule which is suggested to occupy the sixth coordination position of the heme iron is not coordinated to the heme iron at pH 6 but that protonation of the pKa = 5.5 residue induces an appreciable structural change, allowing the coordination of the water molecule to the heme iron.     

Resonance Raman spectroscopy of oxoiron(IV) porphyrin pi-cation radical and oxoiron(IV) hemes in peroxidase intermediates

The catalytic cycle intermediates of heme peroxidases, known as compounds I and II, have been of long standing interest as models for intermediates of heme proteins, such as the terminal oxidases and cytochrome P450 enzymes, and for non-heme iron enzymes as well. Reports of resonance Raman signals for compound I intermediates of the oxo-iron(IV) porphyrin pi-cation radical type have been sometimes contradictory due to complications arising from photolability, causing compound I signals to appear similar to those of compound II or other forms. However, studies of synthetic systems indicated that protein based compound I intermediates of the oxoiron(IV) porphyrin pi-cation radical type should exhibit vibrational signatures that are different from the non-radical forms. The compound I intermediates of horseradish peroxidase (HRP), and chloroperoxidase (CPO) from Caldariomyces fumago do in fact exhibit unique and characteristic vibrational spectra. The nature of the putative oxoiron(IV) bond in peroxidase intermediates has been under discussion in the recent literature, with suggestions that the Fe(IV)O unit might be better described as Fe(IV)-OH. The generally low Fe(IV)O stretching frequencies observed for proteins have been difficult to mimic in synthetic ferryl porphyrins via electron donation from trans axial ligands alone. Resonance Raman studies of iron-oxygen vibrations within protein species that are sensitive to pH, deuteration, and solvent oxygen exchange, indicate that hydrogen bonding to the oxoiron(IV) group within the protein environment contributes to substantial lowering of Fe(IV)O frequencies relative to those of synthetic model compounds.     

Resonance Raman investigation of CO-ligated monomeric insect hemoglobins. Direct evidence for reciprocal changes in iron-axial ligand bonds induced by allosteric transitions

The resonance Raman spectra of the two affinity states of the CO-ligated monomeric insect hemoglobins, Chironomus thummi thummi (CTT) III ad IV, have been investigated. We have identified (via 54Fe/57Fe and 13C18O/12C16O isotope exchange) the Fe-N epsilon(His) stretching mode at approximately 317 cm-1. This stretching mode changes from 329 (pH 5.5) to 317 cm-1 (pH 9.5) reflecting the pH-induced t in equilibrium with r conformational transition. The Fe-CO stretching mode is also pH-sensitive changing from 483 (pH 5.2) to 485 cm-1 (pH 9.2) in 57Fe CTT III . 13C18O complex. However the C-O stretching mode is pH-insensitive. The nonallosteric monomeric insect hemoglobin CTT I does not exhibit a pH-dependence of these vibrational modes. pH-Induced effects were also observed for a vinyl bending mode at 379 cm-1 (pH 9.5) in CTT III deuterated at the beta-carbons of the vinyls in position 2 and 4. It shifts to 390 cm-1 at pH 5.5. The other vinyl vibration at 573 cm-1 exhibits intensity enhancement via through-space coupling with the Fe-C-O bending mode. Our resonance Raman data provide the first direct evidence that the trans-effect is operative as a trigger mechanism for ligand-binding in monomeric allosteric insect hemoglobins. In going from the low-affinity to the high-affinity state, the Fe-N epsilon(His) bond becomes weaker, whereas the Fe-CO bond becomes stronger.     

An iron hydroxide moiety in the 1.35 Å resolution structure of hydrogen peroxide derived myoglobin compound II at pH 5.2

The biological conversions of O(2) and peroxides to water as well as certain incorporations of oxygen atoms into small organic molecules can be catalyzed by metal ions in different clusters or cofactors. The catalytic cycle of these reactions passes through similar metal-based complexes in which one oxygen- or peroxide-derived oxygen atom is coordinated to an oxidized form of the catalytic metal center. In haem-based peroxidases or oxygenases the ferryl (Fe(IV)O) form is important in compound I and compound II, which are two and one oxidation equivalents higher than the ferric (Fe(III)) form, respectively. In this study we report the 1.35 A structure of a compound II model protein, obtained by reacting hydrogen peroxide with ferric myoglobin at pH 5.2. The molecular geometry is virtually unchanged compared to the ferric form, indicating that these reactive intermediates do not undergo large structural changes. It is further suggested that at low pH the dominating compound II resonance form is a hydroxyl radical ferric iron rather than an oxo-ferryl form, based on the short hydrogen bonding to the distal histidine (2.70 A) and the Fe...O distance. The 1.92 A Fe...O distance is in agreement with an EXAFS study of compound II in horseradish peroxidase.     

