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.

Mutation of residues critical for benzohydroxamic acid binding to horseradish peroxidase isoenzyme C.

Aromatic substrate binding to peroxidases is mediated through hydrophobic and hydrogen bonding interactions between residues on the distal side of the heme and the substrate molecule. The effects of perturbing these interactions are investigated by an electronic absorption and resonance Raman study of benzohydroxamic acid (BHA) binding to a series of mutants of horseradish peroxidase isoenzyme C (HRPC). In particular, the Phe179 --> Ala, His42 --> Glu variants and the double mutant His42 --> Glu:Arg38 --> Leu are studied in their ferric state at pH 7 with and without BHA. A comparison of the data with those previously reported for wild-type HRPC and other distal site mutants reaffirms that in the resting state mutation of His42 leads to an increase of 6-coordinate aquo heme forms at the expense of the 5-coordinate heme state, which is the dominant species in wild-type HRPC. The His42Glu:Arg38Leu double mutant displays an enhanced proportion of the pentacoordinate heme state, similar to the single Arg38Leu mutant. The heme spin states are insensitive to mutation of the Phe179 residue. The BHA complexes of all mutants are found to have a greater amount of unbound form compared to the wild-type HRPC complex. It is apparent from the spectral changes induced on complexation with BHA that, although Phe179 provides an important hydrophobic interaction with BHA, the hydrogen bonds formed between His42 and, in particular, Arg38 and BHA assume a more critical role in the binding of BHA to the resting state.

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.

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.

Cationic ascorbate peroxidase isoenzyme II from tea: structural insights into the heme pocket of a unique hybrid peroxidase.

The novel class III ascorbate peroxidase isoenzyme II from tea leaves (TcAPXII), with an unusually high specific ascorbate peroxidase activity associated with stress response, has been characterized by resonance Raman (RR), electronic absorption, and Fourier transform infrared (FT-IR) spectroscopies. Ferric and ferrous forms and the complexes with fluoride, cyanide, and CO have been studied at various pH values. The overall blue shift of the electronic absorption spectrum, the high RR frequencies of the core size marker bands, similar to those of 6-coordinate low-spin heme, and the complex RR spectrum in the low-frequency region of ferric TcAPXII indicate that this protein contains an unusual 5-coordinate quantum mechanically mixed-spin heme. The spectra of both the fluoride and the CO adducts suggest that these exogenous ligands are strongly hydrogen-bonded with a residue that appears to be unique to this peroxidase. Electronic absorption spectra also emphasize structural differences between the benzhydroxamic acid binding sites of TcAPXII and horseradish peroxidases (HRPC). It is concluded that TcAPXII is a paradigm peroxidase since it is the first example of a hybrid enzyme that combines spectroscopic signatures, structural elements, and substrate specificities previously reported only for distinct class I and class III peroxidases.

A novel heme protein, the Cu,Zn-superoxide dismutase from Haemophilus ducreyi.

Haemophilus ducreyi, the causative agent of the genital ulcerative disease known as chancroid, is unable to synthesize heme, which it acquires from humans, its only known host. Here we provide evidence that the periplasmic Cu,Zn-superoxide dismutase from this organism is a heme-binding protein, unlike all the other known Cu,Zn-superoxide dismutases from bacterial and eukaryotic species. When the H. ducreyi enzyme was expressed in Escherichia coli cells grown in standard LB medium, it contained only limited amounts of heme covalently bound to the polypeptide but was able efficiently to bind exogenously added hemin. Resonance Raman and electronic spectra at neutral pH indicate that H. ducreyi Cu,Zn-superoxide dismutase contains a 6-coordinated low spin heme, with two histidines as the most likely axial ligands. By site-directed mutagenesis and analysis of a structural model of the enzyme, we identified as a putative axial ligand a histidine residue (His-64) that is present only in the H. ducreyi enzyme and that was located at the bottom of the dimer interface. The introduction of a histidine residue in the corresponding position of the Cu,Zn-superoxide dismutase from Haemophilus parainfluenzae was not sufficient to confer the ability to bind heme, indicating that other residues neighboring His-64 are involved in the formation of the heme-binding pocket. Our results suggest that periplasmic Cu,Zn-superoxide dismutase plays a role in heme metabolism of H. ducreyi and provide further evidence for the structural flexibility of bacterial enzymes of this class.

Structure of soybean seed coat peroxidase: a plant peroxidase with unusual stability and haem-apoprotein interactions.

