Peroxidase-catalyzed co-oxidation of 3,3',5,5'-tetramethylbenzidine in the presence of substituted phenols and their polydisulfides.

The steady-state kinetics of the horseradish peroxidase (HRP)-catalyzed oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) has been studied in the presence of 2-amino-4-nitrophenol (ANP), gallic acid (GA) or 4,4'-dihydroxydiphenylsulfone (DDS) and their polydisulfides poly(ADSNP), poly(DSGA), poly(DSDDS) at 20 degrees C in 10 mM phosphate buffer, pH 6.4, supplemented with 5-10% dimethylformamide. The second-order rate constants for the reactions of ANP, GA, poly(DSGA) and poly(DSDDS) with HRP-Compound I (k2) and Compound II (k3) have been determined at 25 degrees C in 10 mM phosphate buffer, pH 6.0 by stopped-flow spectrophotometry. ANP, GA and their polydisulfides strongly inhibited HRP-catalyzed TMB oxidation. Inhibition constants (Ki) and stoichiometric coefficients of inhibition (f) have been determined for these reactions. The most effective inhibitor was poly(DSGA) (Ki=1.3 microM, f=35.6). The oxidation of substrate pairs by HRP, i.e., TMB-DDS and TMB-poly(DSDDS) at pH 7.2 resulted in a approximately 8- and approximately 12-fold stimulation of TMB oxidation rates, respectively. The mechanisms of the HRP-catalyzed co-oxidation of TMB-phenol pairs are discussed.

Mechanism of reaction of hydrogen peroxide with horseradish peroxidase: identification of intermediates in the catalytic cycle.

The mechanism of the reaction of horseradish peroxidase isoenzyme C (HRPC) with hydrogen peroxide to form the reactive enzyme intermediate compound I has been studied using electronic absorbance, rapid-scan stopped-flow, and electron paramagnetic resonance (EPR) spectroscopies at both acid and basic pH. The roles of the active site residues His42 and Arg38 in controlling heterolytic cleavage of the H(2)O(2) oxygen-oxygen bond have been probed with site-directed mutant enzymes His42 --> Leu (H42L), Arg38 --> Leu (R38L), and Arg38 --> Gly (R38G). The biphasic reaction kinetics of H42L with H(2)O(2) suggested the presence of an intermediate species and, at acid pH, a reversible second step, probably due to a neutral enzyme-H(2)O(2) complex and the ferric-peroxoanion-containing compound 0. EPR also indicated the formation of a protein radical situated more than approximately 10 A from the heme iron. The stoichiometry of the reaction of the H42L/H(2)O(2) reaction product and 2,2'-azinobis(3-ethylbenzothiazolinesulfonic acid) (ABTS) was concentration dependent and fell from a value of 2 to 1 above 0.7 mM ABTS. These data can be explained if H(2)O(2) undergoes homolytic cleavage in H42L. The apparent rate of compound I formation by H42L, while low, was pH independent in contrast to wild-type HRPC where the rate falls at acid pH, indicating the involvement of an ionizable group with pK(a) approximately 4. In R38L and R38G, the apparent pK(a) was shifted to approximately 8 but there is no evidence that homolytic cleavage of H(2)O(2) occurs. These data suggest that His42 acts initially as a proton acceptor (base catalyst) and then as a donor (acid catalyst) at neutral pH and predict the observed slower rate and lower efficiency of heterolytic cleavage observed at acid pH. Arg38 is influential in lowering the pK(a) of His42 and additionally in aligning H(2)O(2) in the active site, but it does not play a direct role in proton transfer.

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.

Phenol-oxidizing peroxidases contribute to the protection of plants from ultraviolet radiation stress.

We have studied the mechanism of UV protection in two duckweed species (Lemnaceae) by exploiting the UV sensitivity of photosystem II as an in situ sensor for radiation stress. A UV-tolerant Spirodela punctata G.F.W. Meyer ecotype had significantly higher indole-3-acetic acid (IAA) levels than a UV-sensitive ecotype. Parallel work on Lemna gibba mutants suggested that UV tolerance is linked to IAA degradation rather than to levels of free or conjugated IAA. This linkage is consistent with a role for class III phenolic peroxidases, which have been implicated both in the degradation of IAA and the cross-linking of various UV-absorbing phenolics. Biochemical analysis revealed increased activity of a specific peroxidase isozyme in both UV-tolerant duckweed lines. The hypothesis that peroxidases play a role in UV protection was tested in a direct manner using genetically modified tobacco (Nicotiana sylvestris). It was found that increased activity of the anionic peroxidase correlated with increased tolerance to UV radiation as well as decreased levels of free auxin. We conclude that phenol-oxidizing peroxidases concurrently contribute to UV protection as well as the control of leaf and plant architecture.