A kinetic study of the endogenous reduction of the oxidized sites in the primary cytochrome c peroxidase-hydrogen peroxide compound.

The primatry compound formed in the reaction between H2O2 and cytochrome c peroxidase is oxidized two equivalents above the native enzyme. The two oxidized sites are thought to be an Fe(IV) and an amino acid radical. In the absence of oxidizable substrate, the Fe(IV) and radical sites decay by apparent first-order processes but at different rates. It is likely that the decay involves both intra- and intermolecular electron-transfer reactions. The reduction of the Fe(IV) site depends upon the pH with a minimum reduction rate of 2.9-10(-5)s(-1) at pH 6. At pH 4 and 6, the reduction of the Fe(IV) site is facilitated by prior oxidation of amino acid residues in the protein.     

Localization and orientation of functional water molecules in bacteriorhodopsin as revealed by polarized Fourier transform infrared spectroscopy.

Linear dichroic difference Fourier transform infrared spectra upon formation of the M photointermediate were recorded with oriented purple membranes. The purpose was to determine the angle of the directions of the dipole moments of 1) the water molecule whose O-H stretching vibration appears at 3643 cm-1 for the unphotolyzed state and 3671 cm-1 for the M intermediate, and 2) the C=O bond of protonated Asp85 in the M intermediate. The angle of 36 degrees we find for the C=O of the protonated Asp85 in the M intermediate is not markedly different from 26 degrees for unprotonated Asp85 in the model based on cryoelectron diffraction, indicating the absence of gross orientation changes in Asp85 upon its protonation. The O-H band at 3671 cm-1 of a water molecule in the M intermediate, although its position has not determined, is fixed almost parallel to the membrane plane. For the unphotolyzed state the angle of the water O-H to the membrane normal was determined to be 60 degrees. On the basis of these data and the structural model, we place the water molecule in the unphotolyzed state at a position where it forms hydrogen bonds with the Schiff base, Asp85, Asp212, and Trp86.     

Mössbauer identification of a protonated ferryl species in catalase from Proteus mirabilis: density functional calculations on related models

The Proteus mirabilis catalase is one of the most efficient heme-containing catalase and forms a relatively stable compound II. Samples of compound II were prepared from PMC enriched in (57)Fe. For the first time, two different forms of compound II, namely low pH compound II (LpH II) (43%) and high pH compound II (HpH II) (25%), have been characterized by M?ssbauer spectroscopy at pH 8.3. The ratio LpH II/HpH II increases irreversibly with decreasing pH. The large quadrupole splitting value of LpH II (DeltaE(Q)=2.29 (2) mm/s, with delta(/Fe)=0.03 (2) mm/s), compared to that of HpH II (DeltaE(Q)=1.47 (2) mm/s, with delta(/Fe)=0.07 (2) mm/s), reflects the protonation of the ferryl group. Quadrupole splitting values of 1.46 and 2.15mm/s have been computed by DFT for optimized models of the ferryl compound II (model 1) and the protonated ferryl compound II (model 2), respectively, starting from the Fe(IV)O model initially published by Rovira and Fita [C. Rovira, I. Fita, J. Phys. Chem. B 107 (2003) 5300-5305]. Therefore, we attribute the LpH II compound to a protonated ferryl Fe(IV)-OH complex, whereas the HpH II compound corresponds to the classical ferryl Fe(IV)O complex.     

Observation of the Fe4+ = O stretching Raman band for cytochrome oxidase compound B at ambient temperature

Resonance Raman and visible absorption spectra were simultaneously observed for cytochrome oxidase reaction intermediates at 5 degrees C by using the artificial cardiovascular system (Ogura, T., Yoshikawa, S., and Kitagawa, T. (1989) Biochemistry 28, 8022-8027) and a device for Raman/absorption simultaneous measurements (Ogura, T., and Kitagawa, T. (1988) Rev. Sci. Instrum. 59, 1316-1320). The Fe4+ = O stretching (nu FeO) Raman band was observed at 788 cm-1 for compound B for the first time. This band showed the 16O/18O isotopic frequency shift (delta nu FeO) by 40 cm-1, in agreement with that for horseradish peroxidase compound II (nu FeO = 787 cm-1 and delta nu FeO = 34 cm-1). In the time region when the FeII-O2 stretching band for compound A and the nu FeO band for compound B were coexistent, a Raman band assignable to the Fe3+-O-O-Cu2+ linkage was not recognized.     

