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.     

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.     

Coordination structures and reactivities of compound II in iron and manganese horseradish peroxidases. A resonance Raman study

Resonance Raman investigations on compound II of native, diacetyldeuteroheme-, and manganese-substituted horseradish peroxidase (isozyme C) revealed that the metal-oxygen linkage in the compound differed from one another in its bond strength and/or structure. Fe(IV) = O stretching frequency for compound II of native enzyme was pH sensitive, giving the Raman line at 772 and 789 cm-1 at pH 7 and 10, respectively. The results confirmed the presence of a hydrogen bond between the oxo-ligand and a nearby amino acid residue (Sitter, A. J., Reczek, C. M., and Terner, J. (1985) J. Biol. Chem. 260, 7515-7522). The Fe(IV) = O stretch for compound II of diacetylheme-enzyme was located at 781 cm-1 at pH 7 which was 9 cm-1 higher than that of native enzyme compound II. At pH 10, however, the Fe(IV) = O stretch was found at 790 cm-1, essentially the same frequency as that of native enzyme compound II. The pK value for the pH transition, 8.5, was also the same as that of native compound II. Unlike in native enzyme, D2O-H2O exchange did not cause a shift of the Fe(IV) = O frequency of diacetylheme-enzyme. Thus, the metal-oxygen bond at pH 7 was stronger in diacetylheme-enzyme due to a weaker hydrogen bonding to the oxo-ligand, while the Fe(IV) = O bond strength became essentially the same between both enzymes at alkaline pH upon disruption of the hydrogen bond. A much lower reactivity of the diacetylheme-enzyme compound II was accounted to be due to the weaker hydrogen bond. Compound II of manganese-substituted enzyme exhibited Mn(IV)-oxygen stretch about 630 cm-1, which was pH insensitive but down-shifted by 18 cm-1 upon the D2O-H2O exchange. The finding indicates that its structure is in Mn(IV)-OH, where the proton is exchangeable with a water proton. These results establish that the structure of native enzyme compound II is Fe(IV) = O but not Fe(IV)-OH.     

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.     

Chemical and transient state kinetic studies on the formation and decomposition of horseradish peroxidase compounds XI and XII

Previous studies on the chlorination reaction catalyzed by horseradish peroxidase using chlorite as the source of chlorine detected the formation of a chlorinating intermediate that was termed Compound X (Shahangian, S., and Hager, L.P. (1982) J. Biol. Chem. 257, 11529-11533). These studies indicated that at pH 10.7, the optical absorption spectrum of Compound X was similar to the spectrum of horseradish peroxidase Compound II. Compound X was shown to be quite stable at alkaline pH values. This study was undertaken to examine the relationship between the oxidation state of the iron protoporphyrin IX heme prosthetic group in Compound X and the chemistry of the halogenating intermediate. The experimental results show that the optical absorption properties and the oxidation state of the heme prosthetic group in horseradish peroxidase are not directly related to the presence of the activated chlorine atom in the intermediate. The oxyferryl porphyrin heme group in alkaline Compound X can be reduced to a ferric heme species that still retains the activated chlorine atom. Furthermore, the reaction of chlorite with horseradish peroxidase at acidic pH leads to the secondary formation of a green intermediate that has the spectral properties of horseradish peroxidase Compound I (Theorell, H. (1941) Enzymologia 10, 250-252). The green intermediate also retains the activated chlorine atom. By analogy to peroxidase Compound I chemistry, the heme prosthetic group in the green chlorinating intermediate must be an oxyferryl porphyrin pi-cation radical species (Roberts, J. E., Hoffman, B. M., Rutter, R. J., and Hager, L. P. (1981) J. Am. Chem. Soc. 103, 7654-7656). To be consistent with traditional peroxidase nomenclature, the red alkaline form of Compound X has been renamed Compound XII, and the green acidic form has been named Compound XI. The transfer of chlorine from the chlorinating intermediate to an acceptor molecule follows an electrophilic (rather than a free radical) path. A mechanism for the reaction is proposed in which the activated chlorine atom is bonded to a heteroatom on an active-site amino acid side chain. Transient state kinetic studies show that the initial intermediate, Compound XII, is formed in a very fast reaction. The second-order rate constant for the formation of Compound XII is approximately 1.1 x 10(7) M-1 s-1. The rate of formation of Compound XII is strongly pH-dependent. At pH 9, the second-order rate constant for the formation of Compound XII drops to 1.5 M-1 s-1. At acidic pH values, Compound XII undergoes a spontaneous first-order decay to yield Compound XI.(ABSTRACT TRUNCATED AT 400 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.     