Reactivity of horseradish peroxidase compound II toward substrates: kinetic evidence for a two-step mechanism.

Transient kinetic analysis of biphasic, single turnover data for the reaction of 2,2'-azino-bis[3-ethylbenzthiazoline-6-sulfonic acid] (ABTS) with horseradish peroxidase (HRPC) compound II demonstrated preequilibrium binding of ABTS (k(+5) = 7.82 x 10(4) M(-)(1) s(-)(1)) prior to rate-limiting electron transfer (k(+6) = 42.1 s(-)(1)). These data were obtained using a stopped-flow method, which included ascorbate in the reaction medium to maintain a low steady-state concentration of ABTS (pseudo-first-order conditions) and to minimize absorbance changes in the Soret region due to the accumulation of ABTS cation radicals. A steady-state kinetic analysis of the reaction confirmed that the reduction of HRPC compound II by this substrate is rate-limiting in the complete peroxidase cycle. The reaction of HRPC with o-diphenols has been investigated using a chronometric method that also included ascorbate in the assay medium to minimize the effects of nonenzymic reactions involving phenol-derived radical products. This enabled the initial rates of o-diphenol oxidation at different hydrogen peroxide and o-diphenol concentrations to be determined from the lag period induced by the presence of ascorbate. The kinetic analysis resolved the reaction of HRPC compound II with o-diphenols into two steps, initial formation of an enzyme-substrate complex followed by electron transfer from the substrate to the heme. With o-diphenols that are rapidly oxidized, the heterolytic cleavage of the O-O bond of the heme-bound hydrogen peroxide (k(+2) = 2.17 x 10(3) s(-)(1)) is rate-limiting. The size and hydrophobicity of the o-diphenol substrates are correlated with their rate of binding to HRPC, while the electron density at the C-4 hydroxyl group predominantly influences the rate of electron transfer to the heme.

Studies with substrate and cofactor analogues provide evidence for a radical mechanism in the chorismate synthase reaction.

Chorismate synthase catalyzes the conversion of 5-enolpyruvylshikimate 3-phosphate (EPSP) to chorismate. The strict requirement for a reduced FMN cofactor and a trans-1,4-elimination are unusual. (6R)-6-Fluoro-EPSP was shown to be converted to chorismate stoichiometrically with enzyme-active sites in the presence of dithionite. This conversion was associated with the oxidation of FMN to give a stable flavin semiquinone. The IC(50) of the fluorinated substrate analogue was 0.5 and 250 microm with the Escherichia coli enzyme, depending on whether it was preincubated with the enzyme or not. The lack of dissociation of the flavin semiquinone and chorismate from the enzyme appears to be the basis of the essentially irreversible inhibition by this analogue. A dithionite-dependent transient formation of flavin semiquinone during turnover of (6S)-6-fluoro-EPSP has been observed. These reactions are best rationalized by radical chemistry that is strongly supportive of a radical mechanism occurring during normal turnover. The lack of activity with 5-deaza-FMN provides additional evidence for the role of flavin in catalysis by the E. coli enzyme.

Time-resolved infrared spectroscopy reveals a stable ferric heme-NO intermediate in the reaction of Paracoccus pantotrophus cytochrome cd1 nitrite reductase with nitrite.

Cytochrome cd(1) is a respiratory enzyme that catalyzes the physiological one-electron reduction of nitrite to nitric oxide. The enzyme is a dimer, each monomer containing one c-type cytochrome center and one active site d(1) heme. We present stopped-flow Fourier transform infrared data showing the formation of a stable ferric heme d(1)-NO complex (formally d(1)Fe(II)-NO(+)) as a product of the reaction between fully reduced Paracoccus pantotrophus cytochrome cd(1) and nitrite, in the absence of excess reductant. The Fe-(14)NO nu(NO) stretching mode is observed at 1913 cm(-1) with the corresponding Fe-(15)NO band at 1876 cm(-1). This d(1) heme-NO complex is still readily observed after 15 min. EPR and visible absorption spectroscopic data show that within 4 ms of the initiation of the reaction, nitrite is reduced at the d(1) heme, and a cFe(III) d(1)Fe(II)-NO complex is formed. Over the next 100 ms there is an electron redistribution within the enzyme to give a mixed species, 55% cFe(III) d(1)Fe(II)-NO and 45% cFe(II) d(1)Fe(II)-NO(+). No kinetically competent release of NO could be detected, indicating that at least one additional factor is required for product release by the enzyme. Implications for the mechanism of P. pantotrophus cytochrome cd(1) are discussed.

