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1.
Biochemistry ; 40(37): 11013-21, 2001 Sep 18.
Article in English | MEDLINE | ID: mdl-11551197

ABSTRACT

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.


Subject(s)
Peroxidases/chemistry , Arabidopsis/enzymology , Catalytic Domain , Coumaric Acids/metabolism , Crystallography, X-Ray , Horseradish Peroxidase/chemistry , Horseradish Peroxidase/classification , Horseradish Peroxidase/metabolism , Models, Molecular , Peroxidases/classification , Peroxidases/metabolism , Phenols/metabolism , Plant Proteins/chemistry , Plant Proteins/classification , Plant Proteins/metabolism , Recombinant Proteins , Spectrum Analysis, Raman , Substrate Specificity
2.
J Biol Chem ; 276(44): 40704-11, 2001 Nov 02.
Article in English | MEDLINE | ID: mdl-11546788

ABSTRACT

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.


Subject(s)
Calcium/metabolism , Horseradish Peroxidase/metabolism , Chromatography, Gel , Heme/metabolism , Horseradish Peroxidase/chemistry , Models, Molecular , Protein Conformation , Spectrum Analysis, Raman
3.
J Biol Chem ; 276(32): 30326-34, 2001 Aug 10.
Article in English | MEDLINE | ID: mdl-11369755

ABSTRACT

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.


Subject(s)
Haemophilus ducreyi/enzymology , Heme/chemistry , Superoxide Dismutase/chemistry , Superoxide Dismutase/metabolism , Amino Acid Sequence , Dimerization , Electrophoresis, Polyacrylamide Gel , Escherichia coli/metabolism , Hemin/pharmacology , Hydrogen-Ion Concentration , Ligands , Models, Molecular , Mutagenesis, Site-Directed , Plasmids/metabolism , Protein Binding , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , Software , Spectrophotometry , Spectrum Analysis, Raman , Superoxide Dismutase/isolation & purification
4.
Protein Sci ; 10(1): 108-15, 2001 Jan.
Article in English | MEDLINE | ID: mdl-11266599

ABSTRACT

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.


Subject(s)
Glycine max/enzymology , Peroxidase/chemistry , Binding Sites , Crystallization , Crystallography, X-Ray , Models, Molecular , Peroxidase/metabolism , Protein Conformation , Protein Folding , Recombinant Proteins/chemistry , Seeds/enzymology
5.
Biochemistry ; 39(28): 8234-42, 2000 Jul 18.
Article in English | MEDLINE | ID: mdl-10889031

ABSTRACT

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.


Subject(s)
Cytochrome c Group/chemistry , Methylophilus methylotrophus/chemistry , Animals , Circular Dichroism , Cytochrome c Group/metabolism , Enzyme Stability , Horses , Hydrogen-Ion Concentration , Ligands , Methylophilus methylotrophus/enzymology , Myocardium/enzymology , Spectrum Analysis, Raman
6.
J Inorg Biochem ; 79(1-4): 25-30, 2000 Apr.
Article in English | MEDLINE | ID: mdl-10830843

ABSTRACT

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.


Subject(s)
Horseradish Peroxidase/chemistry , Horseradish Peroxidase/metabolism , Anions , Hydrochloric Acid/pharmacology , Hydrogen-Ion Concentration , Isoenzymes/chemistry , Isoenzymes/metabolism , Kinetics , Phosphates/pharmacology , Protein Conformation/drug effects , Spectrophotometry
7.
J Inorg Biochem ; 79(1-4): 269-74, 2000 Apr.
Article in English | MEDLINE | ID: mdl-10830877

ABSTRACT

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.


Subject(s)
Glycine max/enzymology , Peroxidases/chemistry , Binding Sites , Electron Spin Resonance Spectroscopy/methods , Freezing , Heme/chemistry , Imidazoles/chemistry , Porphyrins/chemistry , Protein Conformation , Quantum Theory , Recombinant Proteins/chemistry , Spectrophotometry/methods , Spectrum Analysis, Raman/methods , Thermodynamics
8.
J Biol Inorg Chem ; 5(2): 227-35, 2000 Apr.
Article in English | MEDLINE | ID: mdl-10819468

ABSTRACT

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.


