Prostaglandin H synthase and hydroperoxides: peroxidase reaction and inactivation kinetics

The reaction kinetics of the peroxidase activity of prostaglandin H synthase have been examined with 15-hydroperoxyeicosatetraenoic acid and hydrogen peroxide as substrates and tetramethylphenylenediamine as cosubstrate. The apparent Km and Vmax values for both hydroperoxides were found to increase linearly with the cosubstrate concentration. The overall reaction kinetics could be interpreted in terms of an initial reaction of the synthase with hydroperoxide to form an intermediate equivalent to horseradish peroxidase Compound I, followed by reduction of this intermediate by cosubstrate to regenerate the resting enzyme. The rate constants estimated for the generation of synthase Compound I were 7.1 X 10(7) M-1 s-1 with the lipid hydroperoxide and 9.1 X 10(4) M-1 s-1 with hydrogen peroxide. The rate constants estimated for the rate-determining step in the regeneration of resting enzyme by cosubstrate were 9.2 X 10(6) M-1 s-1 in the case of the reaction with lipid hydroperoxide and 3.5 X 10(6) M-1 s-1 in the case of reaction with hydrogen peroxide. The intrinsic affinities of the synthase peroxidase for substrate (Ks) were estimated to be on the order of 10(-8) M for lipid hydroperoxide and 10(-5) M for hydrogen peroxide. These affinities are quite similar to the reported affinities of the synthase for these hydroperoxides as activators of the cyclooxygenase. The peroxidase activity was found to be progressively inactivated during the peroxidase reaction. The rate of inactivation of the peroxidase was increased by increases in hydroperoxide level, and decreased by increases in peroxidase cosubstrate. The inactivation of the peroxidase appeared to occur by a hydroperoxide-dependent process, originating from synthase Compound I or Compound II.     

Quantitative studies of hydroperoxide reduction by prostaglandin H synthase. Reducing substrate specificity and the relationship of peroxidase to cyclooxygenase activities

The peroxidase activity of prostaglandin H (PGH) synthase catalyzes reduction of 5-phenyl-4-pentenyl hydroperoxide to 5-phenyl-4-pentenyl alcohol with a turnover number of approximately 8000 mol of 5-phenyl-4-pentenyl hydroperoxide/mol of enzyme/min. The kinetics and products of reaction establish PGH synthase as a classical heme peroxidase with catalytic efficiency similar to horseradish peroxidase. This suggests that the protein of PGH synthase evolved to facilitate peroxide heterolysis by the heme prosthetic group. Comparison of an extensive series of phenols, aromatic amines, beta-dicarbonyls, naturally occurring compounds, and nonsteroidal anti-inflammatory drugs indicates that considerable differences exist in their ability to act as reducing substrates. No correlation is observed between the ability of compounds to support peroxidatic hydroperoxide reduction and to inhibit cyclooxygenase. In addition, the resolved enantiomers of MK-410 and etodolac exhibit dramatic enantiospecific differences in their ability to inhibit cyclooxygenase but are equally potent as peroxidase-reducing substrates. This suggests that there are significant differences in the orientation of compounds at cyclooxygenase inhibitory sites and the peroxidase oxidation site(s). Comparison of 5-phenyl-4-pentenyl hydroperoxide reduction by PGH synthase and horseradish peroxidase reveals considerable differences in reducing substrate specificity. Both the cyclooxygenase and peroxidase activities of PGH synthase inactivate in the presence of low micromolar amounts of hydroperoxides and arachidonic acid. PGH synthase was most sensitive to arachidonic acid, which exhibited an I50 of 0.6 microM in the absence of all protective agents. Inactivation by hydroperoxides requires peroxidase turnover and can be prevented by reducing substrates. The I50 values for inactivation by 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid are 4.0 and 92 microM, respectively, in the absence and presence of 500 microM phenol, a moderately good reducing substrate. The ability of compounds to protect against hydroperoxide-induced inactivation correlates directly with their ability to act as reducing substrates. Hydroquinone, an excellent reducing substrate, protected against hydroperoxide-induced inactivation when present in less than 3-fold molar excess over hydroperoxide. The presence of a highly efficient hydroperoxide-reducing activity appears absolutely essential for protection of the cyclooxygenase capacity of PGH synthase. The peroxidase activity is, therefore, a twin-edged sword, responsible for and protective against hydroperoxide-dependent inactivation of PGH synthase.(ABSTRACT TRUNCATED AT 400 WORDS)     