Stopped-flow and steady-state study of the diphenolase activity of mushroom tyrosinase.

The reaction of mushroom (Agaricus bisporus) tyrosinase with dioxygen in the presence of several o-diphenolic substrates has been studied by steady-state and transient-phase kinetics in order to elucidate the rate-limiting step and to provide new insights into the mechanism of oxidation of these substrates. A kinetic analysis has allowed for the first time the determination of individual rate constants for several of the partial reactions that comprise the catalytic cycle. Mushroom tyrosinase rapidly reacts with dioxygen with a second-order rate constant k(+8) = 2.3 x 10(7) M(-)(1) s(-)(1), which is similar to that reported for hemocyanins [(1.3 x 10(6))-(5.7 x 10(7)) M(-)(1) s(-)(1)]. Deoxytyrosinase binds dioxygen reversibly at the binuclear Cu(I) site with a dissociation constant K(D)(O)()2 = 46.6 microM, which is similar to the value (K(D)(O)()2 = 90 microM) reported for the binding of dioxygen to Octopus vulgaris deoxyhemocyanin [Salvato et al. (1998) Biochemistry 37, 14065-14077]. Transient and steady-state kinetics showed that o-diphenols such as 4-tert-butylcatechol react significantly faster with mettyrosinase (k(+2) = 9.02 x 10(6) M(-)(1) s(-)(1)) than with oxytyrosinase (k(+6) = 5.4 x 10(5) M(-)(1) s(-)(1)). This difference is interpreted in terms of differential steric and polar effects that modulate the access of o-diphenols to the active site for these two forms of the enzyme. The values of k(cat) for several o-diphenols are also consistent with steric and polar factors controlling the mobility, orientation, and thence the reactivity of substrates at the active site of tyrosinase.

Reversible alkaline inactivation of lignin peroxidase involves the release of both the distal and proximal site calcium ions and bishistidine co-ordination of the haem.

Phanerochaete chrysosporium lignin peroxidase isoenzyme H2 (LiP H2) exhibits a transition to a stable, inactive form at pH 9.0 with concomitant spectroscopic changes. The Söret peak intensity decreases some 55% with a red shift from 408 to 412 nm; the bands at 502 nm and 638 nm disappear and the peak at 536 nm increases. The EPR spectrum changes from a signal typical of high spin ferric haem to an exclusively low spin spectrum with g=2.92, 2.27, 1.50. These data indicate that the active pentaco-ordinated haem is converted into a hexaco-ordinated species at alkaline pH. Room temperature near-IR MCD data coupled with the EPR spectrum allow us to assign the haem co-ordination of alkali-inactivated enzyme as bishistidine. Re-acidification of the alkali-inactivated enzyme to pH 6 induces further spectroscopic changes and generates an irreversibly inactivated species. By contrast, a pH shift from 9.0 to 6.0 with simultaneous addition of 50 mM CaCl(2) results in the recovery of the initial activity together with the spectroscopic characteristics of the native ferric enzyme. Incubating with 50 mM CaCl(2) at a pH between 6.0 and 9.0 can also re-activate the enzyme. Divalent metals other than Ca(2+) do not result in restoration of activity. Experiments with (45)Ca indicate that two tightly bound calcium ions per enzyme monomer are lost during inactivation and reincorporated during subsequent re-activation, consistent with the presence of two structural Ca(2+) ions in LiP H2. It is concluded that both the structural Ca(2+) ions play key roles in the reversible alkaline inactivation of LiP H2.

Evidence for a major structural change in Escherichia coli chorismate synthase induced by flavin and substrate binding.

