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

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

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.     

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)     

Resonance Raman spectra of horseradish peroxidase and bovine liver catalase compound I species. Evidence for predominant 2A2u pi-cation radical ground state configurations.

The nature of the porphyrin pi-cation radicals in the horseradish peroxidase and bovine liver catalase (BLC) compound I species have been investigated by studying their resonance Raman spectra. A variety of laser excitation and sample interrogation procedures have been employed in order to minimize previously documented problems arising from photoinduced conversions. With Soret band excitation, the spectra obtained for both species resemble that of a compound II-like photoproduct unless the samples are excited with residence times in the microsecond regime with very low (approximately 1 milliwatt) powers. When these precautions are taken, spectra attributable to the compound I species themselves are obtained. The spectrum for horseradish peroxidase compound I is similar to that reported by Paeng and Kincaid (Paeng, K.-J., and Kincaid, J. R. (1988) Am. Chem. Soc. 110, 7913-7915) using a similar approach. Both horseradish peroxidase and BLC compound I exhibit frequency shifts relative to their compound II species that are in the direction observed for model pi-cation radicals with predominant 2A2u character. The magnitudes of these shifts are smaller than those observed for heme models that lack aromatic axial ligands, but agree well with those observed on formation of the compound I analog of N alpha-acetyl microperoxidase-8 that has His as a proximal ligand. This observation is consistent with partial delocalization of the radical density onto the proximal His-170 and Tyr-357 ligands in horseradish peroxidase and BLC, respectively. The strong ligand field provided by these ligands on the proximal side and oxo ligand on the distal side of the heme group is apparently sufficient to reverse the 2A1u radical ground state preference observed for heme-like porphyrin species (e.g. octaethylporphyrins) with weak axial fields. Enhancement of several bands assigned to the Tyr-357 ligand has also been observed for BLC compound I with 406.7-nm excitation. This is attributed either to resonance with a tyrosinate----Fe(IV) charge transfer band or to the coupling provided by radical spin delocalization onto the tyrosinate ligand.     

Resonance Raman spectroscopy of polynucleotides

Resonance Raman Spectroscopy allows a selective study of the bases of DNA and therefore of the interactions of these bases with ligands. This technique is also sensitive to structural modifications. We show here that, first, the structures of native poly(dA-dT).poly(dA-dT) and poly(dA).poly(dT) are not the same and that, secondly, it is possible to characterize the B----Z transition of poly(dG-dC).poly(dG-dC). The study of the Raman hypochromism during the thermal denaturation of the polynucleotides reveals that the stacking of the adenines in poly(dA).poly(dT) is near that observed in poly(rA) but differs of this stacking in poly(dA-dT).poly(dA-dT). The enhancement of the intensity of the guanine line at 1193 cm-1 and of the cytosine lines at 780 cm-1, 1 242 cm-1 and 1268 cm-1 as well as the shift of the guanine line at low frequency should allow to characterize a small proportion of base pairs in Z form in any DNA.     

Resonance Raman spectroscopy of cytochrome c peroxidase single crystals on a variable-temperature microscope stage

Good quality resonance Raman (RR) spectra have been obtained for cytochrome c peroxidase single crystals (0.2 x 0.5 x 1 mm) lying on their 110 faces on a microscope stage. Crystal orientation and polarization effects are observed which differentiate the RR bands on the basis of the symmetries of the porphyrin vibrational modes. The measured depolarization ratios are accurately calibrated for isolated bands of both totally symmetric and non totally symmetric modes by using a model of D4h chromophores in an oriented gas using the crystal structure atomic coordinates. The calculations indicate that the electronic transition moments are approximately along the lines connecting the methine bridges, suggesting an electronic steering effect of the vinyl groups. Deviations are observed for bands associated with the porphyrin v10 and the vinyl C = C stretching modes, which may be due to their near-resonant interaction. The band frequencies correspond to those of a five-coordinate high-spin FeIII heme, as previously observed in solution, consistent with the X-ray structure showing the Fe atom to be out of the heme plane on the proximal side with a distal water molecule located at a nonbonded distance, 2.4 A. The temperature dependence of the RR spectrum was determined with a Joule-Thompson cryostat on crystals sealed in glass capillaries. As the temperature is lowered, the spectrum converts to one characteristic of a low-spin FeIII heme. The conversion, which is readily reversible, is quite gradual. It is detectable at -50 degrees C but is incomplete even at -190 degrees C. A temperature effect on the protein structure is proposed which permits the Fe atom to approach the heme plane and bind the distal water molecule, or the distal histidine.     

