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.     

Myeloperoxidase-catalyzed taurine chlorination: initial versus equilibrium rate

Myeloperoxidase (MPO) catalyzes the two-electron oxidation of chloride, thereby producing hypochlorous acid (HOCl). Taurine (2-aminoethane-sulfonic acid, Tau) is thought to act as a trap of HOCl forming the long-lived oxidant monochlorotaurine [(N-Cl)-Tau], which participates in pathogen defense. Here, we amend and extend previous studies by following initial and equilibrium rate of formation of (N-Cl)-Tau mediated by MPO at pH 4.0-7.0, varying H(2)O(2) concentration. Initial rate studies show no saturation of the active site under assay conditions (i.e. [H(2)O(2)] > or = 2000 [MPO]). Deceleration of Tau chlorination under equilibrium is quantitatively described by the redox equilibrium established by H(2)O(2)-mediated reduction of compound I to compound II. At equilibrium regime the maximum chlorination rate is obtained at [H(2)O(2)] and pH values around 0.4mM and pH 5. The proposed mechanism includes known acid-base and binding equilibria taking place at the working conditions. Kinetic data ruled out the currently accepted mechanism in which a proton participates in the molecular step (MPO-I+Cl(-)) leading to the formation of the chlorinating agent. Results support the formation of a chlorinating compound I-Cl(-) complex (MPO-I-Cl) and/or of ClO(-), through the former or even independently of it. ClO(-) diffuses away and rapidly protonates to HOCl outside the heme pocket. Smaller substrates will be chlorinated inside the enzyme by MPO-I-Cl and outside by HOCl, whereas bulkier ones can only react with the latter.     

Pre-steady-state kinetics of intermediate formation in the deuteroferriheme-hydrogen peroxide system.

The pH dependence of formation of a peroxidatic intermediate from the reaction of deuteroferriheme with hydrogen peroxide has been determined for the region pH 8.7-10.1 from stopped-flow kinetic studies in which absorbancy changes are observed at heme monomer-dimer isosbestic points. Results are interpreted primarily in terms of the attainment of double "steady-state" concentrations of Michaelis-Menten complex I and peroxidatic intermediate I'. A linear correlation of observed first-order rate constants with alpha, the degree of dissociation of heme dimer, has been demonstrated and nonzero intercepts are obtained. Slopes and intercepts show a linear logarithmic dependence on pH which is interpreted in terms of HO2-participation both in the formation and subsequent (catalatic) decomposition of a peroxidatically active intermediate. General acid catalysis of intermediate formation is indicated from studies in phosphate, arsenate, and citrate buffer at pH 7.4-9.3. It is suggested that such catalysis may be responsible for anomalously high rates of H2O2 decomposition previously observed in phosphate buffer solution.     

Chloroperoxidase, a janus enzyme

Chloroperoxidase is a versatile fungal heme-thiolate protein that catalyzes a variety of one-electron and two-electron oxidations. We report here that the alkylation of an essential histidine residue showed no effect on the one-electron peroxidations but inhibited two-electron oxidations. The pH profiles of different peroxidative substrates showed optimal activities at varying pH values for the same enzyme. 2-Allylphenol and substituted ortho-phenolics showed efficient peroxidations. Also, substrates excluded from the active site (or with no favorable positioning at the heme center or heme edge) were converted in the peroxidation reaction. While hydrogen peroxide serves as the superior activator in the two-electron oxidations, small alkylhydroperoxides give much better rates for peroxidation reactions. All the above observations indicate that one-electron oxidations are mechanistically quite different from the two-electron oxidations catalyzed by chloroperoxidase. We propose that the peroxidatic substrates interact predominantly outside the heme active site, presumably at the surface of the enzyme.     

