Ferric Chloroperoxidase Research Articles

Low temperature photo-oxidation of chloroperoxidase Compound II

Oxidation of the heme-thiolate enzyme chloroperoxidase (CPO) from Caldariomyces fumago with peroxynitrite (PN) gave the Compound II intermediate, which was photo-oxidized with 365 nm light to give a reactive oxidizing species. Cryo-solvents at pH ≈ 6 were employed, and reactions were conducted at temperatures as low as − 50 °C. The activity of CPO as evaluated by the chlorodimedone assay was unaltered by treatment with PN or by production of the oxidizing transient and subsequent reaction with styrene. EPR spectra at 77 K gave the amount of ferric protein at each stage in the reaction sequence. The PN oxidation step gave a 6:1 mixture of Compound II and ferric CPO, the photolysis step gave an approximate 1:1 mixture of active oxidant and ferric CPO, and the final mixture after reaction with excess styrene contained ferric CPO in 80% yield. In single turnover reactions at − 50 °C, styrene was oxidized to styrene oxide in high yield. Kinetic studies of styrene oxidation at − 50 °C displayed saturation kinetics with an equilibrium constant for formation of the complex of K bind = 3.8 × 10 4 M − 1 and an oxidation rate constant of k ox = 0.30 s − 1 . UV–Visible spectra of mixtures formed in the photo-oxidation sequence at ca. − 50 °C did not contain the signature Q-band absorbance at 690 nm ascribed to CPO Compound I prepared by chemical oxidation of the enzyme, indicating that different species were formed in the chemical oxidation and the photo-oxidation sequence.

Low-Frequency Dynamics of Caldariomyces fumago Chloroperoxidase Probed by Femtosecond Coherence Spectroscopy

Ultrafast laser spectroscopy techniques are used to measure the low-frequency vibrational coherence spectra and nitric oxide rebinding kinetics of Caldariomyces fumago chloroperoxidase (CPO). Comparisons of the CPO coherence spectra with those of other heme species are made to gauge the protein-specific nature of the low-frequency spectra. The coherence spectrum of native CPO is dominated by a mode that appears near 32-33 cm(-1) at all excitation wavelengths, with a phase that is consistent with a ground-state Raman-excited vibrational wavepacket. On the basis of a normal coordinate structural decomposition (NSD) analysis, we assign this feature to the thiolate-bound heme doming mode. Spectral resolution of the probe pulse ("detuned" detection) reveals a mode at 349 cm(-1), which has been previously assigned using Raman spectroscopy to the Fe-S stretching mode of native CPO. The ferrous species displays a larger degree of spectral inhomogeneity than the ferric species, as reflected by multiple shoulders in the optical absorption spectra. The inhomogeneities are revealed by changes in the coherence spectra at different excitation wavelengths. The appearance of a mode close to 220 cm(-1) in the coherence spectrum of reduced CPO excited at 440 nm suggests that a subpopulation of five coordinated histidine-ligated hemes is present in the ferrous state at a physiologically relevant pH. A significant increase in the amplitude of the coherence signal is observed for the resonance with the 440 nm subpopulation. Kinetics measurements reveal that nitric oxide binding to ferric and ferrous CPO can be described as a single-exponential process, with rebinding time constants of 29.4 +/- 1 and 9.3 +/- 1 ps, respectively. This is very similar to results previously reported for nitric oxide binding to horseradish peroxidase.

Heme active-site structural characterization of chloroperoxidase by resonance Raman spectroscopy.