Reactivity of deoxy- and oxyferrous dehaloperoxidase B from Amphitrite ornata: identification of compound II and its ferrous-hydroperoxide precursor

Dehaloperoxidase (DHP) from the terebellid polychaete Amphitrite ornata is a bifunctional enzyme that possesses both hemoglobin and peroxidase activities. The bifunctional nature of DHP as a globin peroxidase appears to be at odds with the traditional starting oxidation state for each individual activity. Namely, reversible oxygen binding is only mediated via a ferrous heme in globins, and peroxidase activity is initiated from ferric centers and to the exclusion of the oxyferrous oxidation state from the peroxidase cycle. Thus, to address what appears to be a paradox, herein we report the details of our investigations into the DHP catalytic cycle when initiated from the deoxy- and oxyferrous states using biochemical assays, stopped-flow UV-visible, and rapid-freeze-quench electron paramagnetic resonance spectroscopies, and anaerobic methods. We demonstrate the formation of Compound II directly from deoxyferrous DHP B upon its reaction with hydrogen peroxide and show that this occurs both in the presence and in the absence of trihalophenol. Prior to the formation of Compound II, we have identified a new species that we have preliminarily attributed to a ferrous-hydroperoxide precursor that undergoes heterolysis to generate the aforementioned ferryl intermediate. Taken together, the results demonstrate that the oxyferrous state in DHP is a peroxidase competent starting species, and an updated catalytic cycle for DHP is proposed in which the ferric oxidation state is not an obligatory starting point for the peroxidase catalytic cycle of dehaloperoxidase. The data presented herein provide a link between the peroxidase and oxygen transport activities, which furthers our understanding of how this bifunctional enzyme is able to unite its two inherent functions in one system.     

Alkylaryl sulfides as peroxidase reducing substrates for prostaglandin H synthase. Probes for the reactivity and environment of the ferryl-oxo complex

The peroxidase activity of prostaglandin (PGH) synthase catalyzes the reduction of PGG2 and other natural and synthetic hydroperoxides by reducing substrates. Sulfides serve as reductants by incorporating the oxo ligand from the ferryl-oxo complex which represents the higher oxidation state of the peroxidase (Compound I). A series of alkylaryl sulfides and substituted dihydrobenzo[b]thiophenes were synthesized to determine the electronic and steric requirements of PGH synthase for sulfide reducing substrates. Kinetic parameters were determined for most of the molecules by determining their ability to support reduction of 5-phenyl-4-pentenyl-1-hydroperoxide in the presence of PGH synthase purified from ram seminal vesicle microsomes. Electron-donating groups on the aryl moiety para to the sulfide enhanced reducing substrate activity (p = -0.8). As expected from previous results, the major oxidation product of p-methylthioanisole was the corresponding sulfoxide. The presence of a para-amino group increased binding to the enzyme and changed the reduction mechanism from oxygen transfer to electron transfer. The major oxidation product of p-(dimethylamino)thioanisole was identified as p-(methylamino)thioanisole; an equivalent amount of formaldehyde was produced. Increasing the size of the alkyl group attached to sulfur decreased the ability of the sulfide to act as a peroxidase reductant. The maximal turnover for reduction by p-methoxyphenylalkyl sulfides decreased 10-fold on substitution of isopropyl for ethyl. Chiral derivatives of benzo[b]thiophenes demonstrated differences in the ability of the two enantiomers to support reduction. Introduction of a carboxylic acid moiety anywhere in the molecule decreased the maximal turnover for reduction. Esterification of the carboxylate doubled the extent of reduction relative to the free acid. The results are used to develop models for the interaction of sulfides with Compound I of PGH synthase.     

