Comment on "Sensitive marker bands for the detection of spin states of heme in surface-enhanced resonance Raman scattering spectra of metmyoglobin" by Y. Kitahama, M. Egashira, T. Suzuki, I. Tanabe and Y. Ozaki.

We contrast recently reported surface-enhanced resonance Raman spectra (SERRS) of myoglobin on silver nanoparticles with established knowledge about this complex. We conclude that the detected bands are not related to the spin states of the protein cofactor, being rather originated by a heme coordination change induced by the metal surface.

Far- and near-field properties of gold nanoshells studied by photoacoustic and surface-enhanced Raman spectroscopies.

Gold nanoshells, with a silica core and different core and shell dimensions, have been extensively investigated. Optical far-field properties, namely extinction and absorption, have been separately determined by means of spectrophotometry and photoacoustic spectroscopy, respectively, in the 440-900 nm range. The enhancement factor for surface-enhanced Raman scattering, which is related to near-field effects, has been measured from 568 to 920 nm. The absorption contribution to extinction decreases as the overall diameter increases. Moreover, absorption and scattering display different spectral distributions, the latter being red shifted. The Surface Enhanced Raman Scattering enhancement profile, measured using thiobenzoic acid as a Raman probe, is further shifted to the red. The latter result suggests that the enhancement is dominated by the presence of hot spots, which are possibly related to the surface roughness of gold nanoshell particles.

Excited state absorption of fullerenes measured by the photoacoustic calorimetry technique.

Photoacoustic calorimetry (PAC) is used to determine the excited state absorption cross sections in a molecular system showing reverse saturable absorption behavior. PAC experiments on fullerene and fulleropyrrolidine in toluene solutions are performed at 532 nm and 690 nm, with a ns laser source. The PAC signal amplitude displays a superlinear increase when the energy of the applied laser source is increased. This behavior is ascribed to a process of enhanced absorption due to molecules populating the excited electronic states. The PAC signal observed for these chromophores is simulated numerically. The simulations rely on a description of the absorbing molecule as a six-level system, whose molecular parameters (i.e. absorption cross sections and lifetimes) are the ones for a reverse saturable absorber. The time-dependent population in the different energy levels is described through a rate equation system. This kind of model has been widely used by us to reproduce other experimental data such as nonlinear transmittance and Z-scan data. The PAC signal amplitude is the sum of the different contributions to non-radiative relaxation which arise from molecules populating different energy levels. The absorption cross sections for the singlet and triplet excited states of fullerene and fulleropyrrolidine are derived from the simulated PAC signal amplitudes. The values obtained are in good agreement with literature data measured with different techniques.

Differential activity and structure of highly similar peroxidases. Spectroscopic, crystallographic, and enzymatic analyses of lignifying Arabidopsis thaliana peroxidase A2 and horseradish peroxidase A2.

Anionic Arabidopsis thaliana peroxidase ATP A2 was expressed in Escherichia coli and used as a model for the 95% identical commercially available horseradish peroxidase HRP A2. The crystal structure of ATP A2 at 1.45 A resolution at 100 K showed a water molecule only 2.1 A from heme iron [Ostergaard, L., et al. (2000) Plant Mol. Biol. 44, 231-243], whereas spectroscopic studies of HRP A2 in solution at room temperature [Feis, A., et al. (1998) J. Raman Spectrosc. 29, 933-938] showed five-coordinated heme iron, which is common in peroxidases. Presented here, the X-ray crystallographic, single-crystal, and solution resonance Raman studies at room temperature confirmed that the sixth coordination position of heme iron of ATP A2 is essentially vacant. Furthermore, electronic absorption and resonance Raman spectroscopy showed that the heme environments of recombinant ATP A2 and glycosylated plant HRP A2 are indistinguishable at neutral and alkaline pH, from room temperature to 12 K, and are highly flexible compared with other plant peroxidases. Ostergaard et al. (2000) also demonstrated that ATP A2 expression and lignin formation coincide in Arabidopsis tissues, and docking of lignin precursors into the substrate binding site of ATP A2 predicted that coniferyl and p-coumaryl alcohols were good substrates. In contrast, the additional methoxy group of the sinapyl moiety gave rise to steric hindrance, not only in A2 type peroxidases but also in all peroxidases. We confirm these predictions for ATP A2, HRP A2, and HRP C. The specific activity of ATP A2 was lower than that of HRP A2 (pH 4-8), although a steady-state study at pH 5 demonstrated very little difference in their rate constants for reaction with H2O2 (k1 = 1.0 microM(-1) x s(-1). The oxidation of coniferyl alcohol, ferulic, p-coumaric, and sinapic acids by HRP A2, and ATP A2, however, gave modest but significantly different k3 rate constants of 8.7 +/- 0.3, 4.0 +/- 0.2, 0.70 +/- 0.03, and 0.04 +/- 0.2 microM(-1) x s(-1) for HRP A2, respectively, and 4.6 +/- 0.2, 2.3 +/- 0.1, 0.25 +/- 0.01, and 0.01 +/- 0.004 microM(-1) x s(-1) for ATP A2, respectively. The structural origin of the differential reactivity is discussed in relation to glycosylation and amino acid substitutions. The results are of general importance to the use of homologous models and structure determination at low temperatures.