Resonance Raman spectra are reported for the nitric oxide adducts of ferric and ferrous chloroperoxidase and carbon monoxide adducts of ferrous chloroperoxidase. The stretching, v(Fe-NO), and bending, delta(FeNO), modes are detected at 538 and 558 cm-1, respectively, for the ferric nitrosylchloroperoxidase. These two bands shift to 534 and 546 cm-1, respectively, upon substitution by 15N16O. The v(Fe-NO) mode of the nitric oxide adduct of ferrous chloroperoxidase is located at 542 cm-1, which shifts to 528 (15N16O), 540 (14N18O), and 524 (15N18O) cm-1 as the mass of the bound nitric oxide increases by 1 atomic unit. Two distinct states of the carbon monoxide adduct of chloroperoxidase, the acidic and alkaline forms, are found to undergo a reversible pH-induced transition. The v(Fe-CO) mode shifts from 484 to 492 cm-1 and the delta(FeCO) mode at 562 cm-1 disappears as the pH is reduced from 6.0 to 3.3. In addition, two low frequency modes at 382 and 420 cm-1, assignable to the delta(CbC1C2) bending of propionate and vinyl groups, respectively, also show pH sensitivity. The results suggest a peroxidase-like heme active-site environment for chloroperoxidase and indicate a facile conformational change of heme groups accompanying the acid-base transition.

A nuclear magnetic resonance study of axial ligation for the reduced states of chloroperoxidase, cytochrome P-450 cam, and porphinatoiron(II) thiolate complexes

The reduced forms of cytochrome P-450 cam and chloroperoxidase were examined by proton NMR spectroscopy. The pH and temperature dependences of the proton NMR spectra of both ferrous enzymes are reported. A series of alkyl mercaptide complexes of both synthetic and natural-derivative iron(II) porphyrins was also examined. The proton NMR spectra of these complexes facilitated the assignment of resonances due to the axial ligand in the model compounds on the basis of their isotropic shifts and multiplicities. Comparison of model compound data with that for the reduced enzymes supports assignment of the methylene protons for the axial cysteinate of ferrous cytochrome P-450 cam and ferrous chloroperoxidase to proton NMR resonances at 279 and 200 ppm (pH 7.0, 298K), respectively. Differences in the active site structure of the two enzymes are further demonstrated by 15N-NMR spectroscopy of the cyanide complexes of the ferric forms.

Oxygen-17 nuclear magnetic resonance spectroscopic studies of carbonmonoxyperoxidases.

We have obtained oxygen-17 (17O) nuclear magnetic resonance (NMR) spectra of C17O ligands bound to ferrous horseradish peroxidase isozyme A, isozyme C, and ferrous chloroperoxidase, as a function of pH. Our results show that the peroxidases exist in two distinct states, the acidic and alkaline forms, which undergo reversible acid-base-induced transitions characterized by a single pK value. The two forms are characterized spectroscopically in much the same way in all three proteins, suggesting a similar structural origin for the transition process. In particular, the 17O NMR signal of the acidic form is approximately 7 ppm more shielded than that of the alkaline form, and the CO ligand in the acidic form appears to have a smaller 17O nuclear quadrupole coupling constant than that of the alkaline form. We have also obtained the pK values and exchange rates for all three peroxidases. The results indicate that a similar structural change may be involved in the transition process in all three peroxidases.

Ligand and halide binding properties of chloroperoxidase: peroxidase-type active site heme environment with cytochrome P-450 type endogenous axial ligand and spectroscopic properties.

