The oxidation-reduction potentials of compound I/compound II and compound II/ferric couples of horseradish peroxidases A2 and C.

The reversibility of the stepwise reduction of Compound I to the ferric state via Compound II was confirmed in horseradish peroxidases A2 and C. The values of E'o (compound I/Compound II) and E'O (Compound II/ferric) were measured from equilibrium data coupled with the K2IrCl6-K3IrCl6 system in a narrow region of pH near 6.3. The ferric enzymes were also oxidized by ferricyanide to Compound II at alkaline pH and the values of E'O (Compound II/ferric) were measured from the equilibrium data. The pH dependence of E'O (Compound II/ferric) was in accord with the equation: E'O = EO + 0.058 log (Kr[H+] + [H+]2)/(KO + [H+]), where Kr and KO are proton dissociation constants in the ferric enzyme and Compound II, respectively. The pH-E'O (Compound I/Compound II) curves were likewise obtained from the equation, E'O = EO + 0.058 log (Kr + [H+]), where Kr is the proton dissociation constant in Compound II. The forward and backward rate constants were measured in each of one-electron transfer reactions of the peroxidases with the K2IrCl6-K3IrCl6 system at various pH values. The E'O values calculated on the assumption that the ratio of the rate constants equals the equilibrium constant were compared with those obtained from the equilibrium data.     

Proton magnetic resonance studies of peroxidases from turnip and horseradish.

Proton NMR spectra at 270 MHz have been measured for horseradish peroxidase and turnip peroxidase isoenzymes (P1, P2, P3 and P7) in both their high spin ferric native states and as the low spin ferric cyanide complexes. Resonances of amino acids near the heme have been identified and used to investigate variations in the structure of the heme crevice amongst the enzymes. Ligand proton resonances have been resolved in spectra of the cyanide complexes of the peroxidases and these provide information on the heme electronic structure. The electronic structure of the heme and the tertiary structure of the heme crevice are essentially the same in the acidic turnip isoenzymes, P1, P2 and, to a lesser extent, P3 but differ in the basic turnip enzyme, P7. The heme electronic structure and nature of the iron ligands in peroxidases are discussed. Further evidence is presented for histidine as the proximal ligand. A heme-linked ionizable group with a pK of 6.5 has been detected by NMR in the cyanide complex of horseradish peroxidase.     

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.     

Oxidation of horseradish peroxidase compound II to compound I.

In the reaction between equimolar amounts of horseradish peroxidase and chlorite, the native enzyme is oxidized directly to Compound II (Hewson, W.D., and Hager, L.P. (1979) J. Biol. Chem. 254, 3175-3181). At acidic pH but not at alkaline values, this initial reaction is followed by oxidation of Compound II to Compound I. The highly pH-dependent chemistry of Compound II can be readily demonstrated by the reduction of Compound I, with ferrocyanide at acidic, neutral, and alkaline pH values. Titration at low pH yields very little Compound II, whereas at high pH, the yield is quantitative. Similarly, the reaction of horseradish peroxidase and chlorite at low pH yields Compound I while only Compound II is formed at high pH. At intermediate pH values both the ferrocyanide reduction and the chlorite reaction produce intermediate yields of Compound II. This behavior is explained in terms of acidic and basic forms of Compound II. The acidic form is reactive and unstable relative to the basic form. Compound II can be readily oxidized to Compound I by either chloride or chlorine dioxide in acidic solution. The oxidation does not occur in alkaline solution, nor will hydrogen peroxide cause the oxidation of Compound II, even at low pH.     

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 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.     

Resonance Raman spectroscopy and density functional theoretical calculations of manganese corroles. A parallelism between high-valent metallocorroles and metalloporphyrins, relevant to horseradish peroxidase and chloroperoxidase compound I and II intermediates.

