Horseradish peroxidase-catalyzed aerobic oxidation and peroxidation of indole-3-acetic acid. I. Optical spectra.

A study of the indole-3-acetate reaction with horse-radish peroxidase, in the absence or presence of hydrogen peroxide, has been performed, employing rapid scan and conventional spectrophotometry. We present here the first clear spectral evidence, obtained on the millisecond time scale, indicating that at pH 5.0 and for high [enzyme/substrate] ratios peroxidase compound III is formed. Most, if not all, of the compound III is formed by oxygenation of the ferrous peroxidase. There is an inhibitory effect of superoxide dismutase and histidine on compound III formation which indicates the involvement of the active oxygen species superoxide and singlet oxygen. It is concluded that the oxidation of indole-3-acetate by horseradish peroxidase at pH 5.0 proceeds through compound III formation to the catalytically inactive forms P-670 and P-630. A reaction path in which the enzyme is directly reduced by indole-3-acetate might be involved as an initiation step. Rapid scan spectral data, which indicate differences in the formation and decay of enzyme intermediate compounds at pH 7.0, in comparison with those observed at pH 5.0, are also presented. At pH 7.0 compound II is a key intermediate in oxidation--peroxidation of substrate. Mechanisms of reactions consistent with the experimental data are proposed and discussed.     

Kinetics of the oxidation of ferrocyanide by lactoperoxidase compound II

The kinetics of the oxidation of ferrocyanide by lactoperoxidase compound II has been studied over the pH range 5.2-9.9 at 25 degrees C and an ionic strength of 0.11 M. For all pH values, exponential decay curves are obtained for the reaction of compound II in the presence of ferrocyanide which yielded pseudo-first-order rate constants kobs. The spontaneous decay of compound II in the absence of ferrocyanide occurs at an appreciable rate which was measured independently and used in the data analysis. At all pH values two striking effects were observed when the rate of the decay reaction in the presence of ferrocyanide, kobs, was plotted against ferrocyanide concentration: a saturation effect and positive intercepts which are attributable to the spontaneous decay. The plots of kobs versus ferrocyanide concentration were analyzed in terms of the following parameters: a first-order rate constant k3,obs, a Michaelis constant Km,obs and a spontaneous-decay rate constant k4. The parameters k3,obs and Km,obs describe the reaction of compound II with ferrocyanide, independently of the spontaneous decay. The parameter k4 has only a small pH dependence, whereas plots of the logs of k3,obs and Km,obs versus pH have slopes of -1 at high pH. The major part of the pH dependence can be explained by the influence of a single heme-linked acid group in the LPO-compound-II-ferrocyanide complex.     

Kinetic studies on the reaction of compound II of myeloperoxidase with ascorbic acid. Role of ascorbic acid in myeloperoxidase function

Ascorbic acid is known to stimulate leukocyte functions. In a recent publication it was suggested that the role of ascorbic acid is to reduce compound II of myeloperoxidase back to the native enzyme (Bolscher, B. G. J. M., Zoutberg, G. R., Cuperus, R. A., and Wever, R. (1984) Biochim. Biophys. Acta 784, 189-191). In this paper we report rapid spectral scan and transient state kinetic results on the reaction of three myeloperoxidase compounds II, namely, human neutrophil myeloperoxidase, canine myeloperoxidase, and bovine spleen heme protein with ascorbate. We show by rapid scan spectra that compound II does not pass through any other intermediate when ascorbic acid reduces it back to native form. We also show that the reactions of all three compounds II involve a simple binding interaction before enzyme reduction with an apparent dissociation constant of 6.3 +/- 0.9 x 10(-4) to 2.0 +/- 0.3 x 10(-3)M and a first-order rate constant for reduction of 12.6 +/- 0.6 to 18.8 +/- 1.3 s-1. The optimum pH is 4.5, and at this pH the activation energy for the reaction is 13.2 kJ mol-1. Results of this work lend further evidence that the spleen green heme protein is very similar if not identical to leukocyte myeloperoxidase based on a comparison of spectral scans, pH-rate profiles, and kinetic parameters. We demonstrate that chloride cannot reduce compound II whereas iodide reduces compound II to native enzyme at a rate comparable to that of ascorbate. This explains why ascorbate accelerates chlorination but inhibits iodination. Formation of compound II is a dead end for the generation of hypochlorous acid; ascorbate regenerates more native enzyme to enhance the chlorination reaction namely: myeloperoxidase + peroxide----compound I followed by compound I + chloride----HOCl. On the other hand, ascorbate is a competitor with iodide for both compounds I and II and so inhibits iodination.     

Kinetics of the oxidation of p-aminobenzoic acid catalyzed by horseradish peroxidase compounds I and II.