Characterization of equilibrium intermediates in denaturant-induced unfolding of ferrous and ferric cytochromes c using magnetic circular dichroism, circular dichroism, and optical absorption spectroscopies

Protein unfolding during guanidine HCl denaturant titration of the reduced and oxidized forms of cytochrome c is monitored with magnetic circular dichroism (MCD), natural CD, and absorption of the heme bands and far-UV CD of the amide bands. Direct MCD spectral evidence is presented for bis-histidinyl heme ligation in the unfolded states of both the reduced and oxidized protein. For both redox states, the unfolding midpoints measured with MCD, which is an indicator of tertiary structure, are significantly lower than those measured with far-UV CD, an indicator of secondary structure. The disparate titration curves are interpreted in terms of a compound mechanism for denaturant-induced folding and unfolding involving a molten globulelike intermediate state (MG) with near-native secondary structure and nonnative tertiary structure and heme ligation. A comparison of the dependence of the free energy of formation of the MG intermediate on the redox state with the known contributions from heme ligation and solvation suggests that the heme is significantly more accessible to solvent in the MG intermediate than it is in the native state.     

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.     

The energy level scheme for the ferryl heme in compound II of the peroxidase-catalase family as determined from analysis of low-temperature magnetic circular dichroism

The expressions for temperature-dependent magnetic circular dichroism (MCD) of the ferryl heme (Fe(4+)Por, S=1), which is a model of an intermediate product of the catalytic cycle of heme enzymes (compound II), have been derived in the framework of a two-term model. Theoretical predictions for the temperature and magnetic field dependence of MCD intensity of the ferryl heme are compared with those of the high-spin and low-spin ferric heme. Analysis of reported MCD spectra of myoglobin peroxide [Foot et al., Biochem. J. 2651 (1989) 515-522] and compound II of horseradish peroxidase [Browett et al., J. Am. Chem. Soc. 110 (1987) 3633-3640] has shown the presence in the samples of approximately 1% of a low-spin ferric component, which, however, should be taken into account in simulating observed temperature dependences of MCD intensity. The values of two adjustable parameters are estimated from the fit of the observed and simulated plots of MCD intensity against the reciprocal of the absolute temperature. One of them, the energy gap between the ground and excited terms, predetermines the axial zero-field splitting. The other parameter is correlated with the energy of splitting of excited quartets arising from either the porphyrin pi-->pi* transition or the spin-allowed charge-transfer transition.     

Myeloperoxidase-catalyzed chlorination: the quest for the active species

Myeloperoxidase (MPO) is a dominating enzyme of circulating polymorphonuclear neutrophils that catalyzes the two-electron oxidation of chloride, thereby producing the strong halogenating agent hypochlorous acid (ClO⁻/HOCl). In absence of MPO the tripeptide Pro-Gly-Gly reacts with HOCl faster than the amino acid taurine (2-aminoethanesulfonic acid, Tau), while the MPO-mediated chlorination shows reverse order. A comparative study of the enzymatic oxidation of both substrates at pH 4.0–6.0, varying H₂O₂ concentration is presented. Initial and equilibrium rates studies have been carried on, reaction rates in the latter being slower due to the chemical equilibrium between MPO-I and MPO-II–HO₂. A maximum of chlorination rate is observed for Pro-Gly-Gly and Tau when [H₂O₂] ≈ 0.3–0.7 mM and pH ≈ 4.5–5.0. Several mechanistic possibilities are considered, the proposed one implies that chlorination takes place via two pathways. One, for bulkier substrates, involves chlorination by free HOCl outside the heme cavity; ClO⁻ is released from the active center, diffuses away the heme cavity, and undergoes protonation to HOCl. The other implies the existence of compound I–Cl⁻ complex (MPO-I–Cl), capable of chlorinating smaller substrates in the heme pocket. Electronic structure calculations show the size of Pro-Gly-Gly comparable to the available gap in the substrate channel, this tripeptide being unable to reach the active site, and its chlorination is only possible by free HOCl outside the enzyme.     

Magnetic circular dichroism studies of hepatic microsomal cytochrome P-450.