Soret-excited resonance Raman (RR) spectra are reported for the Mn(III) and Mn(IV)Cl derivatives of meso-tris(p-(trifluoromethyl)phenyl)corrole, H(3)T(p-CF(3)-P)Cor, and the Mn(III) derivative of beta-octabromo-meso-tris(p-(trifluoromethyl)phenyl)corrole, H(3)Br(8)T(p-CF(3)-P)Cor. Three high-frequency bands in the RR spectrum of Mn(III)[T(p-CF(3)-P)Cor] at 1465, 1524 and 1615 cm(-1) appear to upshift to 1486, 1528 and 1620 cm(-1) for Mn(IV)[T(p-CF(3)-P)Cor]Cl. This suggests that the electronic character of the corrole ligand is significantly different for these two compounds, which is consistent with electrochemical evidence for partial radical character of the corrole ligand for Mn(IV)[T(p-CF(3)-P)Cor]Cl but not for Mn(III)[T(p-CF(3)-P)Cor]. The observed upshifts are also consistent with DFT calculations showing a shortening of some of the relevant bonds in the Mn(IV)Cl derivative relative to the Mn(III) derivative. The results raise the possibility of an extensive parallelism between the electronic structures of high-valent metallocorroles and metalloporphyrins. Three high-frequency bands in the RR spectrum of Mn(III)[T(p-CF(3)-P)Cor] at 1331, 1465 and 1545 cm(-1) appear to downshift to 1320, 1457 and 1537 cm(-1) for Mn(III)[Br(8)T(p-CF(3)-P)Cor]. This is consistent with the suspected longer carbon-carbon bond lengths in the brominated corrole macrocycle.     

Time-resolved and static resonance Raman spectroscopy of horseradish peroxidase intermediates

By using pulsed and continuous wave laser irradiation in the 350-450-nm region, we have characterized Raman scattering from horseradish peroxidase (HRP) compounds I and II and from iron porphyrin pi-cation radical model compounds. For compound II we support the suggestion [Terner, J., Sitter, A. J., & Reczek, C. M. (1985) Biochim. Biophys. Acta 828, 73-80; Proniewicz, L. M., Bajdor, K., & Nakamoto, K. (1986) J. Phys. Chem. 90, 1760-1766] that resonance enhancement of the FeIV = O vibration proceeds by way of a charge-transfer state. Our excitation profile data locate this state at approximately 400 nm. Compound I was prepared at neutral pH by rapid mixing of the resting enzyme with hydrogen peroxide. Each sample aliquot was excited by a single, 10-ns laser pulse to generate the Raman spectrum; optical spectroscopy following the Raman measurement confirmed that HRP-I was the principal product during the time scale of the measurement. The Raman spectrum of this species, however, is not characteristic of that which we observe from metalloporphyrin pi-cation radicals [Oertling, W. A., Salehi, A., Chung, Y., Leroi, G. E., Chang, C. K., & Babcock, G. T. (1987) J. Phys. Chem. 91, 5887-5898], including the iron porphyrin cation radicals reported here. Instead, the spectrum recorded for HRP-I at neutral pH is suggestive of an oxoferryl heme with the same geometric and electronic structure as that of HRP-II at high pH.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.+].     

A resonance Raman study on the structures of complexes of flavoprotein D-amino acid oxidase

Resonance Raman (RR) spectra were obtained for the purple complexes of D-amino acid oxidase (DAO) with D-lysine or N-methylalanine. RR spectra of a complex of oxidized DAO with the oxidation product of D-lysine or D-proline were also measured. The isotope shifts of the observed bands of the purple complex with D-lysine upon 13C- or 15N-substitution of lysine indicate that the ligand is delta 1-piperideine-2-carboxylate. That the band at 1671 cm-1 for the purple intermediate with N-methylalanine shifts to 1666 cm-1 in D2O solution indicates that the imino acid, N-methyl-alpha-iminopropionate, has a protonated imino group. Many bands due to a ligand in the RR spectra of the complex of oxidized DAO with an oxidation product can be observed below 1000 cm-1, but no band for the purple complex is seen in this frequency region. The band associated with the CO2-symmetric stretching mode of the product, such as delta 1-piperideine-2-carboxylate or delta 1-pyrrolidine-2-carboxylate, complexed with the oxidized DAO shifts in D2O solution. This suggests that the product imino acid interacts with the enzyme through some proton(s).     

