The formation of ferric haem during low-temperature photolysis of horseradish peroxidase Compound I.

Illumination at low temperature of the peroxide compound of horseradish peroxidase (HRP-I) causes partial conversion of the haem electronic structure from a ferryl-porphyrin radical species into a low-spin ferric state. Magnetic-c.d. (m.c.d.) and e.p.r. spectral features of the photolysis product are almost identical with those of the alkaline form of ferric HRP, proposed on the basis of its near-i.r. m.c.d. spectrum to be a Fe(III)-OH species. The ferric product of HRP-I photolysis also contains free-radical e.p.r. signals. Conversion of HRP-I into the Fe(III)-OH species, which requires transfer of a proton and two electrons from the protein, is shown to be a two-step process.     

CO recombination in cytochrome c peroxidase: effect of the local heme environment on CO binding explored through site-directed mutagenesis

CO recombination to the cloned cytochrome c peroxidase [CCP(MI)] and mutants of CCP(MI) prepared by site-directed mutagenesis was examined as a function of pH by flash photolysis. The mutants examined included distal Arg 48----Leu, Lys; proximal Asp 235----Asn; and His 181----Gly. At alkaline pH, ferrous CCP(MI) was converted to a hexacoordinate form by a cooperative two-proton ionization, apparent pK(a) = 8.0. This change was observed in all of the mutants, although in the His 181----Gly mutant, the conversion to the hexacoordinate form was the result of a single-proton ionization, implicating His 181 as one of the two residues deprotonated in this isomerization. The pH-dependent conversion of CO ferrous CCP(MI) from acidic to alkaline forms was also observed and was similar to that reported for cytochrome c peroxidase from bakers' yeast [Iizuka, T., Makino, R., Ishimura, Y., & Yonetani, T. (1985) J. Biol. Chem. 260, 1407-1412]. Photolysis of the acidic form of the CO complex of CCP(MI) produces a kinetic form of the ferrous enzyme (form A) which exhibits the slow rate of CO recombination (l1' approximately 10(3) M-1 s-1) characteristic of peroxidases, while photolysis of the alkaline form of the CO complex produces a second kinetic form (form B), which exhibits a much faster rate of recombination (l2' approximately 10(5) M-1 s-1). Kinetic forms analogous to forms A and B were observed in all of the mutants examined. A third kinetic form (form B*) with a bimolecular rate constant l3' approximately 10(6) M-1 s-1 was also observed in the mutants at alkaline pH. Although the pH dependence for the conversion of form A to form B with increasing pH was altered by changes in the local heme environment, the rate of CO recombination by the respective forms was not dramatically altered in the mutants. Transient spectra of the reaction of CO with ferrous CCP(MI) after photolysis show that equilibrium between penta- and hexacoordinate ferrous enzyme is rapid relative to CO recombination. The presence of the internal sixth ligand has no discernible effect on the observed rate of recombination, however. The results presented indicate that in CCP(MI) the rate of ligand binding is determined primarily by isomerization of the protein from a closed conformation at acidic pH to an open conformation at alkaline pH and that polar effects of proximal Asp 235 and distal Arg 48 are of minor significance in the rate of CO recombination in both conformations.     

Determination of Fe-ligand bond lengths and the Fe-N-O bond angles in soybean ferrous and ferric nitrosylleghemoglobin a using multiple-scattering XAFS analyses