A comparison of the resonance Raman properties of the fast and slow forms of cytochrome oxidase

Resonance Raman (RR) spectra of the "rapid" and "slow" forms (Baker et al., 1987) of resting cytochrome oxidase obtained with Soret excitation at 413.1 nm are reported. There are a number of conspicuous differences between the two forms in the high-frequency region of the RR spectrum which involve changes in Raman intensity arising from a blue shift in the Soret maximum of cytochrome a3 upon conversion to the slow form. In the low-frequency region a peak present at 223 cm-1 in the rapid form shifts to 220 cm-1 in the slow form; this peak is assigned as the cytochrome a3 Fe(III)-N(His-Im) stretch. The slow form of the enzyme possesses greater intensity in RR peaks near 1620 cm-1 which have been previously attributed by others to partial photoreduction of the enzyme. We have quantitated the amount of laser-induced photoreduction in these RR spectra by comparison with the spectra of mixed-valence derivatives of the enzyme and find that these 1620-cm-1 features are unreliable indicators of photoreduction. The spectra of the fast- and slow-reacting species in H2O and D2O have been compared. The fast-reacting form exhibits a 4-cm-1 shift, from 223 to 219 cm-1, upon transferring to D2O in a peak which we assign as the cytochrome a3 Fe(III)-N(His-Im) stretch. There is a parallel shift in the feature at 1651 cm-1 due to the C = O stretch of the formyl group of cytochrome a. These deuterium shifts are not observed in the slow form.(ABSTRACT TRUNCATED AT 250 WORDS)     

Observation of the FeIV=O stretching vibration of ferryl myoglobin by resonance Raman spectroscopy

We have directly observed the oxyferryl group of ferryl myoglobin by resonance Raman spectroscopy. The FeIV = O stretching vibration is observed at 797 cm-1 and confirmed by an 18O-induced isotopic shift to 771 cm-1. The porphyrin center-to-nitrogen distance of ferryl myoglobin is significantly less than that previously observed for horseradish peroxidase compound II, which also contains an FeIV = O heme. The FeIII-CN- stretch of myoglobin (FeIII) cyanide is observed at 454 cm-1, which shifts to 449 cm-1 upon substitution with [13C]cyanide.     

Conformational change and histidine control of heme chemistry in cytochrome c peroxidase: resonance Raman evidence from Leu-52 and Gly-181 mutants of cytochrome c peroxidase.

Resonance Raman (RR) spectra are reported for Fe(III), Fe(II), and Fe(II)CO forms of site-directed mutants of the cytochrome c peroxidase variant CCP(MI), cloned in Escherichia coli. The Fe(II) form is five-coordinate (5-c) and high-spin at low pH, but it is six-coordinate (6-c) and low-spin at high pH except when the distal His-52 residue is replaced with Leu, showing the sixth ligand to be the His-52 imidazole. Although the Leu-52 mutant stays 5-c, it does undergo an alkaline transition, as revealed by upshifts and broadening of bands assigned to vinyl C = C stretching (1620 cm-1) and C beta-vinyl bending (402 cm-1). Similar changes are seen for CCP(MI) and other mutants. Thus the alkaline transition induces a conformational change that affects the vinyl groups, probably through changes in their orientation, and that permits the His-52 imidazole to bind the Fe. The RR band arising from the stretching of the proximal Fe(II)-imidazole bond contains components at ca. 235 and 245 cm-1 for CCP(MI), which are believed to reflect a double well potential for the H-bond between the proximal His-175 imidazole and the Asp-235 carboxylate group. Loss of this H-bond by mutation of Asp-235 to Asn results in the loss of these two bands and their replacement by a single band at 205 cm-1. Although the Fe(II)-imidazole stretching mode cannot be observed in the 6-c alkaline form of the enzyme, the sixth ligand in the alkaline form of CCP(MI) is photolabile, and the status of the Fe(II)-imidazole bond can be determined in the resulting 5-c-photoproduct. For CCP(MI) at alkaline pH, the conformation change induces an increase in the 235/245-cm-1 ratio, reflecting a perturbation of the H-bond potential. In the His-52----Leu mutant, a 205-cm-1 band appears along with the 235/245-cm-1 doublet at alkaline pH, indicating partial loss of the proximal H-bond due to the distal alteration. The effect of mutations that perturb the H-bonding network that extends from the distal to the proximal side of the heme is more dramatic: at alkaline pH, His-181----Gly, Arg-48----Leu, and Trp-51----Phe mutants show an Fe(II)-imidazole stretching mode at 205 cm-1 exclusively, indicating complete loss of the proximal Asp-235-His-175 H-bond.(ABSTRACT TRUNCATED AT 400 WORDS)     