Resonance Raman studies of dioxygen and carbon monoxide binding to imidazole-appended hemes.

Resonance Raman spectroscopy has been employed to probe the effects of proximal base strain on the bonding of O2 and CO in three synthetic hemins with covalently linked imidazole ligands. The strain is introduced by varying the length of the imidazole-containing side chain and by restricting the side chain flexibility with a phenyl ring. These hemins are abbreviated as "long," "short," and "stiff" hemins, respectively. In the deoxy state, the iron-imidazole stretching frequencies [nu(Fe--N epsilon)] for long, short, and stiff hemins are detected at 200, 207, and 204 cm-1, respectively. The strain induced in the iron-imidazole bond by the short hemin results in a higher nu(Fe--N epsilon) frequency, in contrast to the strain induced by sterically hindered 2-methylimidazole or 1,2-dimethylimidazole complexes in which the Fe--N epsilon bond is tilted and lengthened, but the imidazole ring remains perpendicular to the heme plane. However, in the short hemin, the plane of the imidazole ring may not be perpendicular to the plane of the porphyrin, altering the amount of pi-interaction (hence the strength of Fe--N epsilon bond) and the nature of normal mode containing Fe--N epsilon bond stretching. Upon CO binding, we have observed the nu(Fe--CO) stretching frequencies at 497 (long), 499 (short), and 496 cm-1 (stiff), somewhat lower than those reported by Mitchell et al. (Inorg. Chem., 1985, 24:967) for the chelated-heme X CO complexes (i.e., 501-506 cm-1). This is the first report of an iron-oxygen-associated vibration observed in solution for an unprotected heme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Resonance Raman spectroscopy in malaria research

In recent years, the field of Raman spectroscopy has witnessed a surge in technological development, with the incorporation of ultrasensitive, charge-coupled devices, improved laser sources and precision Rayleigh-filter systems. This has led to the development of sensitive confocal micro-Raman spectrometers and imaging spectrometers that are capable of obtaining high spatial-resolution spectra and images of subcellular components within single living cells. This review reports on the application of resonance micro-Raman spectroscopy to the study of malaria pigment (hemozoin), a by-product of hemoglobin catabolization by the malaria parasite, which is an important target site for antimalarial drugs. The review aims to briefly describe recent studies on the application of this technology, elucidate molecular and electronic properties of the malaria pigment and its synthetic analog β-hematin, provide insight into the mechanism of hemozoin formation within the food vacuole of the parasite, and comment on developing strategies for using this technology in drug-screening protocols.     

Resonance Raman spectroscopy in malaria research

In recent years, the field of Raman spectroscopy has witnessed a surge in technological development, with the incorporation of ultrasensitive, charge-coupled devices, improved laser sources and precision Rayleigh-filter systems. This has led to the development of sensitive confocal micro-Raman spectrometers and imaging spectrometers that are capable of obtaining high spatial-resolution spectra and images of subcellular components within single living cells. This review reports on the application of resonance micro-Raman spectroscopy to the study of malaria pigment (hemozoin), a by-product of hemoglobin catabolization by the malaria parasite, which is an important target site for antimalarial drugs. The review aims to briefly describe recent studies on the application of this technology, elucidate molecular and electronic properties of the malaria pigment and its synthetic analog beta-hematin, provide insight into the mechanism of hemozoin formation within the food vacuole of the parasite, and comment on developing strategies for using this technology in drug-screening protocols.     