The NO adducts of leghemoglobin (Lb) are implicated in biological processes, but only the adduct with ferrous Lb (Lb(II)NO) has been characterized previously. We report the first characterization of ferric nitrosylleghemoglobin (Lb(III)NO) and XAS experiments performed on frozen aqueous solutions of Lb(II)NO and Lb(III)NO at 10 K. The XANES and electronic spectra of the NO adducts are similar in shape and energies to the myoglobin (Mb) analogues. The environment of the Fe atom has been refined using multiple-scattering (MS) analyses of the XAFS data. For Lb(II)NO, the MS analysis resulted in an averaged Fe-N(p)(pyrrole) distance of 2.02 A, an Fe-N(epsilon)(imidazole) distance of 1.98 A, an Fe-N(NO) distance of 1.77 A, and an Fe-N-O angle of 147 degrees. The Fe-N(NO) distance and Fe-N-O angle obtained from the analysis of Lb(II)NO are in good agreement with those determined crystallographically for [Fe(TPP)(NO)] (TPP, tetraphenylporphyrinato), with and without 1-methylimidazole (1-MeIm) as the sixth ligand, and the MS XAFS structures reported previously for the myoglobin (Mb(II)NO) analogue and [Fe(TPP)(NO)]. The MS analysis of Lb(III)NO yielded an average Fe-N(p) distance of 2.00 A, an Fe-N(epsilon) distance of 1.89 A, an Fe-N(NO) distance of 1.68 A, and an Fe-N-O angle of 173 degrees. These bond lengths and angles are consistent with those determined previously for the myoglobin analogue (Mb(III)NO) and the crystal structures of the model complexes, [Fe(III)(TPP)(NO)(OH(2))](+) and [Fe(OEP)(NO)](+) (OEP, octaethylporphyrinato). The final XAFS R values were 16.1 and 18.2% for Lb(II)NO and Lb(III)NO, respectively.     

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.     

Iron-histidine stretching vibration in the deoxy state of insect hemoglobins with different O2 affinities and Bohr effects

Resonance Raman spectroscopy has been employed to detect the iron-proximal histidine stretching mode in deoxyhemoglobins from insect larvae of Chironomus thummi thummi (CTT). With the excitation of 413.1 nm, we observe a sharp and intense line in the 220-224 cm-1 region. The assignment of this line to the Fe-N epsilon (His) stretching mode was made on the basis of a 3-cm-1 shift upon 57Fe/54Fe isotope substitution. The Fe-N epsilon (His) vibration is used to monitor the possible changes in the Fe-N epsilon (His) bond strength (hence bone length) in the deoxy state of the monomeric (CTT I, III, and IV) and dimeric (CTT II) insect hemoglobins. As these hemoglobins differ in O2 affinity, off-rate and on-rate constants, and in the Bohr effect, they are excellent model systems for investigating the mechanism of protein control of the heme reactivity. Some of these hemoglobins (CTT III, IV, and II) are allosteric, exhibiting two interconvertible conformational states with high and low O2 affinity at high and low pH, respectively. The Fe-N epsilon (His) stretching frequency does not correlate with the O2 affinity, the on-rate and the off-rate constants for different hemoglobins, for different conformational states, and for modified hemoglobins with different heme peripheral groups. This vibrational mode is insensitive to deuteration of the heme vinyl groups. It is important to note that the Fe-N epsilon (His) bonds in the high pH (high-affinity) and the low pH (low-affinity) states are identical. This implies that the O2 molecule, prior to binding, "sees" identical binding sites. Thus, the difference in free energy changes upon O2 binding is manifested only in the oxy form.     

Heme-linked ionization and ligand binding produce identical changes of proximal heme stereochemistry in reduced horseradish peroxidase. Evidence for existence of two protein conformations

The visible and near infrared magnetic circular dichroism spectra of chemically reduced horseradish peroxidase at neutral and alkaline pH values and 5-coordinate protoheme-(2-methylimidazole) at pH 9.1 were compared at 4.2 K with those of photolysis products of their carbon monoxide complexes. From the results obtained we concluded that: (i) there are two protein conformations of HRP which determine the geometry of the Fe-N(His) bond; (ii) the transition from one conformation (heme stereochemistry) to another can be induced by either heme-linked ionization or ligand binding; (iii) a trigger mechanism for switching between two conformations has to exist.     