Comparative FTIR analysis of the microenvironment of in cyanide-treated, high pH-treated and iron-depleted photosystem II membrane fragments

FTIR difference spectra due to light-induced formation were measured in control PS II membrane fragments and in samples where the magnetic interaction with the non-heme iron center in its high-spin Fe2+ state is eliminated by three different methods, i.e., extraction of the non-heme iron center, treatment with cyanide, and incubation at high pH (pH = 11). The results obtained reveal that (i) the most pronounced band at 1479 cm-1 reflecting the C../--O stretching mode of in the semiquinone anion radical remains invariant to "iron depletion" while it shifts by 4 and 2 cm-1 to lower frequencies upon cyanide and high pH treatments, respectively, (ii) peaks observed in the 2600-3000 cm-1 region which arise from Fermi resonance of harmonics and combinations of the imidazole ring modes with the hydrogen-bonding NH stretching vibrations are not affected upon iron depletion but are lost in cyanide and high pH-treated samples, and (iii) all three treatments give rise to some similar changes in the amide I bands of the protein backbones and in imidazole ring modes of the coupled histidine. These results show that the hydrogen-bonding interaction of is virtually unaffected upon non-heme iron depletion; in particular, the strong hydrogen bond between QA and a histidine side chain (most likely His 215 of the D2 subunit) is not changed. In marked contrast, drastic changes take place in the hydrogen bonding between QA and His upon CN- and high pH treatments. The straightforward interpretation is that the hydrogen bond is lost upon these treatments. Despite the striking difference in the effect of hydrogen-bonding interaction, all three treatments lead to similar structural pertubations on the protein conformational changes due to formation and ring vibrations of the coupled histidine side chain. On the basis of the data presented in the study, it is inferred that, concerning the hydrogen bond interaction, the microenvironment of is close to the native state when a suitable iron depletion is performed. Accordingly, the previously reported conclusion on the hydrogen-bonding pattern of in PS II [MacMillan, F., Lendzian, F., Renger, G., and Lubitz, W. (1995) Biochemistry 34, 8144-8156] studied by using iron-depleted preparations most likely reflects the situation in an intact PS II.     

The reaction between ferrous polyaminocarboxylate complexes and hydrogen peroxide: an investigation of the reaction intermediates by stopped flow spectrophotometry

The reactions of Fe(II)EDTA, Fe(II)DTPA, and Fe(II)HEDTA with hydrogen peroxide near neutral pH have been investigated. All these reactions have been assumed to proceed through an active intermediate, I1, (Formula: see text) where pac is one of the three polyaminocarboxylates mentioned above. I1, whether .OH radical or an iron complex, reacts with ethanol, formate, and other scavengers at rates relative to k2 that, with the exception of t-butanol and benzoate, are similar, but not identical, to those expected for the.OH radical. In contrast, at pH 3, in the absence of ligands the reaction of I1 with Fe2+ was inhibited by ethanol and t-butanol and the reactivity of I1 towards these two scavengers relative to ferrous ion is identical to that exhibited by the hydroxyl radical. When pac = HEDTA, the intermediate of the first reaction reacts with formate ion to form the ferrous HEDTA ligand radical complex, which is characterized by absorption maxima at 295 nm (epsilon = 2,640 M-1 cm-1) and 420 nm (epsilon = 620 M-1 cm-1). For the reaction of Fe(II)HEDTA with H2O2, the following mechanism is proposed: (Formula: see text) where k17 = 4.2 X 10(4) M-1 sec-1 and k19 = 5 +/- 0.2 sec-1.     

Irreversible inactivation of purple acid phosphatase by hydrogen peroxide and ascorbate.