The kinetics of p-aminobenzoic acid oxidation catalyzed by horseradish peroxidase Compounds I and II was investigated intensively as a function of pH at 25 degrees in aqueous solutions of ionic strength 0.11. All of the rate data were collected from single turnover experiments involving reactions of a single enzyme compound. In reactions of both compounds, deviations from first order behavior with respect to the enzyme were observed at high pH values which were explained in terms of a free radical interaction of product with the enzyme. The effect could be eliminated with sufficient excess of substrate. Kinetic behavior which deviated from first order in substrate, observed at low pH, was explained by a mechanism involving an enzyme-substrate complex which reacted with an additional molecule of substrate but at a slower rate. The pH dependence of the second order rate constants for the reaction of p-aminobenzoic acid with free Compounds I and II is similar to results obtained for the comparable reactions of ferrocyanide, suggesting similar proton-transfer mechanisms for both reducing substrates. The reduction of Compound II by p-aminobenzoic acid appeared to be influenced by two ionizable groups on the enzyme which affect the electronic environment of the heme. The lack of influence of substrate ionizable groups on the rate of the Compound II reaction indicated that potential differences in reactivities of NH2C6H4COO- and NH2C6H4COOH were levelled by the diffusion-controlled limit in the acid region of pH. The reduction of Compound I by p-aminobenzoic acid was not diffusion-controlled and the rate-pH profile could be explained in terms of three acid ionizations, two on the substrate and one on Compound I.     

Photochemical reactions of horseradish peroxidase compounds I and II at room temperature and 13 degrees K.

Some photochemical reactions of horseradish peroxidase compounds I and II (HRP-I and HRP-II, respectively) have been studied by electronic absorption spectroscopy over the temperature range 297 degrees K-10 degrees K. In glassy matrices below 80 degrees K HRP-I is rapidly converted to hrp-ii when irradiated with low power white light. The native enzyme and HRP-II are not photochemically active at these temperatures with low power irradiation. At room temperature the spontaneous decay of both HRP-I and HRP-II is catalyzed by irradiation with white light. It is shown that the photolysis is dependent upon light in the region 450-320 nm. It is concluded that the HRP-I and HRP-II conformations are closely related with only a low transition energy in the presence of electrons generated by the light. The conversion of HRP-II to HRP is accompanied by large conformational changes and so is inhibited at low temperatures.     

The use of transition state acid dissociation constants in pH-dependent enzyme kinetics

It is shown that the variation of reaction rate with pH in systems where several protonated forms of the reactants appear to be kinetically active may be expressed most economically in terms of transition state acid dissociation constants. The advantages of this approach are described in relation to the formal analysis of experimental data with regard to both simple and complex reactions and the satisfaction of the principle of microscopic reversibility. Ligand binding to metmyoglobin is used to illustrate the value of the approach in searching for detailed mechanistic explanations.     

Triploid and tetraploid hybrids from diploid x tetraploid crosses in Grindella (compositae)

Crosses between the diploids G. oxylepis var. eligulata Steyermark (Mexico) and G. havardii Steyermark (New Mexico) and the tetraploid G. aphanactis Rydb. (New Mexico) were made. With G. aphanactis as the pistillate parent and G. havardii as the pollen parent a triploid hybrid was obtained in which the maximum meiotic configuration observed was 6m. The plant was 10 % fertile. Two triploid hybrid plants were also obtained when G. aphanactis was used as the pistillate parent and G. oxylepis var. eligulata was the pollen parent. One plant was about 20 % fertile and the other had a maximum configuration of 3_II + 4_III. The reciprocal cross produced a tetraploid which had a maximum configuration of 6_II + 3_IV and was 8 % fertile. The tetraploid plant undoubtedly resulted from the union of an unreduced gamete from the 2n parent and a normally reduced gamete from the tetraploid. Morphology of the hybrids was usually intermediate when compared with the parental species, although some characters in the triploids were those of the diploid parent. Chromosome end arrangements of the respective genomes and putative pairing relationships are presented and phylogenetic implications are discussed. It is concluded that G. aphanactis is more closely related to G. havardii than to G. oxylepis var. eligulata.     

On the mechanism of chlorination by chloroperoxidase

Spectral-scan results obtained on the millisecond time scale are reported for reactions of chloroperoxidase with peracetic acid and chloride ion in both the presence and the absence of monochlorodimedone. A multimixing experiment is performed in which stoichiometric amounts of chloroperoxidase and peracetic acid are premixed for 0.7 s before the resultant compound I is reacted with chloride ion. The combined results show that the only detectable enzyme intermediate species is compound I (except in very late stages of the reaction), that the disappearance of compound I is accelerated by the presence of chloride ion, and that it is further accelerated if both chloride and monochlorodimedone are present. It is concluded that compound I is an obligate intermediate species in the reaction. Experiments are performed on the reaction of monochlorodimedone with hypochlorous acid in both the presence and the absence of added chloride ion, but in the absence of chloroperoxidase. The presence of chloride ion greatly accelerates the reaction rate apparently by setting off a chlorine chain reaction. This reaction would be important in the enzyme-catalyzed reaction if hypochlorous acid were liberated into the solution. A careful analysis of steady-state kinetic results shows that in the chlorination of monochlorodimedone at least, liberation of free hypochlorous acid is not important in the enzyme-catalyzed pathway. Rather the reaction proceeds from compound I to formation of iron(III)-OCl by chloride ion addition to the ferryl oxygen atom. This obligate intermediate species then chlorinates the substrate. It is well described as enzyme-activated hypochlorous acid, in which replacement of the proton in HOCl by the heme iron ion produces a Cl+ species of great potency. Thus the enzyme controls chlorination of monochlorodimedone rather than unleashing an uncontrolled chain reaction in which it would be rapidly destroyed.