Peroxidase self-inactivation in prostaglandin H synthase-1 pretreated with cyclooxygenase inhibitors or substituted with mangano protoporphyrin IX

Self-inactivation imposes an upper limit on bioactive prostanoid synthesis by prostaglandin H synthase (PGHS). Inactivation of PGHS peroxidase activity has been found to begin with Intermediate II, which contains a tyrosyl radical. The structure of this radical is altered by cyclooxygenase inhibitors, such as indomethacin and flurbiprofen, and by replacement of heme by manganese protoporphyrin IX (forming MnPGHS-1). Peroxidase self-inactivation in inhibitor-treated PGHS-1 and MnPGHS-1 was characterized by stopped-flow spectroscopic techniques and by chromatographic and mass spectrometric analysis of the metalloporphyrin. The rate of peroxidase inactivation was about 0.3 s(-)1 in inhibitor-treated PGHS-1 and much slower in MnPGHS-1 (0.05 s(-)1); as with PGHS-1 itself, the peroxidase inactivation rates were independent of peroxide concentration and structure, consistent with an inactivation process beginning with Intermediate II. The changes in metalloporphyrin absorbance spectra during inactivation of inhibitor-treated PGHS-1 were similar to those observed with PGHS-1 but were rather distinct in MnPGHS-1; the kinetics of the spectral transition from Intermediate II to the next species were comparable to the inactivation kinetics in each case. In contrast to the situation with PGHS-1 itself, significant amounts of heme degradation occurred during inactivation of inhibitor-treated PGHS-1, producing iron chlorin and heme-protein adduct species. Structural perturbations at the peroxidase site (MnPGHS-1) or at the cyclooxygenase site (inhibitor-treated PGHS-1) thus can influence markedly the kinetics and the chemistry of PGHS-1 peroxidase inactivation.     

Oxyferryl heme and not tyrosyl radical is the likely culprit in prostaglandin H synthase-1 peroxidase inactivation

Prostaglandin H synthase-1 (PGHS-1) is a bifunctional heme protein catalyzing both a peroxidase reaction, in which peroxides are converted to alcohols, and a cyclooxygenase reaction, in which arachidonic acid is converted into prostaglandin G2. Reaction of PGHS-1 with peroxide forms Intermediate I, which has an oxyferryl heme and a porphyrin radical. An intramolecular electron transfer from Tyr385 to Intermediate I forms Intermediate II, which contains two oxidants: an oxyferryl heme and the Tyr385 radical required for cyclooxygenase catalysis. Self-inactivation of the peroxidase begins with Intermediate II, but it has been unclear which of the two oxidants is involved. The kinetics of tyrosyl radical, oxyferryl heme, and peroxidase inactivation were examined in reactions of PGHS-1 reconstituted with heme or mangano protoporphyrin IX with a lipid hydroperoxide, 15-hydroperoxyeicosatetraenoic acid (15-HPETE), and ethyl hydrogen peroxide (EtOOH). Tyrosyl radical formation was significantly faster with 15-HPETE than with EtOOH and roughly paralleled oxyferryl heme formation at low peroxide levels. However, the oxyferryl heme intensity decayed much more rapidly than the tyrosyl radical intensity at high peroxide levels. The rates of reactions for PGHS-1 reconstituted with MnPPIX were approximately an order of magnitude slower, and the initial species formed displayed a wide singlet (WS) radical, rather than the wide doublet radical observed with PGHS-1 reconstituted with heme. Inactivation of the peroxidase activity during the reaction of PGHS-1 with EtOOH or 15-HPETE correlated with oxyferryl heme decay, but not with changes in tyrosyl radical intensity or EPR line shape, indicating that the oxyferryl heme, and not the tyrosyl radical, is responsible for the self-destructive peroxidase side reactions. Computer modeling to a minimal mechanism was consistent with oxyferryl heme being the source of peroxidase inactivation.     