Heterogeneity and regulation of manganese peroxidases from Phanerochaete chrysosporium.

Lignin and Mn peroxidases are two families of isozymes produced by the lignin-degrading fungus Phanerochaete chrysosporium under nutrient nitrogen or carbon limitation. We purified to homogeneity the three major Mn peroxidase isozymes, H3 (pI = 4.9), H4 (pI = 4.5), and H5 (pI = 4.2). Amino-terminal sequencing of these isozymes demonstrates that they are encoded by different genes. We also analyzed the regulation of these isozymes in carbon- and nitrogen-limited cultures and found not only that the lignin and Mn peroxidases are differentially regulated but also that differential regulation occurs within the Mn peroxidase isozyme family. The isozyme profile and the time at which each isozyme appears in secondary metabolism differ in both nitrogen- and carbon-limited cultures. Each isozyme also responded differently to the addition of a putative inducer, divalent Mn. The stability of the Mn peroxidases in carbon- and nitrogen-limited cultures was also characterized after cycloheximide addition. The Mn peroxidases are more stable in carbon-limited cultures than in nitrogen-limited cultures. They are also more stable than the lignin peroxidases. These data collectively suggest that the Mn peroxidase isozymes serve different functions in lignin biodegradation.     

Non-involvement of lignin peroxidases and manganese peroxidases in 2,4,5-trichlorophenoxyacetic acid degradation byPhanerochaete chrysosporium

Summary Phanerochaete chrysosporium (ME-446) mineralized 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) in high N medium and in malt extract medium in which lignin peroxidases (LIPs) and manganese peroxidases (MNPs) are not produced; furthermore,per mutant of ME-446, which lacks LIPs and MNPs, mineralized 2,4,5-T as well as the wild type. These results indicate that LIPs and MNPs are not required for 2,4,5-T degradation byP. chrysosporium.     

Native azurin and its Ni(II) derivative: a resonance Raman study.

Resonance Raman spectra are reported for native Cu(II) $\text{Pseudomonas}$$\text{aeruginosa}$ azurin and its Ni(II) substituted derivative. The spectrum of the native azurin includes a low frequency feature and bands in the first overtone region not previously reported. The spectrum of the Ni(II) derivative exhibits three major peaks in the metal-ligand stretching region shifted to lower frequency relative to the M-L peaks in the spectrum of native azurin. Resonance enhanced ligand modes are observed which indicate that at least two of the ligands in Ni(II) azurin (cysteine and at least one histidine) are the same as in native azurin. The data also suggest that the disposition of ligands about the metal may be more nearly tetrahedral in the Ni(II) derivative than in native azurin.     

Antenna chlorophyll in photosynthetic membranes. A study by resonance Raman spectroscopy

Raman spectra of antenna chlorophyll a and chlorophyll b were selectively obtained from chloroplasts of green plants and from monocellular algae, using resonance enhancement in the respective Soret bands of these molecules, at 35 K. It is shown that:

Antenna chlorophyll a molecules occur in at least five discrete categories, distinguished by different extramolecular bonding of their 9-keto carbonyl groups.

These vibrational categories are probably identical in nature and number among the different organisms studied, but differ in their relative populations.

Chlorophyll b molecules occur in at least two different categories differing by the strength of the interactions of their 3-formyl C = 0 groups. These vibrational categories also appear as universal.

Most chlorophyll a and b molecules have their magnesium atoms bound to a single foreign ligand, whose nature may depend on the population considered.

Resonance Raman spectra of antenna structures, including those of organisms devoid of chlorophyll b, were compared to resonance Raman spectra of chlorophyll a and b in monomeric, oligomeric and hydrated polymeric states, at room temperature and at 35 K. No sizable amount of antenna chlorophyll a or b occurs as dry or hydrated oligomers, or polymers. The antenna molecules are thus necessarily bound to foreign molecules, probably proteins, through H-bonding on their formyl and/or keto carbonyl groups and through bonding of their magnesium atoms.