Magnetic circular dichroism (MCD) spectra have been measured for cytochrome P-450 (P-450) purified from phenobarbital-induced rabbit liver microsomes. The temperature dependence of some of the MCD spectra has also been determined. The MCD spectrum of oxidized P-450 seems to suggest that it is in a state intermediate between the ferric low-spin states. Model experiments suggest that this anomaly arises from the coordination of a thiolate anion to the heme. Reduced P-450 shows a very peculiar MCD spectrum; the spectrum as well as its temperature dependence suggest that the heme in reduced P-450 is a "mixture" in terms of redox and/or spin states. The MCD spectrum of the CO complex of reduced P-450 exhibits an apparent Faraday A term around 450 nm which consists of about 50% C term and 50% the other terms, indicating that it is not in a purely ferrous low-spin state. The CO complex of reduced cytochrome P-420 (P-420), on the other hand, shows an MCD spectrum characteristic of a ferrous low-spin heme. It is suggested from model experiments that the thiolate anion coordinates to the heme trans to CO in the P-450-CO complex. The Soret region of the MCD spectrum of the EtNC complex of reduced P-450 is characterized by two apparent A terms around 430 and 455 nm, whereas that of the corresponding complex of P-420 has only one apparent A term around 434 nm.     

Calcium-dependent heme structure in the reduced forms of the bacterial cytochrome c peroxidase from Paracoccus pantotrophus

This work reports for the first time a resonance Raman study of the mixed-valence and fully reduced forms of Paracoccus pantotrophus bacterial cytochrome c peroxidase. The spectra of the active mixed-valence enzyme show changes in the structure of the ferric peroxidatic heme compared to the fully oxidized enzyme; these differences are observed upon reduction of the electron-transferring heme and upon full occupancy of the calcium site. For the mixed-valence form in the absence of Ca(2+), the peroxidatic heme is six-coordinate and low-spin on the basis of the frequencies of the structure-sensitive Raman lines: the enzyme is inactive. With added Ca(2+), the peroxidatic heme is five-coordinate high-spin and active. The calcium-dependent spectral differences indicate little change in the conformation of the ferrous electron-transferring heme, but substantial changes in the conformation of the ferric peroxidatic heme. Structural changes associated with Ca(2+) binding are indicated by spectral differences in the structure-sensitive marker lines, the out-of-plane low-frequency macrocyclic modes, and the vibrations associated with the heme substituents of that heme. The Ca(2+)-dependent appearance of a strong gamma 15 saddling-symmetry mode for the mixed-valence form is consistent with a strong saddling deformation in the active peroxidatic heme, a feature seen in the Raman spectra of other peroxidases. For the fully reduced form in the presence of Ca(2+), the resonance Raman spectra show that the peroxidatic heme remains high-spin.     

Modulation of the activities of catalase-peroxidase HPI of Escherichia coli by site-directed mutagenesis

Catalase-peroxidases have a predominant catalatic activity but differ from monofunctional catalases in exhibiting a substantial peroxidatic reaction which has been implicated in the activation of the antitubercular drug isoniazid in Mycobacterium tuberculosis. Hydroperoxidase I of Escherichia coli encoded by katG is a catalase-peroxidase, and residues in its putative active site have been the target of a site directed-mutagenesis study. Variants of residues R102 and H106, on the distal side of the heme, and H267, the proximal side ligand, were constructed, all of which substantially reduced the catalatic activity and, to a lesser extent, the peroxidatic activity. In addition, the heme content of the variants was reduced relative to the wild-type enzyme. The relative ease of heme loss from HPI and a mixture of tetrameric enzymes with 2, 3, and 4 hemes was revealed by mass spectrometry analysis. Conversion of W105 to either an aromatic (F) or aliphatic (I) residue caused a 4-5-fold increase in peroxidatic activity, coupled with a >99% inhibition of catalatic activity. The peroxidatic-to-catalatic ratio of the W105F variant was increased 2800-fold such that compound I could be identified by both electronic and EPR spectroscopy as being similar to the porphyrin cation radical formed in other catalases and peroxidases. Compound I, when generated by a single addition of H(2)O(2), decayed back to the native or resting state within 1 min. When H(2)O(2) was generated enzymatically in situ at low levels, active compound I was evident for up to 2 h. However, such prolonged treatment resulted in conversion of compound I to a reversibly inactivated and, eventually, to an irreversibly inactivated species, both of which were spectrally similar to compound I.     