A comparison of the heme electronic states in equilibrium and nonequilibrium protein conformations of high-spin ferrous hemoproteins. Low temperature magnetic circular dichroism studies

The visible and near infrared magnetic circular dichroism (MCD) spectra of equilibrium high-spin ferrous derivatives of myoglobin, hemoglobin, horseradish peroxidase and mitochondrial cytochrome c oxidase at 15 K are compared with those of the corresponding proteins in nonequilibrium conformations produced by low-temperature photodissociation of CO-complexes of these proteins as well as of O2-complexes of myoglobin and hemoglobin. Over all the spectral region (450-800 nm) the intensities of MCD bands of hemoproteins studied in equilibrium conformation are shown to be strongly temperature-dependent, including a negative band at ca. 630 nm and positive bands at ca. 690 nm and at ca. 760 nm. In contrast to the absorption spectra, the low-temperature MCD spectra of high-spin ferrous hemoproteins differ significantly, reflecting the peculiarities in the heme iron coordination sphere which are created by a protein conformation. The MCD spectra reveal clearly the structural changes in the heme environment which occur on ligand binding. On the basis of assignment of d leads to d and charge-transfer transitions in the near infrared region the correlation is suggested between the wavelength position of the MCD band at approx. 690 nm and the value of iron out-of-plane displacement as well as between the location of the band at approx. 760 nm and the Fe-N epsilon (proximal histidine) bond strength (length) in equilibrium and nonequilibrium conformations of the hemoproteins studied. The high sensitivity of low-temperature MCD spectra to geometry at heme iron is discussed.     

Contribution of protein conformation to heme stereochemistry and reactivity. Low-temperature magnetic circular dichroism data

Visible and near infrared magnetic circular dichroism (MCD) spectra of heme proteins and enzymes as well as those of a protein-free heme bound to 2-methylimidazole were recorded and compared at 4.2 K in unrelaxed metastable and relaxed equilibrium heme stereochemistry. The relaxed and unrelaxed stereochemistries of a 5-coordinate ferrous heme were generated by chemical reduction of iron at room temperature before freezing the sample and by photolysis of CO or O2 complexes at 4.2 K, respectively. The results are discussed in terms of a protein contribution into energies of the Fe-N epsilon(His) and Fe-N(pyrrols) bonds and their change on a ligand binding. We observed and analyzed cases of weak (myoglobin, hemoglobin) and strong (leghemoglobin, peroxidases) constraints imposed by the protein conformation on the proximal heme stereochemistry by comparing the bond energies in proteins with those in the protoheme-(2-methylimidazole) model compound. The role of a protein moiety in modulating the ligand binding properties of leghemoglobin and the heme reactivity of horseradish peroxidase is discussed.     

Magnetic circular dichroism studies of the active site heme coordination sphere of exogenous ligand-free ferric cytochrome c peroxidase from yeast: effects of sample history and pH

Electronic absorption and magnetic circular dichroism (MCD) spectroscopic data at 4 degrees C are reported for exogenous ligand-free ferric forms of cytochrome c peroxidase (CCP) in comparison with two other histidine-ligated heme proteins, horseradish peroxidase (HRP) and myoglobin (Mb). In particular, we have examined the ferric states of yeast wild-type CCP (YCCP), CCP (MKT) which is the form of the enzyme that is expressed in and purified from E. coli, and contains Met-Lys-Thr (MKT) at the N-terminus, CCP (MKT) in the presence of 60% glycerol, lyophilized YCCP, and alkaline CCP (MKT). The present study demonstrates that, while having similar electronic absorption spectra, the MCD spectra of ligand-free ferric YCCP and CCP (MKT) are somewhat varied from one another. Detailed spectral analyses reveal that the ferric form of YCCP, characterized by a long wavelength charge transfer (CT) band at 645 nm, exists in a predominantly penta-coordinate state with spectral features similar to those of native ferric HRP rather than ferric Mb (His/water hexa-coordinate). The electronic absorption spectrum of ferric CCP (MKT) is similar to those of the penta-coordinate states of ferric YCCP and ferric HRP including a CT band at 645 nm. However, its MCD spectrum shows a small trough at 583 nm that is absent in the analogous spectra of YCCP and HRP. Instead, this trough is similar to that seen for ferric myoglobin at about 585 nm, and is attributed (following spectral simulations) to a minor contribution (< or = 5%) in the spectrum of CCP (MKT) from a hexa-coordinate low-spin species in the form of a hydroxide-ligated heme. The MCD data indicate that the lyophilized sample of ferric YCCP (lambda CT = 637 nm) contains considerably increased amounts of hexa-coordinate low-spin species including both His/hydroxide and bis-His species. The crystal structure of a spectroscopically similar sample of CCP (MKT) (lambda CT = 637 nm) solved at 2.0 A resolution is consistent with His/hydroxide coordination. Alkaline CCP (pH 9.7) is proposed to exist as a mixture of hexa-coordinate, predominantly low-spin complexes with distal His 52 and hydroxide acting as distal ligands based on MCD spectral comparisons.     