Chorismate synthase (EC 4.6.1.4) catalyses the conversion of 5-enolpyruvylshikimate 3-phosphate (EPSP) into chorismate, and requires reduced FMN as a cofactor. The enzyme can bind first oxidized FMN and then EPSP to form a stable ternary complex which does not undergo turnover. This complex can be considered to be a model of the ternary complex between enzyme, EPSP and reduced FMN immediately before catalysis commences. It is shown that the binding of oxidized FMN and EPSP to chorismate synthase affects the properties and structure of the protein. Changes in small-angle X-ray scattering data, decreased susceptibility to tryptic digestion and altered Fourier-transform (FT)-IR spectra provide the first strong evidence for major structural changes in the protein. The tetrameric enzyme undergoes correlated screw movements leading to a more overall compact shape, with no change in oligomerization state. The changes in the FT-IR spectrum appear to reflect changes in the environment of the secondary-structural elements rather than alterations in their distribution, because the far-UV CD spectrum changes very little. Changes in the mobility of the protein during non-denaturing PAGE indicate that the ternary complex may exhibit less conformational flexibility than the apoprotein. Increased enzyme solubility and decreased tryptophan fluorescence are discussed in the light of the observed structural changes. The secondary structure of the enzyme was investigated using far-UV CD spectroscopy, and the tertiary structure was predicted to be an alpha-beta-barrel using discrete state-space modelling.

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.

Identification of skatolyl hydroperoxide and its role in the peroxidase-catalysed oxidation of indol-3-yl acetic acid.

Indol-3-yl acetic acid (IAA, auxin) is a plant hormone whose degradation is a key determinant of plant growth and development. The first evidence for skatolyl hydroperoxide formation during the plant peroxidase-catalysed degradation of IAA has been obtained by electrospray MS. Skatolyl hydroperoxide degrades predominantly non-enzymically to oxindol-3-yl carbinol but in part enzymically into indol-3-yl methanol via a peroxidase cycle in which IAA acts as an electron donor. Skatolyl hydroperoxide is degradable by catalase. Horseradish peroxidase isoenzyme C (HRP-C) and anionic tobacco peroxidase (TOP) exhibit differences in their mechanisms of reaction. The insensitivity of the HRP-C-catalysed reaction to catalase is ascribed to the formation of HRP-C Compound III at the initiation step and its subsequent role in radical propagation. This is in contrast with the TOP-catalysed process in which skatolyl hydroperoxide has a key role. Indol-3-yl aldehyde is produced not via the peroxidase cycle but by catalysis involving ferrous peroxidase. Because indol-3-yl aldehyde is one of the main IAA-derived products identified in planta, we conclude that ferrous peroxidases participate in IAA catalytic transformations in vivo. A general scheme for peroxidase-catalysed IAA oxidation is presented.

Redox- and anion-linked protonation sites in horseradish peroxidase: analysis of distal haem pocket mutants.

We have investigated the effects of mutations at residues His-42, Arg-38 and Phe-41 in the distal haem pocket of horseradish peroxidase on the changes in protonation state that accompany redox- and ligand-linked changes to the haem group. The mutations H42L and R38L result in the loss of a characteristic pH dependency in the visible spectrum of the ferrous form and a diminished dependency of the midpoint redox potential of the haem group on pH. The results support the view that His-42, with its pK probably modulated by Arg-38, provides the protonation site on the reduced enzyme that is responsible for these pH dependencies. The mutations H42L and R38L also have major effects on the binding of cyanide to the haem. We have already reported that binding of cyanide to the ferrous forms of these mutants becomes too weak to be measurable [Meunier, Rodriguez-Lopez, Smith, Thorneley and Rich (1995) Biochemistry 34, 14687-14692]. The pH dependency of the rate constants for binding of cyanide to the oxidized form of H42L suggests that CN- is the kinetically active species, in contrast with wild-type horseradish peroxidase, where HCN is the binding form. For the R38L variant, the pH dependency of cyanide binding suggests that the pK of His-42 in the absence of Arg-38 is raised to 7.5-8, in the oxidized form. In contrast with these changes, the mutant F41A exhibits cyanide-binding behaviour that is similar to that of the wild type, both in its oxidized and reduced forms. However, the rate constant for carbon monoxide recombination increases substantially, suggesting that the access route for carbon monoxide, but not for cyanide, is perturbed by this amino acid substitution.

Salicylic acid is a reducing substrate and not an effective inhibitor of ascorbate peroxidase.

This communication describes the interactions of salicylic acid (SA) with plant ascorbate peroxidase (APX). Contrary to a recent report (Durner, J., and Klessig, D. F. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 11312-11316) we show conclusively that ascorbate oxidation by APX is not inhibited by SA (10 mM), but that SA is a slow reducing substrate of this enzyme. The suggestion that SA-dependent inhibition of APX in planta may result in the elevation of H2O2 levels, which in turn acts as a second messenger in systemic acquired resistance signaling, is therefore not tenable. We conclude that APX remains a key antioxidant during systemic acquired resistance following pathogenic infection of plants. The transient products of SA oxidation by APX appear to be SA free radicals that undergo subsequent chemistry. APX-dependent oxidation of SA could be essential for diminishing the detrimental effects of this phenolic acid on plant cells.