Cyclooxygenase inactivation kinetics during reaction of prostaglandin H synthase-1 with peroxide

The peroxidase and cyclooxygenase activities of prostaglandin H synthase-1 (PGHS-1) both become irreversibly inactivated during reaction with peroxide. Sequential stopped-flow absorbance measurements with a chromogenic peroxidase cosubstrate previously were used to evaluate the kinetics of peroxidase inactivation during reaction of PGHS-1 with peroxide [Wu, G., et al. (1999) J. Biol. Chem. 274, 9231-7]. This approach has now been adapted to use a chromogenic cyclooxygenase substrate to analyze the detailed kinetics of cyclooxygenase inactivation during reaction of PGHS-1 with several hydroperoxides. In the absence of added reducing cosubstrates, which maximizes the levels of oxidized enzyme intermediates expected to lead to inactivation, cyclooxygenase activity was lost as fast as, or somewhat faster than, peroxidase activity. Cyclooxygenase inactivation kinetics appeared to be sensitive to the structure of the peroxide used. The addition of reducing cosubstrate during reaction of PGHS-1 with peroxide protected the peroxidase activity to a much greater degree than the cyclooxygenase activity. The results suggest a new concept of PGHS inactivation: that distinct damage can occur at the two active sites during side reactions of Intermediate II, which forms during reaction of PGHS with peroxide and which contains two oxidants, a ferryl heme in the peroxidase site, and a tyrosyl free radical in the cyclooxygenase site.     

Comparison of the peroxidase reaction kinetics of prostaglandin H synthase-1 and -2.

Prostaglandin H synthase isoforms 1 and 2 (PGHS-1 and -2) each have a peroxidase activity and also a cyclooxygenase activity that requires initiation by hydroperoxide. The hydroperoxide initiator requirement for PGHS-2 cyclooxygenase is about 10-fold lower than for PGHS-1 cyclooxygenase, and this difference may contribute to the distinct control of cellular prostanoid synthesis by the two isoforms. We compared the kinetics of the initial peroxidase steps in PGHS-1 and -2 to quantify mechanistic differences between the isoforms that might contribute to the difference in cyclooxygenase initiation efficiency. The kinetics of formation of Intermediate I (an Fe(IV) species with a porphyrin free radical) and Intermediate II (an Fe(IV) species with a tyrosyl free radical, thought to be the crucial oxidant in cyclooxygenase catalysis) were monitored at 4 degrees c by stopped flow spectrophotometry with several hydroperoxides as substrate. With 15-hydroperoxyeicosatetraenoic acid, the rate constant for Intermediate I formation (k1) was 2.3 x 10(7) M-1 s-1 for PGHS-1 and 2.5 x 10(7) M-1 s-1 for PGHS-2, indicating that the isoforms have similar initial reactivity with this lipid hydroperoxide. For PGHS-1, the rate of conversion of Intermediate I to Intermediate II (k2) became the limiting factor when the hydroperoxide level was increased, indicating a rate constant of 10(2)-10(3) s-1 for the generation of the active cyclooxygenase species. For PGHS-2, however, the transition between Intermediates I and II was not rate-limiting even at the highest hydroperoxide concentrations tested, indicating that the k2 value for PGHS-2 was much greater than that for PGHS-1. Computer modelling predicted that faster formation of the active cyclooxygenase species (Intermediate II) or increased stability of the active species increases the resistance of the cyclooxygenase to inhibition by the intracellular hydroperoxide scavenger, glutathione peroxidase. Kinetic differences between the PGHS isoforms in forming or stabilizing the active cyclooxygenase species can thus contribute to the difference in the regulation of their cellular activities.     

Rapid kinetics of tyrosyl radical formation and heme redox state changes in prostaglandin H synthase-1 and -2.

Hydroperoxide-induced tyrosyl radicals are putative intermediates in cyclooxygenase catalysis by prostaglandin H synthase (PGHS)-1 and -2. Rapid-freeze EPR and stopped-flow were used to characterize tyrosyl radical kinetics in PGHS-1 and -2 reacted with ethyl hydrogen peroxide. In PGHS-1, a wide doublet tyrosyl radical (34-35 G) was formed by 4 ms, followed by transition to a wide singlet (33-34 G); changes in total radical intensity paralleled those of Intermediate II absorbance during both formation and decay phases. In PGHS-2, some wide doublet (30 G) was present at early time points, but transition to wide singlet (29 G) was complete by 50 ms. In contrast to PGHS-1, only the formation kinetics of the PGHS-2 tyrosyl radical matched the Intermediate II absorbance kinetics. Indomethacin-treated PGHS-1 and nimesulide-treated PGHS-2 rapidly formed narrow singlet EPR (25-26 G in PGHS-1; 21 G in PGHS-2), and the same line shapes persisted throughout the reactions. Radical intensity paralleled Intermediate II absorbance throughout the indomethacin-treated PGHS-1 reaction. For nimesulide-treated PGHS-2, radical formed in concert with Intermediate II, but later persisted while Intermediate II relaxed. These results substantiate the kinetic competence of a tyrosyl radical as the catalytic intermediate for both PGHS isoforms and also indicate that the heme redox state becomes uncoupled from the tyrosyl radical in PGHS-2.     

