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.

The manner in which the distal heme pocket residues of peroxidases control the reaction mechanism and ligand binding has been investigated further by analysis of the electronic absorption and resonance Raman (RR) spectra of distal site mutants of recombinant horseradish peroxidase (HRP-C*). The roles of the conserved distal histidine and arginine residues, particularly in the context of the catalytic mechanism originally proposed for cytochrome c peroxidase (CCP), have been evaluated by studying the His42 --> Leu, His42 --> Arg, Arg38 --> Gly, and Arg38 --> Leu variants of HRP-C*. Spectra of the ferric forms, their complexes with benzohydroxamic acid (BHA), and the ferrous forms have been recorded at neutral pH. In addition, the ferric forms have been studied at alkaline pH. The relative populations of the three heme spin states characteristic of HRP-C* and its mutants were found to vary markedly from mutant to mutant. This diversity of heme spin state populations among the various mutants has allowed a well-defined set of RR frequencies to be compiled for the three heme spin states. These frequencies support the analysis of wild-type HRP-C* in terms of two heme states, five- (5cHS#) and six-coordinate high-spin (6cHS#), which exhibit anomalous RR frequencies compared to those of model heme systems. The third heme spin state is identified as being six-coordinate high-spin, displaying typical RR frequencies (6cHS). The 6cHS# and the 6cHS heme states are characterized by H bonding between the iron-bound water molecule and the Arg38 residue or the His42 residue, respectively. The proportion of six-coordinate high-spin heme states is at a minimum in the Arg38Leu mutant, indicating that the occupancy of the distal water molecule site is reduced in this mutant. The His42Arg mutant is distinguished from the other mutants by the unexpected presence of an iron-bound hydroxyl group at neutral pH. The spectral changes induced upon complexation with BHA indicate that both the distal histidine and arginine are involved in BHA binding; however, the arginine residue appears to play a more critical role. Measurements at pH 12 suggest there is a concerted involvement of both distal residues in mediating the alkaline transition of HRP-C*. Arg38 appears to be essential for stabilization of the OH- ligand, while His42 acts as a H bond acceptor. A striking similarity between the roles of these residues in the reaction of H2O2 with the enzyme and the alkaline transition is noted. By comparison with the results from corresponding mutants of CCP, it appears that although the hydrogen-bonding network linking the distal and proximal sides of the heme is conserved the distal cavity in HRP-C differs significantly from that of CCP. However, some similarities in the local environment of the distal arginine are suggested.

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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 k 1 ‫؍‬ 3.6 ؋ 10

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.

Browning reactions in fruits and vegetables are a serious problem for the food industry. In mushrooms, the principal enzyme responsible for the browning reaction is polyphenoloxidase (PPO). Microwaves have recently been introduced as an alternative for the industrial blanching of mushrooms. However, the direct application of microwave energy to entire mushrooms is limited by the important temperature gradients generated within the samples during heating, which can produce internal water vaporization and associated damage to the mushrooms texture. A microwave applicator has been developed, whereby irradiation conditions can be regulated and the heating process monitored. Whole edible mushrooms (Agaricus bisporus) were blanched by conventional, microwave, and combined heating methods to optimize the rate of PPO inactivation. A combined microwave and hot-water bath treatment has achieved complete PPO inactivation in a short time. Both the loss of antioxidant content and the increase of browning were minor in the samples treated with this combined method when compared to the control. This reduction in processing time also decreased mushroom weight loss and shrinkage.

The known derivatives from hydroquinone, α and β-arbutin, are used as depigmenting agents. In this work, we demonstrate that the oxy form of tyrosinase (oxytyrosinase) hydroxylates α and β-arbutin in ortho position of the phenolic hydroxyl group, giving rise to a complex formed by met-tyrosinase with the hydroxylated α or β-arbutin. This complex could evolve in two ways: by oxidizing the originated o-diphenol to o-quinone and deoxy-tyrosinase, or by delivering the o-diphenol and met-tyrosinase to the medium, which would produce the self-activation of the system. Note that the quinones generated in both cases are unstable, so the catalysis cannot be studied quantitatively. However, if 3-methyl-2-benzothiazolinone hydrazone hydrochloride hydrate is used, the o-quinone is attacked, so that it becomes an adduct, which can be oxidized by another molecule of o-quinone, generating o-diphenol in the medium. In this way, the system reaches the steady state and originates a chromophore, which, in turn, has a high absorptivity in the visible spectrum. This reaction allowed us to characterize α and β-arbutin kinetically as substrates of tyrosinase for the first time, obtaining a Michaelis constant values of 6.5 ± 0.58 mM and 3 ± 0.19 mM, respectively. The data agree with those from docking studies that showed that the enzyme has a higher affinity for β-arbutin. Moreover, the catalytic constants obtained by the kinetic studies (catalytic constant = 4.43 ± 0.33 s-1 and 3.7 ± 0.29 s-1 for α and β-arbutin respectively) agree with our forecast based on 13 C NMR considerations. This kinetic characterization of α and β-arbutin as substrates of tyrosinase should be taken into account to explain possible adverse effects of these compounds.

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 3 Leu, His-42 3 Arg, Arg-38 3 Leu, Arg-38 3 Lys, Arg-38 3 Ser, and Arg-38 3 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 ؋ 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 3 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 enzymedioxygen complex in order to suppress the formation of the inactive ferrous state.