Soybean seed coat peroxidase (SBP) is a peroxidase with extraordinary stability and catalytic properties. It belongs to the family of class III plant peroxidases that can oxidize a wide variety of organic and inorganic substrates using hydrogen peroxide. Because the plant enzyme is a heterogeneous glycoprotein, SBP was produced recombinant in Escherichia coli for the present crystallographic study. The three-dimensional structure of SBP shows a bound tris(hydroxymethyl)aminomethane molecule (TRIS). This TRIS molecule has hydrogen bonds to active site residues corresponding to the residues that interact with the small phenolic substrate ferulic acid in the horseradish peroxidase C (HRPC):ferulic acid complex. TRIS is positioned in what has been described as a secondary substrate-binding site in HRPC, and the structure of the SBP:TRIS complex indicates that this secondary substrate-binding site could be of functional importance. SBP has one of the most solvent accessible delta-meso haem edge (the site of electron transfer from reducing substrates to the enzymatic intermediates compound I and II) so far described for a plant peroxidase and structural alignment suggests that the volume of Ile74 is a factor that influences the solvent accessibility of this important site. A contact between haem C8 vinyl and the sulphur atom of Met37 is observed in the SBP structure. This interaction might affect the stability of the haem group by stabilisation/delocalisation of the porphyrin pi-cation of compound I.

Haem-linked interactions in horseradish peroxidase revealed by spectroscopic analysis of the Phe-221-->Met mutant.

A gene encoding a Phe-221-to-Met substitution in the haem enzyme horseradish peroxidase has been constructed and expressed in Escherichia coli. In the wild-type enzyme the side chain of Phe-221 is tightly stacked against the imidazole ring of His-170, which provides the only axial ligand to the haem iron atom. The Phe-221-->Met enzyme is active, and forms characteristic complexes with typical peroxidase ligands (CO, cyanide, fluoride), and with benzhydroxamic acid. Significant differences between the mutant and wild-type enzymes can be detected spectroscopically. These include a change in the Fe(III) resting state of the enzyme to an unusual quantum mechanically mixed-spin haem species, a marked decrease in the pK(a) of the alkaline transition and a reduction in enzyme stability at alkaline pH for both Fe(III) and Fe(II) forms. The perturbation of the haem pocket in the mutant can be attributed to several factors, including the increased steric freedom and solvent accessibility of the His-170 ligand, as indicated by (1)H-NMR data, and the loss of the pi-pi interaction between His-170 and Phe-221.

Effect of pH on axial ligand coordination of cytochrome c" from Methylophilus methylotrophus and horse heart cytochrome c.

The effect of protons on the axial ligand coordination and on structural aspects of the protein moiety of cytochrome c' ' from Methylophilus methylotrophus, an obligate methylotroph, has been investigated down to very low pH (i.e., 0.3). The unusual resistance of this cytochrome to very low pH values has been exploited to carry out this study in comparison with horse heart cytochrome c. The experiments were undertaken at a constant phosphate concentration to minimize the variation of ionic strength with pH. The pH-linked effects have been monitored at 23 degrees C in the oxidized forms of both cytochromes by following the variations in the electronic absorption, circular dichroism and resonance Raman spectra. This approach has enabled the conformational changes of the heme surroundings to be monitored and compared with the concomitant overall structural rearrangements of the molecule. The results indicate that horse heart cytochrome c undergoes a first conformational change at around pH 2.0. This event is possibly related to the cleavage of the Fe-Met80 bond and a likely coordination of a H(2)O molecule as a sixth axial ligand. Conversely, in cytochrome c" from M. methylotrophus, a variation of the axial ligand coordination occurs at a pH that is about 1 unit lower. Further, it appears that a concerted cleavage of both His ligands takes place, suggesting indeed that the different axial ligands present in horse heart cytochrome c (Met/His) and in cytochrome c" from M. methylotrophus (His/His) affect the heme conformational changes.

Anion- and pH-linked conformational transition in horseradish peroxidase.

In a previous study we have shown that bringing horseradish peroxidase to pH 3.0 induces a spectroscopic transition (G. Smulevich et al., Biochemistry 36 (1997) 640). We have extended the investigation on this pH-linked conformational change to different experimental conditions, such as (i) in phosphate alone, (ii) in HCl alone and (iii) in phosphate + NaCl. The data obtained allow a number of conclusions to be drawn, namely: (a) the exposure to pH 3.0 under all conditions brings about an alteration of the distal portion of the heme pocket, leading to the rapid formation of a hexa-coordinated species; (b) only in the presence of phosphate is the hexa-coordination followed by a slow cleavage (or severe weakening) of the proximal Fe-His bond, and (c) the rate of this second process is speeded up in the presence of Cl- ions. Such observations underline the presence of a communication pathway between the two opposite sides of the heme pocket, such that any alteration of the structural arrangement on one side of the heme cavity is transmitted to the other, inducing a corresponding conformational change.