(-)-Epicatechin enhances the chlorinating activity of human myeloperoxidase

The heme-containing enzyme myeloperoxidase (MPO) accumulates at inflammatory sites and is able to catalyse one- and two-electron oxidation reactions. Here it is shown that (-)-epicatechin, which is known to have numerous beneficial health effects, in low micromolar concentration enhances the degradation of monochlorodimedon (MCD) or the chlorination of taurine in a concentration-dependent bell-shaped manner whereas at higher concentrations it sufficiently suppresses the release of hypochlorous acid. Presented reaction mechanisms demonstrate the efficiency of micromolar concentrations of the flavan-3-ol in overcoming the accumulation of compound II that does not participate in the chlorination cycle. In case of MCD the mechanism is more complicated since it also acts as peroxidase substrate with very different reactivity towards compound I (3 × 10⁵ M⁻¹ s⁻¹) and compound II (8.8 M⁻¹ s⁻¹) at pH 7. By affecting the chlorinating activity of myeloperoxidase (-)-epicatechin may participate in regulation of immune responses at inflammatory sites.     

Alternatives to the oxoferryl porphyrin cation radical as the proposed reactive intermediate of cytochrome P450: two-electron oxidized Fe(III) porphyrin derivatives

The active intermediates in most heme enzyme-catalyzed oxidations such as epoxidation and hydroxylation have been attributed to the O=Fe(IV) porphyrin ?-cation radical, so-called compound I. This could be correct for many cases, however, alternatives to compound I have been proposed for several oxidations including aliphatic hydroxylation catalyzed by P450. Therefore, two-electron oxidized iron porphyrin complexes other than compound I have been reviewed as candidates for the active species responsible for oxidations catalyzed by heme enzymes.     

The pH dependence of the activity of dehaloperoxidase from Amphitrite ornata

Dehaloperoxidase (DHP) from the terebellid polychaete, Amphitrite ornata, is the first hemoglobin that has peroxidase activity as part of its native function. The substrate 2,4,6-tribromophenol (TBP) is oxidatively debrominated by DHP to form 2,6-dibromoquinone (DBQ) in a two-electron process. There is a well-defined internal binding site for TBP above the heme, a feature not observed in other hemoglobins or peroxidases. A study of the pH dependence of the activity of DHP reveals a substantial difference in mechanism. From direct observation of the Soret band of the heme it is shown that the pKa for heme activation in protein DHP is 6.5. Below this pH the heme absorbance decreases in the presence of H2O2 with or without addition of substrate. The low pH data are consistent with significant heme degradation. Above pH 6.5 addition of H2O2 causes the heme to shift rapidly to a compound II spectrum and then slowly to an unidentified intermediate with an absorbance of 410 nm. However, the pKa of the substrate TBP is 6.8 and the greatest enzyme activity is observed above the pKa of TBP under conditions where the substrate is a phenolate anion (TPBO-). Although the mechanisms may differ, the data show that both neutral TBP and anionic TPBO- are converted to the quinone product. The mechanistic implications of the pH dependence are discussed by comparison other known peroxidases, which oxidize substrates at the heme edge.     

Chlorination of N-acetyltyrosine with HOCl, chloramines, and myeloperoxidase-hydrogen peroxide-chloride system.