Prostaglandin endoperoxide synthase substituted with manganese protoporphyrin IX. Formation of a higher oxidation state and its relation to cyclooxygenase reaction.

The heme in prostaglandin endoperoxide synthase (PGH synthase) was substituted with Mn(III)-protoporphyrin IX. The resulting enzyme, Mn-PGH synthase, showed full cyclooxygenase activity but only 0.9% of the peroxidase activity of the native iron enzyme. During the reaction with exogenous or endogenously produced hydroperoxides, a spectral intermediate of Mn-PGH synthase was observed. The electronic absorption bands of the resting enzyme at 376, 472, and 561 nm decreased, and the intermediate's bands at 417, around 513, and 625 nm appeared. The rate constant of the formation of the intermediate was about 10(4) M-1.s-1 at 22 degrees C, three orders of magnitude lower than with the iron enzyme. Spectral properties, conditions of formation, and the suppressed formation in the presence of electron donors provide evidence for a higher oxidation state of Mn-PGH synthase, tentatively a Mn(IV) species. This species was assigned to an intermediate in the peroxidase reaction of Mn-PGH synthase, the low activity of which was explained by the rate-limiting slow reaction of Mn-PGH synthase with hydroperoxides. The findings and interpretation are consistent with the published properties of other manganese-substituted peroxidases. Although the cyclooxygenase activity was similar to that of Fe-PGH synthase, the cyclooxygenase reaction of Mn-PGH synthase showed distinct differences in comparison with Fe-PGH synthase. A longer activation phase was observed which resembled the time course of the formation of the higher oxidation state. Glutathione peroxidase with glutathione, a hydroperoxide-scavenging system, inhibited the cyclooxygenase of Mn-PGH synthase at concentrations where the activity of Fe-PGH synthase was not affected. It is demonstrated that Mn-PGH synthase requires higher concentrations of hydroperoxides for the activation of the cyclooxygenase. These findings suggest that the substitution of iron with manganese in PGH synthase does not change the mechanism of the enzyme. The main difference is the much lower rate of the reaction with hydroperoxides which affects both the peroxidase activity and the hydroperoxide-dependent activation of the cyclooxygenase. A reaction scheme for Mn-PGH synthase is proposed analogous to that suggested for Fe-PGH synthase (Karthein, R., Dietz, R., Nastainczyk, W., and Ruf, H. H. (1988) Eur. J. Biochem. 171, 313-320).     

Catalase-peroxidase (Mycobacterium tuberculosis KatG) catalysis and isoniazid activation

Resonance Raman spectra of native, overexpressed M. tuberculosis catalase-peroxidase (KatG), the enzyme responsible for activation of the antituberculosis antibiotic isoniazid (isonicotinic acid hydrazide), have confirmed that the heme iron in the resting (ferric) enzyme is high-spin five-coordinate. Difference Raman spectra did not reveal a change in coordination number upon binding of isoniazid to KatG. Stopped-flow spectrophotometric studies of the reaction of KatG with stoichiometric equivalents or small excesses of hydrogen peroxide revealed only the optical spectrum of the ferric enzyme with no hypervalent iron intermediates detected. Large excesses of hydrogen peroxide generated oxyferrous KatG, which was unstable and rapidly decayed to the ferric enzyme. Formation of a pseudo-stable intermediate sharing optical characteristics with the porphyrin pi-cation radical-ferryl iron species (Compound I) of horseradish peroxidase was observed upon reaction of KatG with excess 3-chloroperoxybenzoic acid, peroxyacetic acid, or tert-butylhydroperoxide (apparent second-order rate constants of 3.1 x 10(4), 1.2 x 10(4), and 25 M(-1) s(-1), respectively). Identification of the intermediate as KatG Compound I was confirmed using low-temperature electron paramagnetic resonance spectroscopy. Isoniazid, as well as ascorbate and potassium ferrocyanide, reduced KatG Compound I to the ferric enzyme without detectable formation of Compound II in stopped-flow measurements. This result differed from the reaction of horseradish peroxidase Compound I with isoniazid, during which Compound II was stably generated. These results demonstrate important mechanistic differences between a bacterial catalase-peroxidase and the homologous plant peroxidases and yeast cytochrome c peroxidase, in its reactions with peroxides as well as substrates.     