Equilibrium binding studies of exogenous ligands and halides to the active site heme iron of chloroperoxidase have been carried out from pH 2 to 7. Over twenty ligands have been studied including C, N, O, P, and S donors and the four halides. As judged from changes in the optical absorption spectra, direct binding of the ligands to the heme iron of ferric or ferrous chloroperoxidase occurs in all cases; this has been ascertained for the ferric enzyme in several cases through competition experiments with cyanide. All of the ligands except for the halides, nitrate, and acetate form exclusively low-spin complexes in analogy to results obtained with the spectroscopically related protein, cytochrome P-450-CAM [Sono, M., & Dawson, J.H. (1982) J. Biol. Chem. 257, 5496-5502]. The titration results show that, for the ferric enzyme, (i) weakly acidic ligands (pKa greater than 3) bind to the enzyme in their neutral (protonated) form, followed by deprotonation upon ligation to the heme iron. In contrast, (ii) strongly acidic ligands (pKa less than 0) including SCN-, NO3-, and the halides except for F- likely bind in their anionic (deprotonated) form to the acid form of the enzyme: a single ionizable group on the protein with a pKa less than 2 is involved in this binding. For the ferrous enzyme, (iii) a single ionizable group with the pKa value of 5.5 affects ligand binding. These results reveal that chloroperoxidase, in spite of the previously established close spectroscopic and heme iron coordination structure similarities to the P-450 enzymes, clearly belongs to the hydroperoxidases in terms of its ligand binding properties and active site heme environment. Magnetic circular dichroism studies indicate that the alkaline form (pH 9.5) of ferric chloroperoxidase has an RS-ferric heme-N donor ligand coordination structure with the N donor likely derived from histidine imidazole.

Preparation and properties of ferrous chloroperoxidase complexes with dioxygen, nitric oxide, and an alkyl isocyanide. Spectroscopic dissimilarities between the oxygenated forms of chloroperoxidase and cytochrome P-450.

Extensive spectroscopic investigations of chloroperoxidase and cytochrome P-450 have consistently revealed close similarities between these two functionally distinct enzymes. Although the CO-bound ferrous states were the first to display such resemblance, additional comparisons have focused on the native ferric and ferrous and the ligand-bound ferric derivatives of the enzymes. In order to test the extent to which the spectral properties of the two enzymes match each other, we have prepared the NO, alkyl isocyanide, and O2 adducts of ferrous chloroperoxidase, the latter two for the first time. As expected, the NO adducts of the two proteins have similar UV-visible absorption and magnetic circular dichroism spectra; the same behavior is observed for the alkyl isocyanide complexes. Unexpectedly, the dioxygen adduct of ferrous chloroperoxidase (i.e. Compound III), generated in cryogenic solvents at -30 degrees C by bubbling with O2, is spectrally distinct from oxy-P-450-CAM. Identification of this derivative as oxygenated chloroperoxidase is based on the following criteria: It is EPR-silent at 77 K. The bound O2 is dissociable as judged by the uniform conversion to the CO-bound form. Oxy-chloroperoxidase autoxidizes to form the native ferric enzyme without detectable intermediates at a rate comparable to that determined for oxy-P-450-CAM. Oxy-chloroperoxidase exhibits optical absorption (lambda nm (epsilon mM) = 354 (41), 430 (94), 554 (16.5), 587 (12.5)) and magnetic circular dichroism spectra that are clearly distinct from those of histidine-ligated heme proteins such as oxy-myoglobin or oxy-horseradish peroxidase. Surprisingly, several of its spectral properties, namely the red-shifted Soret peak and discrete alpha peak, are also unlike those of oxy-P-450-CAM. Since considerable evidence has accumulated supporting the ligation of an endogenous thiolate to the heme iron of chloroperoxidase, as has been established for the P-450 enzyme, the observed dissimilarities suggest that the electronic properties of the two dioxygen adducts are quite sensitive to differences in their active site heme environment. This, in turn may be related to the functional differences between the two enzymes.

Oxygen binding to dithionite-reduced chloroperoxidase.

Both the kinetics of ferric chloroperoxidase reduction by dithionite and the binding of molecular oxygen to ferrous chloroperoxidase have been studied. The oxyferrous chloroperoxidase decays spontaneously to the ferric enzyme. In addition the corresponding rapid-scan spectra have been recorded. The reduction reaction is caused by SO-.2 with a rate constant of (7.7 +/- 1.0) X 10(4) M-1 S-1. Oxygen binding occurs with a rate constant of (5.5 +/- 1.0) X 10(5) M-1 S-1 over the pH range 3.5-6. Oxyferrous chloroperoxidase has a Soret absorption peak at 428 nm and two partially resolved peaks at 555 nm and 588 nm. Isosbestic points occur at the following wavelengths: between ferrous and oxyferrous chloroperoxidase at 419, 545, 555 and 580 nm; between oxyferrous and ferric chloroperoxidase at 419, 487, 540, 609 and 682 nm.