N-acetyl-L-tyrosine (N-acTyr), with the alpha amine residue blocked by acetylation, can mimic the reactivity of exposed tyrosyl residues incorporated into polypeptides. In this study chlorination of N-acTyr residue at positions 3 and 5 in reactions with NaOCl, chloramines and the myeloperoxidase (MPO)-H2O2-Cl- chlorinating system were invesigated. The reaction of N-acTyr with HOCl/OCl- depends on the reactant concentration ratio employed. At the OCl-/N-acTyr (molar) ratio 1:4 and pH 5.0 the chlorination reaction yield is about 96% and 3-chlorotyrosine is the predominant reaction product. At the OCl-/N-acTyr molar ratio 1:1.1 both 3-chlorotyrosine and 3,5-dichlorotyrosine are formed. The yield of tyrosine chlorination depends also on pH, amounting to 100% at pH 5.5, 91% at pH 4.5 and 66% at pH 3.0. Replacing HOCl/OCl- by leucine/chloramine or alanine/chloramine in the reaction system, at pH 4.5 and 7.4, produces trace amount of 3-chlorotyrosine with the reaction yield of about 2% only. Employing the MPO-H2O2-Cl- chlorinating system at pH 5.4, production of a small amount of N-acTyr 3-chloroderivative was observed, but the reaction yield was low due to the rapid inactivation of MPO in the reaction system. The study results indicate that direct chlorination of tyrosyl residues which are not incorporated into the polypeptide structure occurs with excess HOCl/OCl- in acidic media. Due to the inability of the myeloperoxidase-H2O2-Cl- system to produce high enough HOCl concentrations, the MPO-mediated tyrosyl residue chlorination is not effective. Semistable amino-acid chloramines also appeared not effective as chlorine donors in direct tyrosyl chlorination.     

Catalase-peroxidase from synechocystis is capable of chlorination and bromination reactions

Catalase-peroxidases (KatGs) are multifunctional heme peroxidases exhibiting an overwhelming catalase activity and a substantial peroxidase activity of broad specificity. Here, we show that catalase-peroxidases are also haloperoxidases capable of oxidizing chloride, bromide, and iodide in a peroxide- and enzyme-dependent manner. Recombinant KatG and the variants R119A, W122F, and W122A from the cyanobacterium Synechocystis PCC 6803 have been tested for their halogenation activity. Halogenation of monochlorodimedon (MCD), formation of triiodide and tribromide, and bromide- and chloride-mediated oxidation of glutathione have been tested. Halogenation of MCD by chloride, bromide, and iodide was shown to be catalyzed by wild-type KatG and the variant R119A. Generally, rates of halogenation increased in the order Cl(-) < Br(-) < I(-) and/or by decreasing pH. The halogenation activity of R119A was about 7-9% that of the wild-type enzyme. Upon exchange of the distal Trp122 by Phe and Ala, both the catalase and halogenation activities were lost but the overall peroxidase activity was increased. The findings suggest that the same redox intermediate is involved in H(2)O(2) and halide oxidation and that distal Trp122 is involved in both two-electron reactions. That halides compete with H(2)O(2) for the same redox intermediate is also emphasized by the fact that the polarographically measured catalase activity is influenced by halides, with bromide being more effective than chloride.     

"Peroxidatic" form of cytochrome oxidase as studied by X-ray absorption spectroscopy

X-ray absorption spectroscopy shows pulsed oxidase to be similar to resting oxidase but to lack the sulfur bridge between iron and copper of active sites (Powers, L., Y. Ching, B. Chance, and B. Muhoberac, 1982, Biophys. J., 37[2, Pt. 2]: 403a. [Abstr.] ) The first shell ligands and bond lengths of the pulsed oxidase active site heme most clearly fit the ferric peroxidases from horseradish and yeast, and the pulsed oxidase cyanide compound resembles the low spin hemoprotein cyanide compounds. The structural results are consistent with an aquo or a peroxo form for pulsed oxidase as is also observed by optical studies. These structural and chemical data are consistent with a role for the pulsed forms in a cyclic peroxidatic side reaction in which the pulsed and pulsed peroxide compounds act as peroxide scavengers. The peroxidatic role of cytochrome oxidase in the nonsulfur bridged form suggests the renaming of the "oxygenated" or "pulsed" forms on a functional basis as "peroxidatic" forms of cytochrome oxidase.     