The hemoglobin of the crossopterygian fish, Latimeria chalumnae (Smith). Subunit structure and oxygen equilibria

There are five oxidation-reduction states of horseradish peroxidase which are interconvertible. These states are ferrous, ferric, Compound II (ferryl), Compound I (primary compound of peroxidase and H₂O₂), and Compound III (oxy-ferrous). The presence of heme-linked ionization groups was confirmed in the ferrous enzyme by spectrophotometric and pH stat titration experiments. The values of pK were 5.87 for isoenzyme A and 7.17 for isoenzymes (B + C). The proton was released when the ferrous enzyme was oxidized to the ferric enzyme while the uptake of the proton occurred when the ferrous enzyme reacted with oxygen to form Compound III. The results could be explained by assuming that the heme-linked ionization group is in the vicinity of the sixth ligand and forms a stable hydrogen bond with the ligand.The measurements of uptake and release of protons in various reactions also yielded the following stoichiometries: Ferric peroxidase + H₂O₂ → Compound I, Compound I + e^? + H⁺ → Compound II, Compound II + e^? + H⁺ → ferric peroxidase, Compound II + H₂O₂ → Compound III, Compound III + 3e^? + 3H⁺ → ferric peroxidase.Based on the above stoichiometries and assuming the interaction between the sixth ligand and heme-linked ionization group of the protein, it was possible to picture simple models showing structural relations between five oxidation-reduction states of peroxidase. Tentative formulae are as follows: [Pr·Po·Fe-(II) $\text{\$}\text{?}$PrH⁺·Po·Fe(II)] is for the ferrous enzyme, Pr·Po·Fe(III)OH₂ for the ferric one, Pr·Po·Fe(IV)OH^? for Compound II, Pr(OH^?)·Po⁺·Fe(IV)OH^? for Compound I, and PrH⁺·Po·Fe(III)O₂^? for Compound III, in which Pr stands for protein and Po for porphyrin. And by Fe(IV)OH^?, for instance, is meant that OH^? is coordinated at the sixth position of the heme iron and the formal oxidation state of the iron is four.     

Reduction of cytochrome c peroxidase compounds I and II by ferrocytochrome c. A stopped-flow kinetic investigation

The oxidation of yeast cytochrome c peroxidase by hydrogen peroxide produces a unique enzyme intermediate, cytochrome c peroxidase Compound I, in which the ferric heme iron has been oxidized to an oxyferryl state, Fe(IV), and an amino acid residue has been oxidized to a radical state. The reduction of cytochrome c peroxidase Compound I by horse heart ferrocytochrome c is biphasic in the presence of excess ferrocytochrome c as cytochrome c peroxidase Compound I is reduced to the native enzyme via a second enzyme intermediate, cytochrome c peroxidase Compound II. In the first phase of the reaction, the oxyferryl heme iron in Compound I is reduced to the ferric state producing Compound II which retains the amino acid free radical. The pseudo-first order rate constant for reduction of Compound I to Compound II increases with increasing cytochrome c concentration in a hyperbolic fashion. The limiting value at infinite cytochrome c concentration, which is attributed to the intracomplex electron transfer rate from ferrocytochrome c to the heme site in Compound I, is 450 +/- 20 s-1 at pH 7.5 and 25 degrees C. Ferricytochrome c inhibits the reaction in a competitive manner. The reduction of the free radical in Compound II is complex. At low cytochrome c peroxidase concentrations, the reduction rate is 5 +/- 3 s-1, independent of the ferrocytochrome c concentration. At higher peroxidase concentrations, a term proportional to the square of the Compound II concentration is involved in the reduction of the free radical. Reduction of Compound II is not inhibited by ferricytochrome c. The rates and equilibrium constant for the interconversion of the free radical and oxyferryl forms of Compound II have also been determined.     