Resonance Raman spectroscopy study of change of iron spin state in horseradish peroxidase C induced by removal of calcium

Resonance Raman spectroscopy is used to probe the effect of calcium depletion on the heme group of horseradish peroxidase C at pH 8. Polarized Raman spectra are recorded with an argon ion laser at eight different wavelengths to provide a sound database for a reliable spectral decomposition. Upon calcium depletion, the spectrum is indicative of a predominantly pentacoordinated high spin state of the heme iron coexisting with small fractions of hexacoordinated high and low spin states. The dominant quantum mixed spin state of native ferric horseradish peroxidase, which is characteristic for class III peroxidases, is not detectable in the spectrum of the enzyme with partial distal Ca(2+) depletion. The quenching of the quantum mixed spin state and the predominance of the pentacoordinated high spin state are likely to arise from distortions induced by distal calcium depletion, which translates into a weaker Fe-N(epsilon)(His) bond and a more tilted imidazole. A correlation is proposed between the lower enzyme activity and the elimination of the pentacoordinated quantum mixed state upon Ca(2+) depletion.     

Purification, characterization and stability of barley grain peroxidase BP 1, a new type of plant peroxidase

The major peroxidase of barley grain (BP 1) has enzymatic and spectroscopic properties that are very differeant from those of other known plant peroxidases (EC 1.11.1.7) and can therefore contribute to the understanding of the many physiological functions ascribed to these enzymes. To study the structure-function relationships of this unique model peroxidase, large-scale and Jaboratory-scale purifications have been developed. The two batches of pure BP 1 obtained were identical in their enzymatic and spectral properties, and confirmed that BP 1 is different from the prototypical horseradish peroxidase isoenzyme C (HRP C). However, when measuring the specific activity of BP 1 at pH 4.0 in the presence of 1 m M C_aCl₂, the enzyme was as competent as HRP C at neutral pH towards a variety of substrates (m M mg⁻¹ min⁻¹): coniferyl alcohol (930±48), caffeic acid (795±53), ABTS (2,2'-azino-di-[3-ethyl-benzothiazoline-(6)-sulfonic acid]) (840±47), ferulic acid (415±20), p -coumaric acid (325±12), and guaiacol (58±3). The absorption spectrum of BP 1 is blue-shifted compared to that of HRP C with a Soret maximum of 399–402 nm, depending on pH. The prosthetic group was shown to be iron-protoporphyrin IX, which is characteristic of plant peroxidases. BP 1 is stable from pH 3 to 11, indicating that its unusual spectral characteristics do not result from enzyme instability. The thermostability is also normal with a melting temperature of 75°C at pH 6.6, and 67°C at pH 4.0 and 8.3. It is clear that the unusual properties of BP 1 are genuine, and reflect a novel regulation of plant peroxidase function.     