The critical role of the proximal calcium ion in the structural properties of horseradish peroxidase.

The extent to which the structural Ca(2+) ions of horseradish peroxidase (HRPC) are a determinant in defining the heme pocket architecture is investigated by electronic absorption and resonance Raman spectroscopy upon removal of one Ca(2+) ion. The Fe(III) heme states are modified upon Ca(2+) depletion, with an uncommon quantum mechanically mixed spin state becoming the dominant species. Ca(2+)-depleted HRPC forms complexes with benzohydroxamic acid and CO which display spectra very similar to those of native HRPC, indicating that any changes to the distal cavity structural properties upon Ca(2+) depletion are easily reversed. Contrary to the native protein, the Ca(2+)-depleted ferrous form displays a low-spin bis-histidyl heme state and a small proportion of high-spin heme. Furthermore, the nu(Fe-Im) stretching mode downshifts 27 cm(-1) upon Ca(2+) depletion revealing a significant structural perturbation of the proximal cavity near the histidine ligand. The specific activity of the Ca(2+)-depleted enzyme is 50% that of the native form. The effects on enzyme activity and spectral features observed upon Ca(2+) depletion are reversible upon reconstitution. Evaluation of the present and previous data firmly favors the proximal Ca(2+) ion as that which is lost upon Ca(2+) depletion and which likely plays the more critical role in regulating the heme pocket structural and catalytic properties.

Effect of low temperature on soybean peroxidase: spectroscopic characterization of the quantum-mechanically admixed spin state.

A spectroscopic study of soybean peroxidase (SBP) has been carried out using electronic absorption, resonance Raman (RR) and electron paramagnetic resonance (EPR) spectroscopy in order to determine the effects of temperature on the heme spin state. Upon lowering the temperature a transition from high spin to low spin is induced in SBP resulting from conformational changes in the heme cavity, including a contraction of the heme core, the reorientation of the vinyl group in position 2 of the porphyrin macrocycle, and the binding of the distal His to the Fe atom. Moreover, the combined analysis of the data derived from the different techniques at both room and low temperatures demonstrates that at low temperature the quantum-mechanically admixed spin state (QS) of SBP has RR frequencies different from those observed for the QS species at room temperature.

Formation of two types of low-spin heme in horseradish peroxidase isoenzyme A2 at low temperature.