How axial ligands control the reactivity of high-valent iron(IV)-oxo porphyrin pi-cation radicals in alkane hydroxylation: a computational study

The push effect of anionic axial ligands of high-valent iron(IV)-oxo porphyrin pi-cation radicals, (Porp)(+.)Fe(IV)(O)(X) (X=OH(-), AcO(-), Cl(-), and CF(3)SO(3)(-)), in alkane hydroxylation is investigated by B3LYP DFT calculations. The electron-donating ability of anionic axial ligands influences the activation energy for the alkane hydroxylation by the iron(IV)-oxo intermediates and the Fe-O bond distance of the iron-oxo species in transition state.     

Formation of porphyrin pi cation radical in zinc-substituted horseradish peroxidase

Zinc-substituted horseradish peroxidase is oxidized by K2IrCl6 to a characteristic state which retains one oxidizing equivalent more than the zinc peroxidase. The oxidized enzyme gives an optical absorption spectrum similar to that of compound I of peroxidase and catalase, and a g = 2 electron paramagnetic resonance signal which has an intensity corresponding to the porphyrin content. It is reduced back to the zinc peroxidase by a stoichiometric amount of ferrocyanide or by a large excess of K3IrCl6. From the equilibrium data, the value of E0' for the zinc peroxidase couple is estimated to be 0.74 V at pH 6. The oxidized zinc peroxidase is also formed by the addition of H2O2 or upon illumination with white light. The rate constants for the oxidation by K2IrCl6 and H2O2 at pH 8.0 are 8 x 10(5) and 8 x 10(2) M-1 s-1, respectively. No essential spectral change can be observed when K2IrCl6 is added to the metal-free peroxidase (protoporphyrin--apoperoxidase complex) or to zinc-substituted sperm whale myoglobin.     

Resonance Raman study of intermediates of the halorhodopsin photocycle

The resonance Raman (RR) study of the retinal protein halorhodopsin (HR₅₇₈) was extended to two of its photoproducts: HR and HR^L₄₁₀ RR spectra of both species were recorded in H₂O and D₂O and compared with the RR spectra of the intermediates L₅₅₀ and M₄₁₂ from the bacteriorhodopsin photocycle. HR₅₂₀ was found to be a protonated Schiff base in the 13-cis configuration and HR^L₄₁₀ a deprotonated Schiff base in the 13-cis configuration.     

Kinetic studies of reactions of iron(IV)-oxo porphyrin radical cations with organic reductants

Iron(IV)-oxo porphyrin radical cations are observed intermediates in peroxidase and catalase enzymes, where they are known as Compound I species, and the putative oxidizing species in cytochrome P450 enzymes. In this work, we report kinetic studies of reactions of iron(IV)-oxo porphyrin radical cations that can be compared to reactions of other metal-oxo species. The iron(IV)-oxo radical cations studied were those produced from 5,10,15,20-tetramesitylporphryinato-iron(III) perchlorate (1), 5,10,15,20-tetramesitylporphryinato-iron(III) chloride (2), both in CH(3)CN solvent, and that from 5,10,15,20-tetrakis(pentafluorophenyl)porphyrinato-iron(III) perchlorate (3) in CH(2)Cl(2) solvent. The substrates studied were alkenes and activated hydrocarbons diphenylmethane and ethylbenzene. For a given organic reductant, various iron(IV)-oxo porphyrin radical cations react in a relatively narrow kinetic range; typically the second-order rate constants vary by less than 1 order of magnitude for the oxidants studied here and the related oxidant 5,10,15,20-tetrakis(pentafluorophenyl)porphyrinato-iron(IV)-oxo porphyrin radical cation in CH(3)CN solvent. Charge transfer in the transition states for epoxidation reactions of substituted styrenes by oxidants 1 and 2, rho(+) values of -1.9 and -0.9, respectively, mirrors results found previously for related species. Competition kinetic reactions with a catalytic amount of porphyrin iron(III) species and a terminal oxidant give relative rate constants for oxidations of competing substrates that are somewhat smaller than the ratios of absolute rate constants. Water in CH(3)CN solutions has an apparent modest stabilizing effect on oxidant 1 as indicated in slightly reduced rate constants for oxidation reactions. The iron(IV)-oxo porphyrin radical cations are orders of magnitude less reactive than porphyrin-manganese(V)-oxo cations and a corrole-iron(V)-oxo species. The small environment effects found here suggest that high energy demanding hydrocarbon oxidation reactions catalyzed by cytochrome P450 enzymes might require highly reactive iron(V)-oxo transients as oxidants instead of the more stable, isomeric iron(IV)-oxo porphyrin radical cations.     