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.

The quantum mixed-spin heme state of barley peroxidase: A paradigm for class III peroxidases.

Electronic absorption and resonance Raman (RR) spectra of the ferric form of barley grain peroxidase (BP 1) at various pH values, at both room temperature and 20 K, are reported, together with electron paramagnetic resonance spectra at 10 K. The ferrous forms and the ferric complex with fluoride have also been studied. A quantum mechanically mixed-spin (QS) state has been identified. The QS heme species coexists with 6- and 5-cHS hemes; the relative populations of these three spin states are found to be dependent on pH and temperature. However, the QS species remains in all cases the dominant heme spin species. Barley peroxidase appears to be further characterized by a splitting of the two vinyl stretching modes, indicating that the vinyl groups are differently conjugated with the porphyrin. An analysis of the currently available spectroscopic data for proteins from all three peroxidase classes suggests that the simultaneous occurrence of the QS heme state as well as the splitting of the two vinyl stretching modes is confined to class III enzymes. The former point is discussed in terms of the possible influences of heme deformations on heme spin state. It is found that moderate saddling alone is probably not enough to cause the QS state, although some saddling may be necessary for the QS state.

Role of the distal phenylalanine 54 on the structure, stability, and ligand binding of Coprinus cinereus peroxidase.

Resonance Raman and electronic absorption spectra obtained at various pH values for the Fe3+ form of distal F54 mutants of Coprinus cinereus peroxidase are reported, together with the Fe2+ form and fluoride and imidazole adducts at pH 6.0, 5.0, and 10.5, respectively. The distal phenylalanine residue has been replaced by the small aliphatic residues glycine and valine and the hydrogen-bonding aromatic residues tyrosine and tryptophan (F54G, -V, -Y, and -W, respectively). These mutations resulted in transitions between ferric high-spin five-coordinate and six-coordinate forms, and caused a decrease of the pKa of the alkaline transition together with a higher tendency for unfolding. The mutations also alter the ability of the proteins to bind fluoride in such a way that those that are six-coordinate at pH 5.0 bind more strongly than both wild-type CIP and F54Y which are five-coordinate at this pH value. The data provide evidence that the architecture of the distal pocket of CIP is altered by the mutations. Direct evidence is provided that the distal phenylalanine plays an important role in controlling the conjugation between the vinyl double bonds and the porphyrin macrocycle, as indicated by the reorientation of the vinyl groups upon mutation of phenylalanine with the small aliphatic side chains of glycine and valine residues. Furthermore, it appears that the presence of the hydrogen-bonding tyrosine or tryptophan in the cavity increases the pKa of the distal histidine for protonation compared with that of wild-type CIP.

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.

Understanding heme cavity structure of peroxidases: comparison of electronic absorption and resonance Raman spectra with crystallographic results.

Electronic absorption and resonance Raman spectra of various peroxidases and selected site-directed mutants are reported. These results and the X-ray crystal structure data are critically analyzed and underline the differences that exist between the crystal and solution states. The effect of the vinyl conjugation on the electronic absorption maxima and the influence of the ligand nature on the wavelength of the charge-transfer (CT1) band are shown to be useful probes of subtle interactions in the heme pocket. The spectroscopic differences observed between the three classes of peroxidases are discussed in terms of their structural diversity.

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.

Spectroscopic characterization of recombinant pea cytosolic ascorbate peroxidase: similarities and differences with cytochrome c peroxidase.

Recombinant pea cytosolic ascorbate peroxidase (APX) has been characterized by resonance Raman (RR) and electronic absorption spectroscopies. The ferric and ferrous forms together with the complexes with fluoride and imidazole have been studied and compared with the corresponding spectra of cytochrome c peroxidase (CCP). Ferric APX at neutral pH is a mixture of 6- and 5-coordinate high-spin and 6-c low-spin hemes, the latter two species being dominant. The results suggest that the low-spin form derives from a water/hydroxo ligand bound to the heme iron and not from a strong internal ligand as observed in CCP at alkaline pH. Two Fe-Im stretching modes are identified, as in CCP, but the RR frequencies confirm a weaker His163-Asp208 hydrogen bond than in CCP, as suggested on the basis of the X-ray structure [Patterson, W. R., and Poulos, T. L. (1995) Biochemistry 34, 4331-4341]. The data show that CCP and APX have markedly different orientations of the vinyl substituents on the heme chromophore resulting from different steric constraints exerted by the protein matrix.