Electronic absorption, resonance Raman and EPR spectra are reported for ferric horseradish peroxidase isoenzyme A2 at neutral and alkaline pH together with its imidazole complex at 12 K. The data are compared with those obtained at room temperature. At neutral pH, lowering the temperature induces conformational changes with the formation of two types of low-spin hemes, a bis-histidyl type and a hydroxo type. The transition induced by lowering the temperature is accompanied by a change in the orientation of a vinyl substituent which appears less conjugated to the porphyrin macrocycle than at room temperature. At low temperature the low-spin hemes coexist with a quantum admixed spin species. All the forms are characterized by extremely high resonance Raman frequencies, indicating a contraction of the core size from that of the room temperature species. At alkaline pH, only one low-spin species is observed at both room and low temperatures, with a hydroxo ligand bound to the heme iron. The v(Fe-OH) stretching mode has been assigned at 512 cm(-1), on the basis of the isotopic shift observed in D2O and H2(18)O. This relatively low frequency, together with the anomalous shift observed in deuterium, indicates that the hydrogen bonds between the oxygen atom and the distal residues are stronger than in metmyoglobin, but weaker than those of horseradish peroxidase isoenzyme C. This is in agreement with the lower tetragonality, determined from the EPR g values, of alkaline horseradish peroxidase isoenzyme A2 than of metmyoglobin.

Peroxidase-benzhydroxamic acid complexes: spectroscopic evidence that a Fe-H2O distance of 2.6 A can correspond to hexa-coordinate high-spin heme.

Resonance Raman (RR) spectra have been obtained for single-crystal horseradish peroxidase isozyme C complexed with benzhydroxamic acid (BHA). The data are compared with those obtained in solution by both RR and electronic absorption spectroscopies at room and low (12-80 K) temperatures. Moreover, the analysis has been extended to Coprinus cinereus peroxidase complexed with BHA. The results obtained for the two complexes are very similar and are consistent with the presence of an aqua six-coordinate high-spin heme. Therefore it can be concluded that despite the rather long Fe-H2O distance of 2.6-2.7 A found by X-ray crystallography in both complexes, the distal water molecule can still coordinate to the heme iron.

Heterotropic effectors exert more significant strain on monoligated than on unligated hemoglobin.

The effect of allosteric effectors, such as inositol hexakisphosphate and/or bezafibrate, has been investigated on the unliganded human adult hemoglobin both spectroscopically (employing electronic absorption, circular dichroism, resonance Raman, and x-ray absorption near-edge spectroscopies) and functionally (following the kinetics of the first CO binding step up to a final 4% ligand saturation degree). All data indicate that the unliganded T-state is not perturbed by the interaction with either one or both effectors, suggesting that their functional influence is only exerted when a ligand molecule is bound to the heme. This is confirmed by the observation that CO dissociation from partially liganded hemoglobin (

Characterization of soybean seed coat peroxidase: resonance Raman evidence for a structure-based classification of plant peroxidases.

Electronic absorption and resonance Raman spectra of ferric and ferrous forms of a peroxidase from soybean seed coat (SBP) at neutral and alkaline pH values together with the spectra of the ferric-fluoride complex are reported. At neutral pH a quantum mechanically mixed spin state, resulting from the admixture of intermediate spin, S = 3/2, and high spin, S = 5/2, configurations, has been identified which coexists with five- and six-coordinate high-spin hemes. A complete conversion to a fluoride-ligated six-coordinate high-spin and a hydroxy-ligated six-coordinate low-spin heme are observed at acid pH in the presence of fluoride and at alkaline pH, respectively. The spectral features suggest that both the fluoride and hydroxo ligands are stabilized by hydrogen-bond interactions with the distal Arg residue and through a water molecule with the distal His residue. The ferrous form shows a single nu(Fe-Im) at 246 cm(-1) at neutral pH. The data indicate that SBP shares many characteristics with peroxidases belonging to class III of the "plant peroxidase" superfamily.

The distal cavity structure of carbonyl horseradish peroxidase as probed by the resonance Raman spectra of His 42 Leu and Arg 38 Leu mutants.