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 spectroscopic evidence for heme iron-hydroxide ligation in peroxidase alkaline forms

Horseradish peroxidase will convert from a five-coordinate high-spin heme at neutral pH to a six-coordinate low-spin heme at alkaline pH. Though alkaline forms of other heme proteins such as hemoglobin and myoglobin are known to contain a heme-ligated hydroxide, alkaline horseradish peroxidase has been considered not to contain a ligated hydroxide. Several alternatives have been proposed which would be stronger field ligands than a hydroxide ion. In this report we provide resonance Raman evidence, using Soret excitation, that alkaline horseradish peroxidase does in fact contain a heme iron-ligated hydroxyl group. The band was located for isoenzymes C and A-1 by its sensitivity to 18O substitution and confirmed with 54Fe, 57Fe, and 2H. An isoenzyme of turnip peroxidase was investigated and found to also contain a ligated hydroxide at alkaline pH. The observed peroxidase Fe(III)-OH frequencies are 15-25 cm-1 higher than the corresponding frequencies of alkaline methemoglobin and metmyoglobin and correlate with changes in spin-state distribution. This is explained in the context of hydrogen bonding to a distal histidine which results in increased ligand field strength facilitating the formation of low-spin hemes. It has been demonstrated that the ferryl/ferric redox potential of horseradish peroxidase is markedly lowered at alkaline pH (Hayashi, Y., and Yamazaki, I. (1979) J. Biol. Chem. 254, 9101-9106). These observations are rationalized in terms of oxidation of a ligated ferric hydroxyl group facilitated through base catalysis by a distal histidine.     

A comparison by magnetic circular dichroism of compound X and compound II of horseradish peroxidase

The chlorite product of horseradish peroxidase, compound X, is shown by magnetic circular dichroism (MCD) spectroscopy in the temperature range 1.6-50 K to have a very similar haem structure to compound II under the same conditions (pH 10.7). Both are concluded to contain the Fe(IV) = 0 group. The MCD spectrum also detects an unusual species, absorbing at wavelengths between 600 and 750 nm, that has magnetic properties different from those of the ferryl haem group. It is suggested that this is a species at the same oxidation level as ferryl haem but with the porphyrin ring having suffered a one-electron oxidation, i.e. [Fe(III) P.+].     

On the status of ferryl protonation

We examine the issue of ferryl protonation in heme proteins. An analysis of the results obtained from X-ray crystallography, resonance Raman spectroscopy, and extended X-ray absorption spectroscopy (EXAFS) is presented. Fe-O bond distances obtained from all three techniques are compared using Badger's rule. The long Fe-O bond lengths found in the ferryl crystal structures of myoglobin, cytochrome c peroxidase, horseradish peroxidase, and catalase deviate substantially from the values predict by Badger's rule, while the oxo-like distances obtained from EXAFS measurements are in good agreement with the empirical formula. Density functional calculations, which suggest that M?ssbauer spectroscopy can be used to determine ferryl protonation states, are presented. Our calculations indicate that the quadrupole splitting (DeltaE(Q)) changes significantly upon ferryl protonation. New resonance Raman data for horse-heart myoglobin compound II (Mb-II, pH 4.5) are also presented. An Fe-O stretching frequency of 790cm(-1) (shifting to 754cm(-1) with (18)O substitution) was obtained. This frequency provides a Badger distance of r(Fe-O)=1.66A. This distance is in agreement with the 1.69A Fe-O bond distance obtained from EXAFS measurements but is significantly shorter than the 1.93A bond found in the crystal structure of Mb-II (pH 5.2). In light of the available evidence, we conclude that the ferryl forms of myoglobin (pKa4), horseradish peroxidase (pKa4), cytochrome c peroxidase (pKa4), and catalase (pKa7) are not basic. They are authentic Fe(IV)oxos with Fe-O bonds on the order of 1.65A.     