The generation of a hyperporphyrin spectrum upon thiol binding to ferric chloroperoxidase. Further evidence of endogenous thiolate ligation to the ferric enzyme.

In spite of numerous spectroscopic similarities between chloroperoxidase and cytochrome P-450 (P-450) which suggest endogenous cysteinate axial ligation in chloroperoxidase as has been established for P-450, assignment of the endogenous axial ligand of chloroperoxidase has remained controversial since no available free sulfhydryl groups have been detected in chemical studies of chloroperoxidase. To help clarify this problem, we have carried out extensive studies of thiol-binding properties of native ferric chloroperoxidase and have compared our new results with those previously obtained in our laboratory on thiol adducts of P-450. We have found that the ligation of exogenous thiols to the heme iron of chloroperoxidase generates hyperporphyrin (split Soret) spectra (lambda max = approximately 372 and approximately 455 nm), consistent with the formation of bisthiolate low-spin ferric heme adducts as has been established for P-450 and its heme models. However, in contrast to the results with P-450, thiols not only coordinate to the ferric heme iron of chloroperoxidase in the thiolate form in competition with cyanide but also bind, presumably in the thiol form, to a site in the heme vicinity other than the heme iron without competition with cyanide. The thiol acidity (pK alpha greater than 7) or medium pH (pH 3-7) have little effect on the spectral and equilibrium properties of the adducts. Thiol binding to the latter site, which is presumably the organic substrate-binding site, causes a red shift of the Soret peak (339 vector approximately 420 nm) of the high-spin ferric enzyme; the resulting thiol adducts are still predominantly high spin. Similar spectral changes are also observed upon binding of other neutral sulfur (sulfides and disulfide) and oxygen (alcohols and ketones) donor ligands to native ferric chloroperoxidase. In conclusion, the generation of hyperporphyrin spectra upon exogenous thiol binding to native ferric chloroperoxidase provides considerable support for the presence of an endogenous thiolate ligand to the heme of the enzyme in its ferric state.

Kinetics of cyanide binding to chloroperoxidase in the presence of nitrate: detection of the influence of a heme-linked acid group by shift in the appa

The kinetics of the binding of cyanide to ferric chloroperoxidase have been studied at 25°C and ionic strength 0.11 M using a stopped-flow apparatus. The dissociation constant ( K CN) of the peroxidase-cyanide complex and both forward ( k +) and reverse ( k −) rate constants are independent of the H + concentration over the pH range 2.7 to 7.1. The values obtained are k cn = (9.5 ± 1.0) × 10 -5 M, k +. = (5.2 ± 0.5) × 10 4 M −1 sec −1 and k - = (5.0± 1.4) sec -1. In the presence of 0 06 M potassium nitrate the affinity of cyanide for chloroperoxidase decreases due to the inhibition of the forward reaction. The dissociation rate is not affected. The nitrate anion exerts its influence by binding to a protonated form of the enzyme, whereas the cyanide binds to the unprotonated form. Binding of nitrate results in an apparent shift towards higher p K a values of the ionization of a crucial heme-linked acid group. Hence the influence of this group can be detected in the accessible pH range. Extrapolation to zero nitrate concentration yields a value of 3.1±0.3 for the p K a of the heme-linked acid group.