Raman and infrared spectra of cytochrome c peroxidase-carbon monoxide adducts in alternative conformational states

Resonance Raman (RR) spectra are reported for CO-bound cytochrome c peroxidase (CCP). At low pH, two forms are observed: form II, with nu Fe-C = 530 cm-1 and delta FeCO = 585 cm-1, and form I, with nu Fe-C = 495 cm-1 and no detectable delta FeCO. They appear to have coincident nu CO infrared bands, at 1922 cm-1. These low-pH forms, similar to those observed for horseradish peroxidase (HRP), are attributed to tilted, H-bonded CO and perpendicular CO, respectively. The frequencies differ between the two proteins, a weaker H bond to CO being indicated for CCP. As with HRP, the equilibrium between forms I and II is shifted toward the latter at increasing CO concentrations, suggesting that secondary binding of CO perturbs the distal residues. At high pH [8.4, tris(hydroxymethyl)aminomethane buffer] the form II fraction converts to another form, II', with nu FeC = 503 cm-1, delta FeCO = 575 cm-1, and nu CO = 1948 cm-1; a tilted, non-H-bonded geometry is suggested. If phosphate buffer is used, however, form II (H bonded) persists at pH 8.4. This result establishes a role for phosphate in stabilizing the H-bonded form of the enzyme; it is suggested that phosphate binds near the distal imidazole and substantially increases its pKa. The conformational state is also influenced by aging. Fresh protein contains purely high spin FeIII heme, as monitored by the high-frequency RR spectrum, and yields form II almost exclusively at elevated CO concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Determination of the nature of the heme environment in nitrosyl indoleamine 2,3-dioxygenase using Multiple-scattering analyses of X-ray absorption fine structure

Multiple-scattering analysis of X-ray absorption fine structure data on the NO adducts of indoleamine 2,3-dioxygenase (IDO) and analysis of X-ray absorption near-edge structure (XANES) have provided the first direct structural information about the iron center for this ubiquitous mammalian metalloprotein. The IDO(II)NO adduct, which is likely to play a physiological role in the immune system, differs from similar adducts such as Mb(II)NO and Lb(II)NO in that the Fe-His bond is essentially broken. At 10 K, the Fe-N(p)(av) bond length = 2.00(2) A, Fe-NO bond length = 1.75 A, and angle = 140 degrees, which are typical of five-coordinate Fe(II)NO species. The XANES is also closer to that of five-coordinate model complexes than six-coordinate species. In addition to the Fe(II)NO species, there was a minor component of the Fe(III)NO adduct because of incomplete reduction of the Fe(II) species. This was also a five-coordinate center and consists of a linear Fe(II)NO(+) moiety with the Fe-N(p)(av) bond length = 2.00(2) A, Fe-NO bond length = 1.63(3) A, and angle = 179 degrees. The results indicate that both the blocking of the heme site to O(2) binding and conformational changes induced by breaking the Fe-N(epsilon) bond may be important mechanisms by which NO inhibits IDO in vitro and in vivo.     

The nature of species prepared by photolysis of half-reduced, fully reduced and fully reduced carbonmonoxy-cytochrome c-551 peroxidase from Pseudomonas aeruginosa.

The half-reduced, fully reduced and fully reduced CO-bound forms of the enzyme cytochrome c-551 peroxidase isolated from Pseudomonas aeruginosa were examined by a combination of low-temperature absorption and magnetic-circular-dichroism spectroscopy. Deliberate low-temperature (4.2K) photolysis of these forms of the enzyme, in all of which the high-potential haem is in the ferrous state, revealed that this haem group, assigned to have a histidine-methionine ligand set, is photosensitive. The photolabile ligand is most likely to be the methionine residue, and the product of photolysis, namely the high-spin (S = 2) ferrous form, is stable at low temperature (4.2K). Warming to approx. 20K allows thermal recombination to occur, restoring the low-spin (S = 0) state. The low-potential haem (bis-histidine ligation) is photoinert in both ferric and ferrous states; however, the photosensitive CO adduct of this centre cannot be maintained as the photolysed (S = 2) product at 4.2K. This surprising observation may be due to quantum-mechanical tunnelling of the CO through the activation barrier even at 4.2K, implying that the activation barrier to thermal recombination is both narrow and low. Low-temperature absorption spectroscopy reveals that the high-potential haem has a very characteristic low-spin ferrous spectrum with intense highly structured beta- and split alpha-bands, whereas the spectrum of the low-potential ferrous haem contains alpha- and beta-bands devoid of fine structure.     