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)     

Contribution of protein conformation to stereochemistry and reactivity of the active center of heme proteins and enzymes. The existence of horseradish peroxidase conformations and their possible role in the catalysis mechanism

In the spectral region 350-800 nm at 4.2 K we measured magnetic circular dichroism (MCD) spectra of the pentacoordinated complex of protcheme with 2-methylimidazole, deoxyleghemoglobin, neutral and alkaline forms of reduced horseradish peroxidase in the equilibrium states, as well as in non-equilibrium states produced by low-temperature photolysis of their carbon monoxide derivatives. Earlier the corresponding results have been obtained for myoglobin, hemoglobin and cytochromes P-450 and P-420. The energies of Fe-N (proximal His) and Fe-N(pyrroles) bonds and their changes upon ligand binding in heme proteins and enzymes were compared with those in the model heme complex thus providing conformational contribution into stereochemistry of the active site. The examples of weak and strong conformational "pressure" on stereochemistry were analysed and observed. If conformational energy contribution into stereochemistry prevails the electronic one the heme stereochemistry remains unchanged on ligand binding as it was observed for leghemoglobin and alkaline horseradish peroxidase. The change of bond energies in myoglobin and hemoglobin on ligand binding are comparable with those in protein free pentacoordinated protoheme, giving an example of weak conformational contribution to heme stereochemistry. The role of protein conformation energy in the modulation of ligand binding properties of heme in leghemoglobin relative to those in myoglobins is discussed. The most striking result were obtained in the study of reduced horseradish peroxidase in the pH region of 6.0-10.2. It was found that such different perturbations as ligand binding and heme-linked ionization of the distal amino acid residue induce identical changes in heme stereochemistry. Neither heme-linked ionization in the carbon monoxide complex nor the geometry of Fe-Co bond affect the heme local structure of photoproducts. These and other findings suggest a very low conformation mobility of horseradish peroxidase whose protein constraints appear to allow only two preferable geometries of specific amino acid residues that form the heme pocket. The role of the two tertiary structure constraints on the heme in the mechanism of horseradish peroxidase function is discussed. It is supposed that one conformation produces a heme environment suitable for two-electron oxidation of the native enzyme to compound I by hydrogen peroxide while another conformation changes the heme stereochemistry in the direction favourable for back reduction of compound I by the substrate to the resting enzyme through two one-electron steps. The switch from one tertiary structure to another is expected to be induced by substrate bind     

Electronic state of heme in cytochrome oxidase. I. Magnetic circular dichroism of the isolated enzyme and its derivatives.

Magnetic circular dichroism (MCD) spectra have been recorded for beef heart cytochrome oxidase and a number of its inhibitor complexes. The resting enzyme exhibits a derivate shape Faraday C term in the Soret region, characteristic of low spin ferric heme, which accounts for 50% of the total oxidase heme a. The remaining heme a (50%) is assigned to the high spin state. MCD temperature studies, comparison with the MCD spectra of heme a-imidazole model compounds, and ligand binding (cyanide, formate) studies are consistent with these spin state assignments in the oxidized enzyme. Furthermore, the ligand binding properties and correlations between optical and MCD parameters indicate that in the resting enzyme the low spin heme a is due solely to cytochrome a3+ and the high spin heme a to cytochrome a33+. The Soret MCD of the reduced protein is interpreted as th sum of two MCD curves: an intense, asymmetric MCD band very similar to that exhibited by deoxymyoglobin which we assign to paramagnetic high spin cytochrome a3(2+) and a weaker, more symmetric MCD contribution, which is attributed to diamagnetic low spin cytochrome a2+. Temperature studies of the Soret MCD intensity support this proposed spin state heterogeneity. Ligand binding (CO, CN-) to the reduced protein eliminates the intense MCD associated with high spin cytochrome a3(2+); however, the band associated with cytochrome a2+ is observed under these conditions as well as in a number of inhibitor complexes (cyanide, formate, sulfide, azide) of the partially reduced protein. The MCD spectra of oxidized, reduced, and inhibitor-complexed cytochrome oxidase show no evidence for heme-heme interaction via spectral parameters. This conclusion is used in conjunction with the fact that ferric, high spin heme exhibits weak MCD intensity to calculate the MCD spectra for the individual cytochromes of the oxidase as well as the spectra for some inhibitor complexes of cytochrome a3. The results are most simply interpreted using the model we have recently proposed to account for the electronic and magnetic properties of cytochrome (Palmer, G., Babcock, F.T., and Vcikery, L.E. (1976) Proc. Natl. Acad. Sci. U. S. A. 73, 2206-2210).