Resonance Raman spectroscopy of horseradish peroxidase derivatives and intermediates with excitation in the near ultraviolet

Resonance Raman enhancement of derivatives and intermediates of horseradish peroxidase in the near ultraviolet (N-band excitation) results in intensity and enhancement patterns that are different from those normally observed within the porphyrin Soret (B-band) and alpha-beta (Q-band) absorptions. In particular it allows the resolution of resonance Raman spectra of horseradish peroxidase compound I. The bands above 1300 cm-1 can be assigned to porphyrin vibrational modes that are characteristically shifted in frequency due to removal of an electron from the porphyrin ring. The resonance Raman frequency shifts follow normal mode compositions. Relative to resonance Raman spectra of compound II, the v4 frequency (primarily Ca-N) exhibits a 20 cm-1 downshift. The v2, v11, and v37 vibrational frequencies whose mode compositions are primarily porphyrin Cb-Cb, exhibit 10-20 cm-1 upshifts. The v3, v10, and v28 frequencies, whose mode compositions are primarily Ca-Cm, exhibit downshifts. The downshifts for v3 and v10 are small, 3-5 cm-1; however, the downshift for v28 is 14 cm-1. These frequency shifts are consistent with those of previously published resonance Raman studies of model compounds. In contrast to reports from other laboratories, the data presented here for horseradish peroxidase compound I can be attributed unambiguously to resonance Raman scattering from a porphyrin pi-cation radical.     

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.     

Identification and properties of an oxoferryl structure in myeloperoxidase compound II

Myeloperoxidase compound II has been characterized by using optical absorption and resonance Raman spectroscopies. Compared to compounds II in other peroxidases, the electronic and vibrational properties of this intermediate are strongly perturbed due to the unusual active-site iron chromophore that occurs in myeloperoxidase. Despite this difference in prosthetic group, however, other properties of myeloperoxidase compound II are similar to those observed for this intermediate in the more common peroxidases (horseradish peroxidase in particular). Two forms of the myeloperoxidase intermediate species, each with distinct absorption spectra, are recognized as a function of pH. We present evidence consistent with interconversion of these two forms via a heme-linked ionization of a distal amino acid residue with a pKa congruent to 9. From resonance Raman studies of isotopically labeled species at pH 10.7, we identify an iron-oxygen stretching frequency at 782 cm-1, indicating the presence of an oxoferryl (O = FeIV) group in myeloperoxidase compound II. We further conclude that the oxo ligand is not hydrogen bonded above the pKa but possibly exhibits oxygen exchange with the medium at pH values below the pKa due to hydrogen bonding of the oxo ligand to the distal protein group.     

Modeling low-pH hemoproteins

A tetracoordinate ferrous heme (iron-porphyrin) has been proposed as an intermediate at low pH (less than 3.0) for respiratory hemoproteins, peroxidases, and model heme complexes. This intermediate is believed to arise via protonation of the N(epsilon) atom of the proximal histidine and consequent cleavage of the Fe-N(epsilon) bond. To establish a spectral signature for the proposed low-pH tetracoordinate species, we have obtained Soret excitation resonance Raman spectra on samples of crystallographically defined, tetracoordinate iron(II)-octaethylporphyrin (Fe.OEP; S = 1). The high-frequency (greater than or equal to 900 cm-1) resonance Raman spectral features of Fe.OEP are clearly distinct from those of high-spin pentacoordinate or low-spin hexacoordinate ferrous hemes. Rather, they are at frequencies more typically observed for low-spin hexacoordinate ferric porphyrins. Comparative spectral analysis of tetracoordinate Fe.OEP and other proposed tetracoordinate ferrous hemes (e.g. iron(II)-protoporphyrin IX) demonstrates little or no macrocycle effect on the resonance Raman frequencies above 900 cm-1. This work thus serves to provide a testable spectral signature by which the existence of the proposed tetracoordinate biological intermediate may be verified and by which its functional significance may be tested.     

Resonance Raman evidence for Fe(IV) in compound II of horseradish peroxidase

Resonance Raman spectra have been obtained for Compound II of horseradish peroxidase. Its prophyrin vibrational frequencies are consistent with a planar low-spin heme containing Fe(IV). The oxidation-state marker band is found at the unprecedentedly high value of 1382 cm^?1. This band was also observed in solutions of myoglobin and cytochrome c peroxidase to which H₂O₂ had been added. No evidence was found for an actual FeO double bond in Compound II.     