Oxidation of horseradish peroxidase compound II to compound I.

In the reaction between equimolar amounts of horseradish peroxidase and chlorite, the native enzyme is oxidized directly to Compound II (Hewson, W.D., and Hager, L.P. (1979) J. Biol. Chem. 254, 3175-3181). At acidic pH but not at alkaline values, this initial reaction is followed by oxidation of Compound II to Compound I. The highly pH-dependent chemistry of Compound II can be readily demonstrated by the reduction of Compound I, with ferrocyanide at acidic, neutral, and alkaline pH values. Titration at low pH yields very little Compound II, whereas at high pH, the yield is quantitative. Similarly, the reaction of horseradish peroxidase and chlorite at low pH yields Compound I while only Compound II is formed at high pH. At intermediate pH values both the ferrocyanide reduction and the chlorite reaction produce intermediate yields of Compound II. This behavior is explained in terms of acidic and basic forms of Compound II. The acidic form is reactive and unstable relative to the basic form. Compound II can be readily oxidized to Compound I by either chloride or chlorine dioxide in acidic solution. The oxidation does not occur in alkaline solution, nor will hydrogen peroxide cause the oxidation of Compound II, even at low pH.     

Preparation and initial characterization of the compound I, II, and III states of iron methylchlorin-reconstituted horseradish peroxidase and myoglobin: models for key intermediates in iron chlorin enzymes

To better understand the spectral properties of high valent and oxyferrous states in naturally occurring iron chlorin-containing proteins, we have prepared the oxoferryl compound I derivative of iron methylchlorin-reconstituted horseradish peroxidase (MeChl-HRP) and the compound II and oxyferrous compound III states of iron MeChl-reconstituted myoglobin. Initial spectral characterization has been carried out with UV-visible absorption and magnetic circular dichroism. In addition, the peroxidase activity of iron MeChl-HRP in pyrogallol oxidation has been found to be 40% of the rate for native HRP. Previous studies of oxoferryl chlorins have employed tetraphenylchlorins in organic solvents at low temperatures; stable oxyferrous chlorins have not been previously examined. The present study describes the compound I, II, and III states of histidine-ligated iron chlorins in a protein environment for the first time.     

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.     

A mechanistic study of self-inactivation of the peroxidase activity in prostaglandin H synthase-1

Prostaglandin H synthase (PGHS) is a self-activating and self-inactivating enzyme. Both the peroxidase and cyclooxygenase activities have a limited number of catalytic turnovers. Sequential stopped-flow measurements were used to analyze the kinetics of PGHS-1 peroxidase self-inactivation during reaction with several different hydroperoxides. The inactivation followed single exponential kinetics, with a first-order rate constant of 0.2-0.5 s-1 at 24 degrees C. This rate was independent of the peroxide species and concentration used, strongly suggesting that the self-inactivation process originates after formation of Compound I and probably with Intermediate II, which contains an oxyferryl heme and a tyrosyl radical. Kinetic scan and rapid scan experiments were used to monitor the heme changes during the inactivation process. The results from both experiments converged to a simple, linear, two-step mechanism in which Intermediate II is first converted in a faster step (0.5-2 s-1) to a new compound, Intermediate III, which undergoes a subsequent slower (0.01-0.05 s-1) transition to a terminal species. Rapid-quench and high pressure liquid chromatography analysis indicated that Intermediate III likely retains an intact heme group that is not covalently linked with the PGHS-1 protein.     