CO ligation to horseradish peroxidase C (HRPC) was studied by means of site-directed mutagenesis and resonance Raman spectroscopy. The CO complexes of HRPC His 42 --> Leu and Arg 38 --> Leu mutants were characterized at pH values ranging from 3.6 to 9.5. The vibrational frequencies of the Fe-C stretching and Fe-C-O bending modes have been identified by isotopic substitution. Both His 42 --> Leu and Arg 38 --> Leu adducts with CO displayed a single Fe-C stretching band, whereas both recombinant and wild-type HRPC-CO have two bands, corresponding to different conformers. This comparison suggests that CO is H-bonded either to the distal Arg or to the distal His in the two conformers. An acid transition, common to the wild-type protein, was observed for both mutants. This indicates that these distal amino acids do not influence the acid transition. On the contrary, an alkaline transition was only observed for the Arg 38 --> Leu mutant, which suggests that distal His is involved in the alkaline transition of HRPC-CO complex. The spectroscopic information is found to be consistent with the X-ray structure of ferric HRPC. A comparison with the CO complexes of cytochrome c peroxidase and myoglobin is performed, which displays the functional significance of the structural differences between peroxidase classes I and III and between peroxidases and globins, respectively.

Resonance Raman study of the active site of Coprinus cinereus peroxidase.

Resonance Raman (RR) spectra for the resting state ferric and the reduced ferrous forms of recombinant Coprinus cinereus peroxidase (CIP), obtained with different excitation wavelengths and in polarized light, are reported. The spectra are compared with those obtained previously for cytochrome c peroxidase expressed in Escherichia coli [(CCP(MI)] and horseradish peroxidase (HRP-C). Although the enzymic properties of CIP and HRP-C are similar, the RR data show that, in terms of the heme cavity structures, CIP and CCP(MI) are much more closely related to each other than to HRP-C. The ferric state of CIP at neutral pH is characteristic mainly of a five-coordinate high spin heme. However, the lower frequency of the v2 mode and a higher frequency of the v(C = C) vinyl stretching modes for CIP as compared to CCP, indicate a higher degree of vibrational coupling between the two modes in CIP. In addition, CIP is rather unstable under low laser power irradiation as an irreversible transition to a six-coordinate high spin heme followed by a second transition to a six-coordinate low spin heme is observed. This instability of CIP as compared to CCP(MI) is proposed to be a consequence of the presence of a distal Phe54 in CIP rather than the homologous Trp51 in CCP, as Trp51 is hydrogen-bonded to a distal water molecule located above the heme Fe thereby preventing its coordination in CCP. In CIP the FeII-His RR band has two components with frequencies at 230 and 211 cm-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Spin state and axial ligand bonding in the hydroxide complexes of metmyoglobin, methemoglobin, and horseradish peroxidase at room and low temperatures.

Absorption and resonance Raman spectra using Soret excitation of alkaline metmyoglobin (metMb), methemoglobin (metHb), and horseradish peroxidase (HRP) were obtained at room and low temperature. At 298 K both metMb and metHb exhibit two isotope-sensitive bands assigned to high- and low-spin nu(Fe-OH) stretching modes, respectively, which are correlated with the spin-state population. The low-spin stretch occurs 60 cm-1 to higher energy than the corresponding high-spin vibration. When the temperature is lowered, only the low-spin species is observed. HRP exhibits at both 298 and 20 K only the low-spin nu(Fe-OH) stretching mode, which occurs 50 cm-1 to lower energy than the corresponding modes observed in the globins. This is explained in the context of a strong hydrogen bond between the hydroxyl ligand and the distal His42 and/or Arg38. Lowering temperature causes in all of the examined proteins a strengthening of the Fe-OH bond and a contraction of the core of about 0.01 A, as determined by the upshifting of the low-spin nu(Fe-OH) stretching mode and the core size marker bands. Both effects are ascribed to an increase of the packing forces.

Inclusion complex formation of 1,8-dihydroxyanthraquinone with cyclodextrins in aqueous solution and in solid state.

Complex formation between cyclodextrins and 1,8-dihydroxyanthraquinone in buffer solution has been investigated using absorption, its second derivative (D2), and fluorescence spectroscopy. The results showed that whereas the self-association process was found for 1,8-dihydroxyanthraquinone alone, the monomeric form is microincluded in beta- and gamma-cyclodextrins. The interaction is more favored as the cavity size of cyclodextrins is larger, the molecule being more tightly bound with gamma- than with beta-cyclodextrin. The complex formation inhibits the excited-state intramolecular proton transfer process that has already been reported for 1,8-dihydroxyanthraquinone alone.