Alternative carbon monoxide binding modes for horseradish peroxidase studied by resonance Raman spectroscopy

Resonance Raman (RR) spectroscopy and infrared spectroscopy have been used to characterize the three vibrational modes, CO and FeC stretching and FeCO bending, for carbon monoxide bound to reduced horseradish peroxidase, with the aid of 13CO and C18O isotope shifts. At high pH, one species, I, is observed, with nu FeC = 490 cm-1 and nu CO = 1932 cm-1. The absence of a band attributable to delta FeCO suggests a linear FeCO unit normal to the heme plane. The data were consistent with I having a strongly H-bonded proximal histidine, as shown by a comparison with imidazole and imidazolate adducts of FeIIPPDME(CO) (PPDME = protoporphyrin IX dimethyl ester), with nu FeC = 497 and 492 cm-1 and nu CO = 1960 and 1942 cm-1. At low pH an additional species, II, is observed, with nu FeC = 537 cm-1, nu CO = 1904 cm-1, and delta FeCO = 587 cm-1; it is attributed to FeCO that is H bonded to a protonated distal histidine, the H bond strongly lowering nu CO and raising nu FeC. The appearance of delta FeCO in the RR spectrum suggests that the FeCO unit in II is tilted with respect to the heme plane. At low pH, the population of I and II depends on the CO concentration. I dominates at low CO/protein levels but is replaced by II as the amount of CO is increased. This behavior is suggested to arise from secondary binding of CO, which induces a conformation change involving the distal residues of the heme pocket.     

Infrared spectroscopic studies of carbonyl horseradish peroxidases.

Infrared difference spectra, FeIIICO vs. FeIII of horseradish peroxidase isoenzymes A2 and C were recorded from 2000 to 1800 cm-1. Under alkaline conditions, pH 9, both isoenzymes exhibit two CO stretching bands, at 1938 and 1925 cm-1 for A2 and at 1933 and 1929 cm-1 for C. As the pH is lowered the low-frequency band for each isoenzyme decreases in intensity with a concommitant appearance and increase in intensity of a band at 1906 and 1905 cm-1 for the A2 and C isoenzymes, respectively. These changes conform to pK values of 6.7 for the A2 and 8.8 for the C isoenzymes of horseradish peroxidase. The interpretation of the infrared results was simplified by the observation that a linear relationship exists between the redox potential, Em7, for the FeIII/FeII system vs. the infrared CO stretching frequency, vCO, for cytochrome a3, hemoglobin, myoglobin, and cytochrome P-450 cam with substrate. This relationship suggests that the primary force altering vCO in these heme proteins is a variation in electron density at the heme iron and not direct protein interactions with the CO ligand. The horseradish peroxidase infrared bands in the 1930-cm-1 region correlate well with this relationship. The large deviation of the 1905-cm-1 band from the linear relationship and its dependence upon hydrogen ion concentration are consistent with horseradish peroxidase having a single CO binding site which can hold in two geometries, one of which contains an amino acid moiety capable of forming a hydrogen bond to the carbonyl oxygen.     

Compound X. An intermediate in enzymatic halogenation.

Previous studies have shown that chlorite serves as a halogenation substrate for horseradish peroxidase. In its substrate role, chlorite serves both as a halogen donor and as a source of oxidizing equivalents in the chlorination reaction. We now show that a new spectral intermediate, which we have termed Compound X, can be detected as the initial product of the reaction of chlorite with horseradish peroxidase. The reaction of chlorite with horseradish peroxidase to form Compound X is a relatively fast reaction especially at acidic pH values. The second order rate constant (Kf) for the formation of Compound X at pH 4.5 (optimum pH) is 0.9 X 10(6) M-1 S-1. Compound X, in the absence of a halogen acceptor, decomposes to Compound I and chloride ion. The first order rate constant (Kd) for the decay of Compound X to Compound I is 0.2 s-1 at pH 4.5. The pH optimum for enzymatic chlorination with chlorite compares favorably with the pH profile for the lifetime of Compound X (Kf/Kd). These observations indicate that Compound X is the halogenating intermediate in the chlorite reaction and that the rate of enzymatic chlorination is directly related to the stability of Compound X. We propose an -OCl ligand on a ferric heme as the most likely structure for Compound X.