Purification and characterization of a novel class III peroxidase isoenzyme from tea leaves.

A novel, basic (isoelectric point > 10), heme peroxidase isoenzyme (TP; relative molecular weight = 34,660 +/- 10, mean +/- SE) that can account for a significant part of the ascorbate peroxidase activity in tea (Camellia sinensis) leaves has been purified to homogeneity. The ultraviolet/visible absorption spectrum is typical of heme-containing plant peroxidases, with a Soret peak at 406 nm (epsilon = 115 mM-1 cm-1) and an A406/A280 value of 3.4. The enzyme has a high specific activity for ascorbate oxidation (151 mumol min-1 mg-1), with a pH optimum in the range of 4.5 to 5.0. Substrate-specificity studies have revealed significant differences between TP and other class III peroxidases, as well as similarities with class I ascorbate peroxidases. TP, like ascorbate peroxidase, exhibits a preference for ascorbate over guaiacol, whereas other class III isoenzymes are characterized by 2-orders-of-magnitude higher activity for guaiacol than for ascorbate. TP also forms an unstable porphyrin pi cation radical-type compound I, which is converted to compound II within approximately 2 min in the absence of added reductant. Amino acid sequence data show TP to be the first example, to our knowledge, of a class III peroxidase with a high specificity for ascorbate as an electron donor.

The inactivation and catalytic pathways of horseradish peroxidase with m-chloroperoxybenzoic acid. A spectrophotometric and transient kinetic study.

The kinetics of the catalytic cycle and irreversible inactivation of horseradish peroxidase C (HRP-C) reacting with m-chloroperoxybenzoic acid (mCPBA) have been studied by conventional and stopped-flow spectrophotometry. mCPBA oxidized HRP-C to compound I with a second order-rate constant k1 = 3.6 x 10(7) M-1 s-1 at pH 7.0, 25 degrees C. Excess mCPBA subsequently acted as a one-electron reducing substrate, converting compound I to compound II and compound II to resting, ferric enzyme. In both of these reactions, spectrally distinct, transient forms of the enzyme were observed (lambdamax = 411 nm, epsilon = 45 mM-1 cm-1 for compound I with mCPBA, and lambdamax = 408 nm, epsilon = 77 mM-1 cm-1 for compound II with mCPBA). The compound I-mCPBA intermediate (shown by near infrared spectroscopy to be identical to P965) decayed either to compound II in a catalytic cycle (k3 = 6.4 x 10(-3) s-1) or, in a competing inactivation reaction, to verdohemoprotein (ki = 3.3 x 10(-3) s-1). Thus, a partition ratio of r = 2 is obtained for the inactivation of ferric HRP-C by mCPBA. The intermediate formed from compound II with mCPBA is not part of the inactivation pathway and only decays via the catalytic cycle to give resting, ferric enzyme (k5 = 1.0 x 10(-3) s-1). The data are compared with those from earlier steady-state kinetic studies and demonstrate the importance of single turnover experiments. The results are discussed in terms of the physiologically relevant reactions of plant peroxidases with hydrogen peroxide.

Effect of distal cavity mutations on the binding and activation of oxygen by ferrous horseradish peroxidase.

Mutations have been introduced at residues Arg-38 or His-42 in horseradish peroxidase isoenzyme C (HRPC) in order to probe the role of these key distal residues in the reaction of ferrous HRPC with dioxygen. The association and dissociation rate constants for dioxygen binding to His-42 --> Leu, His-42 --> Arg, Arg-38 --> Leu, Arg-38 --> Lys, Arg-38 --> Ser, and Arg-38 --> Gly variants have been measured using stopped-flow spectrophotometry. Replacement of His-42 by Leu or Arg increases the oxygen binding rate constant by less than an order of magnitude, whereas changing the polar distal Arg-38 causes increases of more than 2 orders. These results demonstrate that His-42 and Arg-38 impede the binding of dioxygen to ferrous HRPC, presumably by steric and/or electrostatic interactions in the distal heme cavity. Recombinant HRPC oxyperoxidase reverted slowly to the ferric state with no spectrophotometrically detectable intermediates and with an apparent first-order rate constant of 9.0 x 10(-3) s(-1), which is essentially the same as that for the native, glycosylated enzyme. This reaction was accelerated when His-42 was replaced by Leu or Arg (k(decay) = 0.10 and 0.07 s(-1), respectively) presumably due to the loss of the hydrogen bond between the His-42 imidazole and the bound dioxygen. Substitution of Arg-38 by Leu, Lys, or Gly also produced a less stable oxyperoxidase (k(decay) = 0.22, 0.20, and 0.58 s(-1), respectively). However, with the Arg-38 --> Ser variant, a transient intermediate, proposed to be a ferric-superoxide complex, was detected by rapid-scan stopped-flow spectrophotometry during the conversion of oxyperoxidase to the ferric state. This variant also exhibits an unusually high affinity for dioxygen. It is proposed that Arg-38 interacts with the bound dioxygen to promote superoxide character, thereby stabilizing the oxyperoxidase state and making the binding of dioxygen to ferrous HRPC essentially irreversible. We conclude that Arg-38 and His-42 not only promote the heterolytic cleavage of bound hydrogen peroxide to form compound I but also decrease the lability of the ferrous enzyme-dioxygen complex in order to suppress the formation of the inactive ferrous state.