The active sites of chloroperoxidase and cytochrome P-450-CAM: Comparative spectroscopic and ligand binding properties

Spectral similarities between chloroperoxidase (CPO) and cytochrome P-450 (P-450) have previously been used to suggest endogenous thiolate axial ligation to the CPO heme iron [1–6] as has been established for P-450. Both enzymes exhibit unique hyperporphyrin (‘split Soret’) spectra in their ferrous CO forms [1]. However, no free sulfhydryl group available for ligation to the heme iron have been found in chemical studies of CPO [7]. The t001. Soret Absorption Maxima (nm) and Spin States of the Ligand Complexes of Ferric CPO and P-450-CAM.aLigandCPObP-450-CAMcλ(ϵmM)Spin Statedλ(ϵmM)Spin StatedSH−449(e)ls467(43)flCH3COS−446(62)gls459elsCN−439(92)ls439(78)lsPyridine439(82)l421(111)lsNO437(114)ls430.5(105)lsCH3(CH2)3NC435(105)ls429.5(98)lsN−3432(110)ls427(81)lsSeCN−432(80)ls425(90)lsSCN−429(112)ls425(98)lsImidazole429(100)ls425(98)lsNo−2427.5(101)lseOCN−∼427els418.5(98)lsHCOO−425(121)ls419(106)lsHOCH2CH2SH418(69)ms464(58)fls417 (∼60)ms417(∼60)CH3CH2CH2SH416(74)ms464(∼33)flsCH3SCH3416(93)ms424(91)lsThioxane416(88)ms418(97)lsCH3SSCH3416(93)ms418(98)lsCH3COO−413(97)ms420(98)lsF−409(98)ghsno bindinga0.1 M K+ Phospate, 4 °C.bpH 6.0.cpH 7.0, (Refs.9–11).dls, low spin; ms, high-low mixed spin; hs, high spin.eUnstable.fRed Soret of hyperporphyrin spectrum (Ref. 11).gpH 3.0.t002. Soret Absorption Maxima (nm) of the Ligand Complexes of Ferrous CPO and P-450-CAM.LigandCPOa2 λ(ϵmM)P-450-CAMb2 λ(ϵmM)Me2PhPc2459d2460(98)CN−454(∼140)Not observedCH3(CH2)3NC452(141)452(106)CO444(167)446(120)NO440(114)438(83)a2pH 6.0.b2pH 7.0.c2Dimenthyllphenylphosphine.d2Not determined. MCD [4] and EPR [6] spectroscopic properties of analogous CPO and P-450 derivatives are generally similar, although some differences do exist. To obtain additional information about the active site structure of CPO, we have carried out extensive optical and MCD spectroscopic studies of both ferric and ferrous CPO in the presence of various exogenous ligands. The results obtained with CPO have been compared with those obtained with P-450 [9 - 11, this work] in order to obtain a better understanding of the electronic structure of the heme iron in both proteins.

A kinetic study of the binding of carbon monoxide to ferrous chloroperoxidase.

The binding of carbon monoxide to ferrous chloroperoxidase in the pH range 4-6.5 is influenced by a titratable group on the enzyme having a pKA of 5.5 +/- 0.2 at 20 degrees C. The basic form of the enzyme reacts much faster with carbon monoxide than does the protonated form of the enzyme. The delta H degrees for the ionization of the functional group in the enzyme involved in carbon monoxide binding is about 8 kcal mol-1, and the delta S degrees is approximately 1 cal mol-1 K-1. These pKA and delta H degrees values suggest that this functional group is an imidazole ring associated with a histidine residue situated at the active site of the enzyme. The rates of the reaction for the formation and dissociation of the complex suggest that this histidine residue is not directly liganded to the iron atom of the heme prosthetic group. The relatively good agreement between the various kinetic approaches with several methods of experimentation, data collection, and data analysis lends strength to a proposed model in which the histidine occupies a distal site close to the sixth axial ligand position of the heme iron atom.

Oxidation-reduction potential measurements on chloroperoxidase and its complexes.