Some properties of human eosinophil peroxidase, a comparison with other peroxidases

Eosinophil peroxidase (donor:hydrogen peroxide oxidoreductase, EC 1.11.1.7) was isolated from outdated human white blood cells. The purified enzyme has a molecular weight of 71000 +/- 1000. The enzyme is composed of two subunits, of Mr 58000 and 14000, in a 1:1 stoichiometry. Amino-acid analyses showed that eosinophil peroxidase has a high content of the amino acids arginine, leucine and aspartic acid. The millimolar absorbance coefficient of the Soret band at 412 nm of eosinophil peroxidase was determined. Three independent methods yield a value for epsilon 412nm of 110 +/- 4 mm-1 X cm-1. Purified eosinophil peroxidase showed a homogeneous high-spin EPR signal with rhombic symmetry (gx = 6.50; gy = 5.40; gz = 1.982) for the haem group. EPR spectroscopy of low-spin cyanide and azide derivatives of eosinophil peroxidase, lactoperoxidase, myeloperoxidase and catalase revealed that the haem-ligand structure of eosinophil peroxidase is closely related to lactoperoxidase, whereas that of myeloperoxidase shows great resemblance to catalase.     

CO dissociation in cytochrome c peroxidase: site-directed mutagenesis shows that distal Arg 48 influences CO dissociation rates

To investigate the molecular basis for the 100-fold slower rate of CO dissociation in ferrous peroxidases relative to myoglobin, CO dissociation rates were measured as a function of pH in the cloned cytochrome c peroxidase from yeast [CCP(MI)] and in several mutants in the heme binding pocket prepared by site-directed mutagenesis. The mutants included Asp 235----Asn; Arg 48----Lys, Leu; and His 181----Gly. Changes in the absorption spectrum with pH are consistent with conversion of the CO-ferrous CCP(MI) complex from acidic to alkaline forms by a two-proton cooperative ionization, with an apparent pKa = 7.6, analogous to that described for CCP from bakers' yeast [Iizuka, T., Makino, R., Ishimura, Y., & Yonetani, T. (1985) J. Biol. Chem. 260, 1407-1412]. The rate of CO dissociation (koff) was increased 11-fold (from 0.7 x 10(-4) to 8.0 x 10(-4) s-1) by conversion of the acidic to the alkaline form. Analogous acidic and alkaline forms of the CO complex were also observed in the mutants of CCP(MI) examined here. In the acidic form, koff was increased 5- and 20-fold when Arg 48 was replaced with Lys and Leu, respectively, while in the acidic form of mutants that possess Arg 48, koff was similar to that observed in CCP(MI). Conversion of the CO complex from the acidic to alkaline form increased koff in all the mutants, and the pH-dependent increase in koff correlated with a two-proton cooperative ionization, except in the case of His 181----Gly. In this mutant, pH-dependent increase in koff correlated with a single-proton ionization, implicating His 181 as one of the two residues that is deprotonated in the conversion of CO-ferrous CCP(MI) from acidic to alkaline forms. Only a 2.5-fold variation was observed for koff between the alkaline form of CCP(MI) and the Arg 48----Leu mutant, suggesting that the influence of Arg 48 on the rate of CO dissociation is decreased in the alkaline form by a conformational change.(ABSTRACT TRUNCATED AT 400 WORDS)     

A novel mechanism of heme-heme interaction in the homodimeric hemoglobin from Scapharca inaequivalvis as manifested upon cleavage of the proximal Fe-N epsilon bond at low pH

The CO-binding kinetics and the optical spectra of the NO derivative of the homodimeric hemoglobin from Scapharca inaequivalvis have been investigated over the range between pH 7.0 and 2.0. In the deoxygenated derivative, protonation of the proximal imidazole at very low pH values and the consequent cleavage of the Fe-N epsilon bond result in a approximately 50-fold enhancement of the rate constant for CO binding, as found in other hemoproteins. However, in the case of the hemoglobin from S. inaequivalvis, the pH profile displays a cooperative behavior (n = 1.8 +/- 0.1), a unique feature that differentiates this protein from any other hemoprotein investigated thus far. Cleavage of the proximal bond in the NO derivative of S. inaequivalvis hemoglobin likewise displays a very steep pH transition. The mode of assembly of the homodimer, in which the heme-carrying E and F helices provide the subunit interface and bring the hemes at a much shorter distance (18.4 A) than in vertebrate hemoglobins, is likely to provide the structural basis for this unique behavior.     