Resonance Raman spectroscopy of iron-oxygen vibrations in hemerythrin

Resonance Raman spectra are reported for oxyhemerythrin and 15 anionic adducts of methemerythrin. All methemerythrin derivatives except sulfidomethemerythrin contain a Raman band near 510 cm^?1 which is assigned to an iron-oxygen stretching vibration. The effect of H₂¹⁸O on the frequency of this vibration was studied extensively. On the basis of the exchange results, the vibration is assigned to OH^?, H₂O, or a μ-oxo bridge between the irons.     

Resonance Raman spectroscopy of pyridoxal Schiff bases

Resonance Raman (RR) spectra are reported for amino acid and amine adducts of pyridoxal 5'-phosphate (PLP) and 5'-deoxypyridoxal (5'-dPL) in aqueous solution. For the valine adducts, a detailed study has been carried out on solutions at pH and pD 5, 9, and 13, values at which the pyridine and imine protons are successively ionized, and on the adducts formed from 15N-valine, alpha-deuterovaline, and N-methyl-PLP. Good quality spectra were obtained, despite the strong fluorescence of pyridoxal Schiff bases, by adding KI as a quencher, and by exciting the molecules on the blue side of their absorption bands: 406.7 nm (cw Kr+ laser) for the pH 5 and 9 species (lambda max = 409 and 414 nm), and 354.7 nm (pulsed YAG laser, third harmonic) for the pH 13 species (lambda max = 360 nm). A prominent band at 1646 cm-1 is assigned to the imine C=N stretch via its 13 cm-1 15N shift. A 12 cm-1 down-shift of the band in D2O confirms that the Schiff base linkage is protonated at pH 9. Deprotonation at pH 13 shifts VC = N from 1646 to 1629 cm-1, values typical of conjugated Schiff bases. The strongest band in the spectrum, at 1338 cm-1, shifts to 1347 cm-1 upon pyridine protonation at pH 5, and is assigned to a ring mode with a large component of phenolate C-O stretch. A shoulder on its low-frequency side is assigned to the C4-C4' stretch. Large enhancements of these modes can be understood qualitatively in terms of the dominant resonance structures contributing to the ground and resonant excited states. A number of weaker bands are observed, and assigned to pyridine ring modes. These modes gain significantly in intensity, while the exocyclic modes diminish, when the spectra are excited at 266 nm (YAG laser, fourth harmonic) in resonance with ring-localized electronic transitions.     

Resonance Raman spectroscopy shows different temperature-dependent coordination equilibria for native horseradish and cytochrome c peroxidase

Resonance Raman spectra are reported for native horseradish peroxidase (HRP) and cytochrome c peroxidase (CCP) at 290, 77 and 9 K, using 406.7 nm excitation, in resonance with the Soret electronic transition. The spectra reveal temperature-dependent equilibria involving changes in coordination or spin state. At 290 K and pH 6.5, CCP contains a mixture of 5- and 6-coordinate high-spin Fe^III heme while at 9 K the equilibrium is shifted entirely to the 6-coordinate species. The spectra indicate weak binding of H₂O to the heme Pe, consistent with the long distance, 2.4 Å, seen in the crystal structure. At 290 K HRP also contains a mixture of high-spin Fe^III hemes with the 5-coordinate form predominant. At low temperature, a small 6-coordinate high-spin component remains but the 5-coordinate high-spin spectrum is replaced by another which is characteristic either of 6-coordinate low-spin or 5-coordinate intermediate spin heme. The latter species is definitely indicated by previous EPR studies at low temperature. This behavior implies that, in contrast to CCP, the distal coordination site of HRP is only partially occupied by H₂O at any temperature and that lowering the temperature significantly weakens the Fe-proximal imidazole bond. Consistent with this inference, the 77 K spectrum of reduced HRP shows an appreciable fraction of molecules having an Fe-imidazole stretching frequency of 222 cm⁻¹, a value indicating weakened H-bonding of the proximal imidazole.Resonance Roman spectroscopyHorseradish peroxidaseCytochrome c peroxidaseCoordination equilibrium     