Prostaglandin H synthase: Resolved and unresolved mechanistic issues

The cyclooxygenase and peroxidase activities of prostaglandin H synthase (PGHS)-1 and -2 have complex kinetics, with the cyclooxygenase exhibiting feedback activation by product peroxide and irreversible self-inactivation, and the peroxidase undergoing an independent self-inactivation process. The mechanistic bases for these complex, non-linear steady-state kinetics have been gradually elucidated by a combination of structure/function, spectroscopic and transient kinetic analyses. It is now apparent that most aspects of PGHS-1 and -2 catalysis can be accounted for by a branched chain radical mechanism involving a classic heme-based peroxidase cycle and a radical-based cyclooxygenase cycle. The two cycles are linked by the Tyr385 radical, which originates from an oxidized peroxidase intermediate and begins the cyclooxygenase cycle by abstracting a hydrogen atom from the fatty acid substrate. Peroxidase cycle intermediates have been well characterized, and peroxidase self-inactivation has been kinetically linked to a damaging side reaction involving the oxyferryl heme oxidant in an intermediate that also contains the Tyr385 radical. The cyclooxygenase cycle intermediates are poorly characterized, with the exception of the Tyr385 radical and the initial arachidonate radical, which has a pentadiene structure involving C11-C15 of the fatty acid. Oxygen isotope effect studies suggest that formation of the arachidonate radical is reversible, a conclusion consistent with electron paramagnetic resonance spectroscopic observations, radical trapping by NO, and thermodynamic calculations, although moderate isotope selectivity was found for the H-abstraction step as well. Reaction with peroxide also produces an alternate radical at Tyr504 that is linked to cyclooxygenase activation efficiency and may serve as a reservoir of oxidizing equivalent. The interconversions among radicals on Tyr385, on Tyr504, and on arachidonate, and their relationships to regulation and inactivation of the cyclooxygenase, are still under active investigation for both PGHS isozymes.     

Different suicide inactivation processes for the peroxidase and cyclooxygenase activities of prostaglandin endoperoxide H synthase-1.

Prostaglandin endoperoxide H synthases (PGHSs)-1 and -2 have a cyclooxygenase (COX) activity involved in forming prostaglandin G2 (PGG2) from arachidonic acid and an associated peroxidase (POX) activity that reduces PGG2 to PGH2. Suicide inactivation processes are observed for both POX and COX reactions. Here we report COX reaction conditions for PGHS-1 under which complete COX inactivation occurs but with > or = 60% retention of POX activity. The rates of POX inactivation were compared for native oPGHS-1 versus Y385F oPGHS-1, a mutant that cannot form the Tyr385 radical of COX Intermediate II; the rates were the same for both native and Y385F oPGHS-1. Our data indicate that a COX Intermediate II/acyl or product complex is the precursor in COX inactivation. However, another species, probably an Intermediate II-like species but with a radical centered on a tyrosine other than Tyr385, is the immediate precursor for POX inactivation.     

Role of Asn-382 and Thr-383 in activation and inactivation of human prostaglandin H synthase cyclooxygenase catalysis

Cyclooxygenase catalysis by prostaglandin H synthase-1 and -2 (PGHS-1 and -2) requires activation of the normally latent enzyme by peroxide-dependent generation of a free radical at Tyr-385 (PGHS-1 numbering) in the cyclooxygenase active site; the Tyr-385 radical has also been linked to self-inactivation processes that impose an ultimate limit on cyclooxygenase catalysis. Cyclooxygenase activation is more resistant to suppression by cytosolic glutathione peroxidase in PGHS-2 than in PGHS-1. This differential response to peroxide scavenging enzymes provides a basis for the differential catalytic regulation of the two PGHS isoforms observed in vivo. We sought to identify structural differences between the isoforms, which could account for the differential cyclooxygenase activation, and used site-directed mutagenesis of recombinant human PGHS-2 to focus on one heme-vicinity residue that diverges between the two isoforms, Thr-383, and an adjacent residue that is conserved between the isoforms, Asn-382. Substitutions of Thr-383 (histidine in most PGHS-1) with histidine or aspartate decreased cyclooxygenase activation efficiency by about 40%, with little effect on cyclooxygenase specific activity or self-inactivation. Substitutions of Asn-382 with alanine, aspartate, or leucine had little effect on the cyclooxygenase specific activity or activation efficiency but almost doubled the cyclooxygenase catalytic output before self-inactivation. Asn-382 and Thr-383 mutations did not appreciably alter the Km value for arachidonate, the cyclooxygenase product profile, or the Tyr-385 radical spectroscopic characteristics, confirming the structural integrity of the cyclooxygenase site. The side chain structures of Asn-382 and Thr-383 in PGHS-2 thus selectively influence two important aspects of cyclooxygenase catalytic regulation: activation by peroxide and self-inactivation.     