Subject(s)
Heme/chemistry , Horseradish Peroxidase/chemistry , Arginine/chemistry , Cold Temperature , Electron Spin Resonance Spectroscopy , Histidine/chemistry , Hydrogen Bonding , Hydrogen-Ion Concentration , Isoenzymes/chemistry , Protein Conformation , Spectrophotometry, Infrared
9.
J Biol Inorg Chem ; 4(1): 39-47, 1999 Feb.
Article in English | MEDLINE | ID: mdl-10499101

ABSTRACT

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.


Subject(s)
Horseradish Peroxidase/chemistry , Horseradish Peroxidase/metabolism , Hydroxamic Acids/chemistry , Hydroxamic Acids/metabolism , Coprinus/enzymology , Crystallization , Heme , Iron/chemistry , Iron/metabolism , Isoenzymes/chemistry , Isoenzymes/metabolism , Metmyoglobin/chemistry , Metmyoglobin/metabolism , Peroxidase/chemistry , Peroxidase/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Solutions , Spectrum Analysis, Raman , Temperature , Water
10.
Biochemistry ; 38(24): 7819-27, 1999 Jun 15.
Article in English | MEDLINE | ID: mdl-10387022

ABSTRACT

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.


Subject(s)
Coprinus/enzymology , Fungal Proteins/chemistry , Peroxidase/chemistry , Phenylalanine/chemistry , Binding Sites/genetics , Coprinus/genetics , Enzyme Stability/genetics , Fungal Proteins/genetics , Fungal Proteins/metabolism , Glycine/genetics , Hydrogen-Ion Concentration , Imidazoles/chemistry , Ligands , Mutagenesis, Site-Directed , Peroxidase/genetics , Peroxidase/metabolism , Phenylalanine/genetics , Phenylalanine/metabolism , Sodium Fluoride/chemistry , Sodium Fluoride/metabolism , Spectrophotometry , Spectrum Analysis, Raman , Structure-Activity Relationship , Titrimetry , Tryptophan/genetics , Tyrosine/genetics , Valine/genetics
11.
Eur J Biochem ; 251(3): 830-8, 1998 Feb 01.
Article in English | MEDLINE | ID: mdl-9490058

ABSTRACT

Heme peroxidases of prokaryotic, plant and fungal origin share the essential His and Arg catalytic residues of the distal cavity and a proximal His bound to heme iron. Spectroscopic techniques, in contrast to X-ray crystallography, are well suited to detect the precise structure, spin and coordination states of the heme as influenced by its near environment. Resonance Raman and electronic absorption spectra obtained at various pH values for Fe3+ and Fe2+ forms of distal Arg51 mutants of the fungal Coprinus cinereus peroxidase are reported, together with the fluoride adducts at pH 5.0. This basic catalytic residue has been replaced by the aliphatic residue Leu, the polar residues Asn and Gln and the basic residue Lys (Arg51-->Leu, Asn, Gln, and Lys, respectively). These mutations cause changes in the coordination and spin states of the heme iron, and in the v(Fe-Im) stretching frequency. The variations are explained in terms of pH-dependent changes, charge location, size and hydrogen-bonding acceptor/donor properties of the residue at position 51. The present work indicates that the hydrogen-bond capability of the residue in position 51 influences the occupancy of water molecules in the distal cavity and the ability to form stable complexes between anionic ligands and the heme Fe atom.


Subject(s)
Arginine , Coprinus/enzymology , Peroxidases/chemistry , Protein Conformation , Amino Acid Sequence , Amino Acid Substitution , Binding Sites , Crystallography, X-Ray , Fluorides/pharmacology , Heme/metabolism , Histidine , Iron/metabolism , Ligands , Models, Molecular , Mutagenesis, Site-Directed , Recombinant Proteins/chemistry , Spectrophotometry , Spectrum Analysis, Raman
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