Resonance Raman study on cytochrome c peroxidase and its intermediate. Presence of the Fe(IV) = O bond in compound ES and heme-linked ionization

Resonance Raman spectra of ferrous and ferric cytochrome c peroxidase and Compound ES and their pH dependences were investigated in resonance with Soret band. The Fe(IV) = O stretching Raman line of Compound ES was assigned to a broad band around 767 cm-1, which was shifted to 727 cm-1 upon 18O substitution. The 18O-isotopic frequency shift was recognized for Compound ES derived in H218O, but not in H216O. This clearly indicated occurrence of an oxygen exchange between the Fe(IV) = O heme and bulk water. The Fe(IV) = O stretching Raman band was definitely more intense and of higher frequency in D2O than in H2O as in Compound II of horseradish peroxidase, but in contrast with this its frequency was unaltered between pH 4 and 11. The Fe(II)-histidine stretching Raman line was assigned on the basis of the frequency shift observed for 54Fe isotopic substitution. From the intensity analysis of this band, the pKa of the heme-linked ionization of ferrocytochrome c peroxidase was determined to be 7.3. The Raman spectrum of ferricytochrome c peroxidase strongly suggested that the heme is placed under an equilibrium between the 5- and 6-coordinate high-spin structures. At neutral pH it is biased to the 5-coordinate structure, but at the acidic side of the transition of pKa = 5.5 the 6-coordinate heme becomes dominant. F- was bound to the heme iron at pH 6, but Cl- was bound only at acidic pH. Acidification by HNO3, H2SO4, CH3COOH, HBr, or HI resulted in somewhat different populations of the 5- and 6-coordinate forms when they were compared at pH 4.3. Accordingly, it is inferred that a water molecule which is suggested to occupy the sixth coordination position of the heme iron is not coordinated to the heme iron at pH 6 but that protonation of the pKa = 5.5 residue induces an appreciable structural change, allowing the coordination of the water molecule to the heme iron.     

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.     

Resonance Raman spectroscopic characterization of compound III of lignin peroxidase

Resonance Raman (RR) spectra of several compounds III of lignin peroxidase (LiP) have been measured at 90 K with Soret and visible excitation wavelengths. The samples include LiPIIIa (or oxyLiP) prepared by oxygenation of the ferrous enzyme, LiPIIIb generated by reaction of the native ferric enzyme with superoxide, LiPIIIc prepared from native LiP plus H2O2 followed by removal of excess peroxide with catalase, and LiPIII* made by addition of excess H2O2 to the native enzyme. The RR spectra of these four products appear to be similar and, thus, indicate that the environments of these hexacoordinate, low-spin ferriheme species must also be very similar. Nonetheless, the Soret absorption band of LiPIII* is red-shifted by 5 nm from the 414-nm maximum common to LiPIIIa, -b, and -c [Wariishi, H., & Gold, M.H. (1990) J. Biol. Chem. 265, 2070-2077]. Analysis of the iron-porphyrin vibrational frequencies indicates that the electronic structures for the various compounds III are consistent with an FeIIIO2.-formulation. The spectral changes observed between the oxygenated complex and the ferrous heme of lignin peroxidase are similar to those between oxymyoglobin and deoxymyoglobin. The contraction in the core sizes in compound III relative to the native peroxidase is analyzed and compared with that of other heme systems. EPR spectra confirm that the high-spin ferric form of the native enzyme, with an apparent g = 5.83, is converted into the EPR-silent LiPIII* upon addition of excess H2O2. Its magnetic behavior may be explained by anti-ferromagnetic coupling between the low-spin FeIII and the superoxide ligand.(ABSTRACT TRUNCATED AT 250 WORDS)