Anionic tobacco peroxidase is active at extremely low pH: veratryl alcohol oxidation with a pH optimum of 1.8.

Tobacco peroxidase (36 kDa, pI 3.5) exhibits unique catalytic and spectral properties that are modulated by pH, calcium and magnesium ions. It catalyses the oxidation of veratryl alcohol by hydrogen peroxide over a wide pH range (1.5-5.0) in the presence of these metal ions with a pH optimum of 1.8. This is the only example of a holoperoxidase described so far that is active and comparatively stable at such a low pH. The enhancement of tobacco peroxidase activity by magnesium ions is to our knowledge the first example of a magnesium-induced peroxidase activation. UV/visible spectra of tobacco peroxidase showed that the Soret band shifted and its absorption coefficient increased upon the addition of calcium or magnesium ions and on lowering the pH. The tobacco peroxidase spectrum at pH 1.85, in the presence of calcium chloride (> 50 mM), is similar to that of lignin peroxidase at pH 6.0, with the Soret band shifting from 403 to 409 nm and the molar absorption coefficient increasing from 108,000 to 148,000 +/- 2000 M-1.cm-1 (results given +/- S.E.M.; n = 3). The data provide evidence for a low-affinity site for bivalent metal ion binding in addition to the two constitutive calcium sites that are present in all plant peroxidases. The presence of a glutamic acid residue (Glu-141) at the entrance to the haem-binding pocket, analogous to Glu-146 in lignin peroxidase and not present in other plant peroxidases, may account for these novel properties.

Studies with flavin analogs provide evidence that a protonated reduced FMN is the substrate-induced transient intermediate in the reaction of Escherichia coli chorismate synthase.

Chorismate synthase catalyzes the 1,4-elimination of phosphate and the C-(6-pro-R) hydrogen from 5-enolpyruvylshikimate 3-phosphate (EPSP) to generate chorismate. Although this reaction does not involve an overall change in redox state, the enzyme requires reduced FMN. To investigate the role of the flavin in catalysis we have employed chemically modified flavins: 1- and 5-deaza-, 2- and 4-thio-, 6-hydroxy-, 8-nor-6-methyl-, 8-methyl-sulfonyl-, 8-chloro-, 8-fluoro-, 8-nor-methyl-, 8-S-methyl-, 8-methoxy, 8-mercapto- and 8-amino-FMN. Photoreduction of 4-thio-FMN in the presence of chorismate synthase at pH 7.5 produced a reduced flavin species with an absorbance maximum at lambda = 410 nm indicative of monoanionic, reduced 4-thio-FMN. Binding of 8-mercapto- and 6-hydroxy-FMN to chorismate synthase in the presence of EPSP or (6R)-6-fluoro-EPSP resulted in an increase of the flavin analogs' pKa values by 4 and 1 pH units, respectively. On the basis of these findings it is concluded that chorismate synthase preferentially binds neutral flavin species, including the protonated reduced form, rather than anionic flavin species in the presence of EPSP or the 6-fluoro-substrate analog. Further support for this conclusion was obtained using 5-deaza- and 4-thio-FMN. Addition of EPSP to enzyme-bound, reduced 5-deaza-FMN produced spectral changes consistent with protonation of the flavin. Photoreduction of 4-thio-FMN in the presence of enzyme and the (6R)-6-fluoro-EPSP generated a reduced flavin species with absorbance properties of a neutral, reduced 4-thio-flavin. These results and their implications for the nature and kinetic properties of an observed flavin intermediate are discussed in the context of a possible role of reduced flavin as an electron donor to bound EPSP.