Treatment of the Cu(II)-Fe(III) derivative of pig allantoic fluid acid phosphatase with hydrogen peroxide caused irreversible inactivation of the enzyme and loss of half of the intensity of the visible absorption spectrum. Phosphate, a competitive inhibitor, protected against this inactivation, suggesting that it occurred as a result of a reaction at the active site. The native Fe(II)-Fe(III) enzyme was irreversibly inactivated by H2O2 to a much smaller extent than the Cu(II)-Fe(III) derivative, whereas the Zn(II)-Fe(III) derivative was stable to H2O2 treatment. The rates of inactivation of the Cu(II)-Fe(III) and Fe(II)-Fe(III) enzymes in the presence of H2O2 were increased by addition of ascorbate. These results suggest involvement of a Fenton-type reaction, generating hydroxyl radicals which react with essential active site groups. Experiments carried out on the Fe(II)-Fe(III) enzyme showed that irreversible inactivation by H2O2 in the presence of ascorbate obeyed pseudo first-order kinetics. A plot of kobs for this reaction against H2O2 concentration (at saturating ascorbate) was hyperbolic, giving kobs(max) = 0.41 +/- 0.025 min-1 and S0.5(H2O2) = 1.16 +/- 0.18 mM. A kinetic scheme is presented to describe the irreversible inactivation, involving hydroxyl radical generation by reaction of H2O2 with Fe(II)-Fe(III) enzyme, reduction of the product Fe(III)-Fe(III) enzyme by ascorbate and reaction of hydroxyl radical with an essential group in the enzyme.     

Crystallographic and spectroscopic studies of peroxide-derived myoglobin compound II and occurrence of protonated FeIV O

High resolution crystal structures of myoglobin in the pH range 5.2-8.7 have been used as models for the peroxide-derived compound II intermediates in heme peroxidases and oxygenases. The observed Fe-O bond length (1.86-1.90 A) is consistent with that of a single bond. The compound II state of myoglobin in crystals was controlled by single-crystal microspectrophotometry before and after synchrotron data collection. We observe some radiation-induced changes in both compound II (resulting in intermediate H) and in the resting ferric state of myoglobin. These radiation-induced states are quite unstable, and compound II and ferric myoglobin are immediately regenerated through a short heating above the glass transition temperature (<1 s) of the crystals. It is unclear how this influences our compound II structures compared with the unaffected compound II, but some crystallographic data suggest that the influence on the Fe-O bond distance is minimal. Based on our crystallographic and spectroscopic data we suggest that for myoglobin the compound II intermediate consists of an Fe(IV)-O species with a single bond. The presence of Fe(IV) is indicated by a small isomer shift of delta = 0.07 mm/s from M?ssbauer spectroscopy. Earlier quantum refinements (crystallographic refinement where the molecular-mechanics potential is replaced by a quantum chemical calculation) and density functional theory calculations suggest that this intermediate H species is protonated.     

Mn(II) oxidation is the principal function of the extracellular Mn-peroxidase from Phanerochaete chrysosporium

The manganese peroxidase (MnP), from the lignin-degrading fungus Phanerochaete chrysosporium, an H2O2-dependent heme enzyme, oxidizes a variety of organic compounds but only in the presence of Mn(II). The homogeneous enzyme rapidly oxidizes Mn(II) to Mn(III) with a pH optimum of 5.0; the latter was detected by the characteristic spectrum of its lactate complex. In the presence of H2O2 the enzyme oxidizes Mn(II) significantly faster than it oxidizes all other substrates. Addition of 1 M equivalent of H2O2 to the native enzyme in 20 mM Na-succinate, pH 4.5, yields MnP compound II, characterized by a Soret maximum at 416 nm. Subsequent addition of 1 M equivalent of Mn(II) to the compound II form of the enzyme results in its rapid reduction to the native Fe3+ species. Mn(III)-lactate oxidizes all of the compounds which are oxidized by the enzymatic system. The relative rates of oxidation of various substrates by the enzymatic and chemical systems are similar. In addition, when separated from the polymeric dye Poly B by a semipermeable membrane, the enzyme in the presence of Mn(II)-lactate and H2O2 oxidizes the substrate. All of these results indicate that the enzyme oxidizes Mn(II) to Mn(III) and that the Mn(III) complexed to lactate or other alpha-hydroxy acids acts as an obligatory oxidation intermediate in the oxidation of various dyes and lignin model compounds. In the absence of exogenous H2O2, the Mn-peroxidase oxidized NADH to NAD+, generating H2O2 in the process. The H2O2 generated by the oxidation of NADH could be utilized by the enzyme to oxidize a variety of other substrates.