The oxidation-reduction potential of chloroperoxidase, an enzyme which catalyzes peroxidative chlorination, bromination, and iodination reactions, has been investigated. In addition to catalyzing biological halogenation reactions, chloroperoxidase is unusual in that the carbon monoxide complex of ferrous chloroperoxidase shows the typical long wavelength Soret absorption associated with P-450 hemoproteins. The pH dependence of the chloroperoxidase oxidation-reduction potential shows a discontinuity around pH 4.7. Similarly, measurements of the affinity of ferrous chloroperoxidase for carbon monoxide monitored both by spectroscopic and potentiometric titration exhibit a discontinuity in the pH 4.7 region. Oxidation-reduction potential measurements on chloroperoxidase in a CO atmosphere also show a discontinuous pH profile. These results suggest that ferrous chloroperoxidase undergoes reversible modification at low pH and that these changes are reflected in the oxidation-reduction potential. The oxidation-reduction potential of chloroperoxidase at pH 6.9 is - 140 mV, close to that measured for cytochrome P-450cam in the presence of substrate. The oxidation-reduction potential of chloroperoxidase at pH 2.7, the pH optimum for enzymatic chlorination, is +150 mV. The oxidation-reduction potentials of the halide complexes of chloroperoxidase (chloride, bromide, and iodide) are essentially identical with the potential measurements on the native enzyme. These observations suggest that, although halide anions bind to the enzyme, they probably do not bind as an axial ligand to the heme ferric iron.

Chloroperoxidase. P-450 type absorption in the absence of sulfhydryl groups

The oxidation state of the two half-cystine residues in the native ferric form of chloroperoxidase and in the reduced ferrous chloroperoxidase has been examined in order to evaluate the role of sulfhydryl groups as determinants of P-450 type spectra. Mössbauer and optical spectroscopy studies indicate that the ferrous forms of P-450cam and chloroperoxidase have very similar or identical heme environments. Model studies have suggested that sulfhydryl groups may function as axial ligands for developing P-450 character. However, chemical studies involving both sulfhydryl reagents and amperometric titrations show that neither the ferric nor the chemically produced ferrous forms of chloroperoxidase contain a sulfhydryl group. These results rule out the hypothesis that sulfhydryl groups are unique components for P-450 absorption characteristics. The optical and electron paramagnetic resonance (EPR) spectra of the nitric oxide complex of chloroperoxidase have been obtained and compared to those of myoglobin, hemoglobin, and cytochrome c and horseradish peroxidase. The EPR spectrum of the NO-ferrous chloroperoxidase complex, which is similar to that of cytochrome P-450cam, does not show the extra nitrogen hyperfine structure which appears to be characteristic of those hemoproteins which have a nitrogen atom as an axial heme ligand.

The P-450 Nature of the Carbon Monoxide Complex of Ferrous Chloroperoxidase

The absorption spectrum of the carbon monoxide complex of ferrous chloroperoxidase from Caldariomyces fumago has been shown to be quite similar to the characteristic spectrum of CO complexes of cytochromes of the P-450 type. Comparison of other spectral properties of chloroperoxidase and cytochrome P-450cam reveals a striking resemblance between the two proteins. The Soret absorption maxima for native, reduced, cyanide, nitrous oxide, and N-phenylimidazole complexes are quite similar. N-Phenylimidazole, a potent inhibitor of the cytochrome P-450cam-catalyzed hydroxylation of camphor, is a very effective inhibitor of a chloroperoxidase-catalyzed peroxidation reaction. Like P-450, chloroperoxidase undergoes characteristic spectral changes in the presence of substrates and nitrogenous compounds. Type I and type II spectral changes have been observed. Carefully controlled denaturation of chloroperoxidase resulted in the formation of a species having a spectrum essentially identical with that of cytochrome P-420, the denatured form of P-450. The spectral similarities described here indicate that both proteins provide quite similar environments for the heme prosthetic group. Both proteins also compare favorably with respect to physical properties such as molecular weight, high content of acidic amino acids, and low isoelectric point.