Pre-steady-state kinetics and modelling of the oxygenase and cyclooxygenase reactions of prostaglandin endoperoxide synthase

The pre-steady-state kinetics of the prostaglandin endoperoxide synthase oxygenase reaction with eicosadienoic acids and the cyclooxygenase reaction with arachidonic acid were investigated by stopped-flow spectrophotometry at 426 nm, an isosbestic point between native enzyme and compound I. A similar reaction mechanism for both types of catalysis is defined from combined kinetic experiments and numerical simulations. In the first step a fatty acid hydroperoxide reacts with the native enzyme to form compound I and the fatty acid hydroxide. In the second step the fatty acid reduces compound I to compound II and a fatty acid carbon radical is formed. This is followed by two fast steps: (1) the addition of either one molecule of oxygen (the oxygenase reaction) or two molecules of oxygen (the cyclooxygenase reaction) to the fatty acid carbon radical to form the corresponding hydroperoxyl radical, and (2) the reaction of the hydroperoxyl radical with compound II to form the fatty acid hydroperoxide and a compound I-protein radical. A unimolecular reaction of the compound I-protein radical to reform the native enzyme is assumed for the last step in the cycle. This is a slow reaction not significantly affecting steps 1 and 2 under pre-steady-state conditions. A linear dependence of the observed pseudo-first-order rate constant, k(obs), on fatty acid concentration is quantitatively reproduced by the model for both the oxygenase and cyclooxygenase reactions. The simulated second order rate constants for the conversion of native enzyme to compound I with arachidonic or eicosadienoic acids hydroperoxides as a substrate are 8 x 10(7) and 4 x 10(7) M(-1) s(-1), respectively. The simulated and experimentally obtained second-order rate constants for the conversion of compound I to compound II with arachidonic and eicosadienoic acids as a substrate are 1.2 x 10(5) and 3.0 x 10(5) M(-1) s(-1), respectively.     

Spectral studies with lactoperoxidase and thyroid peroxidase: interconversions between native enzyme, compound II, and compound III

Spectral scans in both the visible (650-450 nm) and the Soret (450-380 nm) regions were recorded for the native enzyme, Compound II, and Compound III of lactoperoxidase and thyroid peroxidase. Compound II for each enzyme (1.7 microM) was prepared by adding a slight excess of H2O2 (6 microM), whereas Compound III was prepared by adding a large excess of H2O2 (200 microM). After these compounds had been formed it was observed that they were slowly reconverted to the native enzyme in the absence of exogenous donors. The pathway of Compound III back to the native enzyme involved Compound II as an intermediate. Reconversion of Compound III to native enzyme was accompanied by the disappearance of H2O2 and generation of O2, with approximately 1 mol of O2 formed for each 2 mol of H2O2 that disappeared. A scheme is proposed to explain these observations, involving intermediate formation of the ferrous enzyme. According to the scheme, Compound III participates in a reaction cycle that effectively converts H2O2 to O2. Iodide markedly affected the interconversions between native enzyme, Compound II, and Compound III for lactoperoxidase and thyroid peroxidase. A low concentration of iodide (4 microM) completely blocked the formation of Compound II when lactoperoxidase or thyroid peroxidase was treated with 6 microM H2O2. When the enzymes were treated with 200 microM H2O2, the same low concentration of iodide completely blocked the formation of Compound III and largely prevented the enzyme degradation that otherwise occurred in the absence of iodide. These effects of iodide are readily explained by (i) the two-electron oxidation of iodide to hypoiodite by Compound I, which bypasses Compound II as an intermediate, and (ii) the rapid oxidation of H2O2 to O2 by the hypoiodite formed in the reaction between Compound I and iodide.     