Horseradish peroxidase C

Summary Horseradish peroxidase C (HRP; ferric) reacts with H₂O₂ to form Compound I, with an equilibrium constant of about 10¹⁴ M^–1. Two-step reduction of Compound I to Compound II and further to the ferric enzyme occurs reversibly at E_o values of 0.90 and 0.93 V (pH 7.0), respectively. The pH dependence of E_o values for each one-electron step, ferrous ferric Compound II Compound I indicates the presence of redox-linked ionization at pK_a values of 7.3 in the ferrous state, 11.0 in the ferric and 8.6 in Compound II. Zinc-substituted HRP C is oxidized to its free-radical form at an E_o value of 0.74 (pH 6.0). Comparison of oxidized zinc HRP C with Compound I shows that Compound I contains a porphyrin -cation radical. The flash photolysis study on the NO-ferric HRP C complex clearly indicates that the iron is pentacoordinated in HRP C while it is hexacoordinated in metmyoglobin. From the kinetic analysis of the acid-alkaline conversion of HRP C, the second-order rate constants of the reactions with H⁺ and HO^– are estimated to be 1.5 × 10¹⁰ and 6.7 × 10⁴ M^–1s^–1, respectively. The latter rate constant greatly varies with the kind of hemoproteins. In the presence of HRP C and O₂, indole-3-acetate is oxidized to its hydroperoxide form, which reacts effectively with HRP C to form Compound I and further converts Compound I to a verdohemoprotein.Abbreviations HRP horseradish peroxidase (HRP without subgroup letter denotes a classical preparation consisting of HRP B and HRP C) - EPR electron paramagnetic resonance - NMR nuclear magnetic resonance     

Reversible acidic-alkaline transition of the carbon monoxide complex of cytochrome c peroxidase

The Soret absorption band of the ferrous carbon monoxide (CO) complex of cytochrome c peroxidase exhibited a blue shift from 423.7 to 420 nm upon an increase in pH from 6.5 to 8.5. The spectral change was reversible with an isosbestic point at 422 nm. The pH dependence of this spectral change gave a sigmoidal curve fitted well to a theoretical curve of a cooperative release of two protons with a pK value of 7.5, indicating the existence of the acidic and alkaline forms of the ferrous CO enzyme. Upon irradiation of light flash (100 J of power and 30-microseconds), the heme-bound CO was readily dissociated in both acidic and alkaline forms with a quantum yield of approximately unity. On the other hand, the rate of recombination of the dissociated CO with the heme iron was significantly different between these two forms; the recombination rate constants were 1.1 X 10(3) and 3.0 X 10(4) M-1 S-1 at 25 degrees C for the acidic and alkaline forms, respectively. At intermediate pH values, kinetics of recombination were biphasic, consisting of the slow and fast processes with the appropriate rate constants mentioned above. When the fraction of the fast process was plotted against pH, the pH profile coincided with the spectrophotometric pH titration curve described above. Thus, it was concluded that the acidic and alkaline forms of the enzyme were responsible for the slow and fast processes, respectively. In infrared spectroscopy, the acidic form showed a narrow CO stretching band at 1922 cm-1 with a half-band width of 12.5 cm-1, while the alkaline form exhibited a broad CO-stretching band at 1948 cm-1 with a half-band width of 33 cm-1. Significance of these results are discussed in relation to the structure of the heme vicinity on the CO complex of cytochrome c peroxidase.     

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.