Properties and function of the two hemes in Pseudomonas cytochrome c peroxidase

The oxidation-reduction potentials of the two c-type hemes of Pseudomonas aeruginosa cytochrome c peroxidase (ferrocytochrome c:hydrogen-peroxide oxidoreductase EC 1.11.1.5) have been determined and found to be widely different, about +320 and -330 mV, respectively. The EPR spectrum at temperatures below 77 K reveals only low-spin signals (gz 3.24 and 2.93), whereas optical spectra at room temperature indicate the presence of one high-spin and one low-spin heme in the enzyme. Optical absorption spectra of both resting and half-reduced enzyme at 77 K lack features of a high-spin compound. It is concluded that the heme ligand arrangement changes on cooling from 298 to 77 K with a concomitant change in the spin state. The active form of the peroxidase is the half-reduced enzyme, in which one heme is in the ferrous and the other in the ferric state (low-spin below 77 K with gz 2.84). Reaction of the half-reduced enzyme with hydrogen peroxide forms Compound I with the hemes predominantly in the ferric (gz 3.15) and the ferryl states. Compound I has a half-life of several seconds and is converted into Compound II apparently having a ferric-ferric structure, characterized by an EPR peak at g 3.6 with unusual temperature and relaxation behavior. Rapid-freeze experiments showed that Compound II is formed in a one-electron reduction of Compound I. The rates of formation of both compounds are consistent with the notion that they are involved in the catalytic cycle.     

Reactions of the NAD radical with higher oxidation states of horseradish peroxidase

The reactions of the NAD radical (NAD.) with ferric horseradish peroxidase and with compounds I and II were investigated by pulse radiolysis. NAD. reacted with the ferric enzyme and with compound I to form the ferrous enzyme and compound II with second-order rate constants of 8 X 10(8) and 1.5 X 10(8) M-1 s-1, respectively, at pH 7.0. In contrast, no reaction of NAD. with native compound II at pH 10.0 nor with diacetyldeutero-compound II at pH 5.0-8.0 could be detected. Other reducing species generated by pulse radiolysis, such as hydrated electron (eaq-), superoxide anion (O2-), and benzoate anion radical, could not reduce compound II of the enzyme to the ferric state, although the methylviologen radical reduced it. The results are discussed in relation to the mechanism of catalysis of the one-electron oxidation of substrates by peroxidase.     

Higher oxidation states of prostaglandin H synthase. Rapid electronic spectroscopy detected two spectral intermediates during the peroxidase reaction with prostaglandin G2

The reaction of prostaglandin H synthase with prostaglandin G2, the physiological substrate for the peroxidase reaction, was examined by rapid reaction techniques at 1 degree C. Two spectral intermediates were observed and assigned to higher oxidation states of the enzymes. Intermediate I was formed within 20 ms in a bimolecular reaction between the enzyme and prostaglandin G2 with k1 = 1.4 x 10(7) M-1 s-1. From the resemblance to compound I of horseradish peroxidase, the structure of intermediate I was assigned to [(protoporphyrin IX)+.FeIVO]. Between 10 ms and 170 ms intermediate II was formed from intermediate I in a monomolecular reaction with k2 = 65 s-1. Intermediate II, spectrally very similar to compound II of horseradish peroxidase or complex ES of cytochrome-c peroxidase, was assigned to a two-electron oxidized state [(protoporphyrin IX)FeIVO] Tyr+. which was formed by an intramolecular electron transfer from tyrosine to the porphyrin-pi-cation radical of intermediate I. A reaction scheme for prostaglandin H synthase is proposed where the tyrosyl radical of intermediate II activates the cyclooxygenase reaction.     

Reduced affinity for Isoniazid in the S315T mutant of Mycobacterium tuberculosis KatG is a key factor in antibiotic resistance

Catalase-peroxidase (KatG) from Mycobacterium tuberculosis is responsible for the activation of the antitubercular drug isonicotinic acid hydrazide (INH) and is important for survival of M. tuberculosis in macrophages. Characterization of the structure and catalytic mechanism of KatG is being pursued to provide insights into drug (INH) resistance in M. tuberculosis. Site-directed mutagenesis was used to prepare the INH-resistant mutant KatG[S315T], and the overexpressed enzyme was characterized and compared with wild-type KatG. KatG[S315T] exhibits a reduced tendency to form six-coordinate heme, because of coordination of water to iron during purification and storage, and also forms a highly unstable Compound III (oxyferrous enzyme). Catalase activity and peroxidase activity measured using t-butylhydroperoxide and o-dianisidine were moderately reduced in the mutant compared with wild-type KatG. Stopped-flow spectrophotometric experiments revealed a rate of Compound I formation similar to wild-type KatG using peroxyacetic acid to initiate the catalytic cycle, but no Compound I was detected when bulkier peroxides (chloroperoxybenzoic acid, t-butylhydroperoxide) were used. The affinity of resting (ferric) KatG[S315T] for INH, measured using isothermal titration calorimetry, was greatly reduced compared with wild-type KatG, as were rates of reaction of Compound I with the drug. These observations reveal that although KatG[S315T] maintains reasonably good steady state catalytic rates, poor binding of the drug to the enzyme limits drug activation and brings about INH resistance.     

Tyrosine 385 of prostaglandin endoperoxide synthase is required for cyclooxygenase catalysis

There are spectral and biochemical data suggesting that a tyrosine group(s) is involved in the cyclooxygenase reaction catalyzed by prostaglandin endoperoxide (PGH) synthase. Treatment with tetranitromethane, a reagent which nitrates tyrosine residues, abolishes cyclooxygenase activity, but this inactivation can be largely prevented by competitive cyclooxygenase inhibitors such as ibuprofen and indomethacin. To identify sites of nitration, native PGH synthase and indomethacin-pretreated PGH synthase were incubated with tetranitromethane, and the sequences of peptides containing nitrotyrosine were determined. Three unique tyrosines (Tyr-355, Tyr-385, and Tyr-417) were nitrated in the native enzyme but not in the indomethacin-treated PGH synthase. Using site-directed mutagenesis of sheep PGH synthase, each of these tyrosines, as well as two other tyrosine residues selected as controls (Tyr-254 and Tyr-262), were replaced with phenylalanine; cos-1 cells were transfected with constructs containing cDNAs coding for the native PGH synthase and each of the five phenylalanine mutants, and microsomes from these cells were assayed for cyclooxygenase and hydroperoxidase activities. The Phe-385 mutant of PGH synthase lacked cyclooxygenase activity but retained peroxidase activity; all other mutants expressed both enzyme activities. Our results establish that Tyr-385 is essential for the cyclooxygenase activity of PGH synthase and that nitration of this residue can be prevented by indomethacin. We conclude that Tyr-385 is at or near the cyclooxygenase active site of PGH synthase and could be the tyrosine residue proposed to be involved in the first step of the cyclooxygenase reaction, abstraction of the 13-proS hydrogen from arachidonate.     

Proton stoichiometry of the cytochrome c peroxidase mechanism as a function of pH.

The proton stoichiometry for the oxidation of cytochrome c peroxidase (ferrocytochrome c: hydrogen-peroxide oxidoreductase, EC 1.11.1.5) to cytochrome c peroxidase Compound I by H2O2, for the reduction of cytochrome c peroxidase Compound I to cytochrome c peroxidase Compound II by ferrocyanide, and for the reduction of cytochrome c peroxidase Compound II to the native enzyme by ferrocyanide has been determined as a function of pH between pH 4 and 8. The basic stoichiometry for the reaction is that no protons are required for the oxidation of the native enzyme to Compound I, while one proton is required for the reduction of Compound I to Compound II, and one proton is required for the reduction of Compound II to the native enzyme. Superimposed upon the basic stoichiometry is a contribution due to the perturbation of two ionizable groups in the enzyme by the redox reactions. The pKa values for the two groups are 4.9 +/- 0.3 and 5.7 +/- 0.2 in the native enzyme, 4.1 +/- 0.4 and 7.8 +/- 0.2 in Compound I, and 4.3 +/- 0.4 and 6.7 +/- 0.2 in Compound II.