Kinetic study of the slow cyanide binding to Glycera dibranchiata monomer hemoglobin components III and IV

Compared to other monomeric heme proteins and the heme peroxidases, the Glycera dibranchiata monomer hemoglobin components III and IV exhibit very slow cyanide binding kinetics. This is agreement with the previously reported behavior of component II. Similar to component II, components III and IV have been studied under pseudo-first-order conditions at pH 6.0, 7.0, 8.0, and 9.0 by using a 100-250-fold excess of potassium cyanide at each pH. At 20 degrees C with micromolar protein concentrations, kobs for component III varies between 7.08 x 10(-5) s-1 at pH 6.0 and 100-fold cyanide excess and 1.06 x 10(-2) s-1 at pH 9.0 and 250-fold cyanide excess. For component IV, the values are 2.03 x 10(-4) s-1 for 100-fold cyanide excess at pH 6.0 and 4.13 x 10(-2) s-1 for 250-fold cyanide excess at pH 9.0. In comparison to other heme proteins, our analysis shows that the bimolecular rate constant (klapp) is small. For example, at pH 7.0, it is 3.02 x 10(-1) M-1 s-1 for component III and 1.82 M-1 s-1 for component IV, compared to 400 M-1 s-1 for sperm whale metmyoglobin, 692 M-1 s-1 for soybean metleghemoglobin a, 111 M-1 s-1 for guinea pig methemoglobin, and 1.1 x 10(5) M-1 s-1 for cytochrome c peroxidase. Our results also show that the dissociation rates (k-lapp) are extremely slow and no larger than 10(-6) s-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Heme catalyzes tyrosine 385 nitration and inactivation of prostaglandin H2 synthase-1 by peroxynitrite

The mechanism by which the inflammatory enzyme prostaglandin H(2) synthase-1 (PGHS-1) deactivates remains undefined. This study aimed to determine the stabilizing parameters of PGHS-1 and identify factors leading to deactivation by nitric oxide species (NO(x)). Purified PGHS-1 was stabilized when solubilized in beta-octylglucoside (rather than Tween-20 or CHAPS) and when reconstituted with hemin chloride (rather than hematin). Peroxynitrite (ONOO(-)) activated the peroxidase site of PGHS-1 independently of the cyclooxygenase site. After ONOO(-) exposure, holoPGHS-1 could not metabolize arachidonic acid and was structurally compromised, whereas apoPGHS-1 retained full activity once reconstituted with heme. After incubation of holoPGHS-1 with ONOO(-), heme absorbance was diminished but to a lesser extent than the loss in enzymatic function, suggesting the contribution of more than one process to enzyme inactivation. Hydroperoxide scavengers improved enzyme activity, whereas hydroxyl radical scavengers provided no protection from the effects of ONOO(-). Mass spectral analyses revealed that tyrosine 385 (Tyr 385) is a target for nitration by ONOO(-) only when heme is present. Multimer formation was also observed and required heme but could be attenuated by arachidonic acid substrate. We conclude that the heme plays a role in catalyzing Tyr 385 nitration by ONOO(-) and the demise of PGHS-1.     

Substitution of tyrosine for the proximal histidine ligand to the heme of prostaglandin endoperoxide synthase 2: implications for the mechanism of cyclooxygenase activation and catalysis

Prostaglandin H(2) synthesis by prostaglandin endoperoxide synthase (PGHS) requires the heme-dependent activation of the protein's cyclooxygenase activity. The PGHS heme participates in cyclooxygenase activation by accepting an electron from Tyr385 located in the cyclooxygenase active site. Two mechanisms have been proposed for the oxidation of Tyr385 by the heme iron: (1) ferric enzyme oxidizes a hydroperoxide activator and the incipient peroxyl radical oxidizes Tyr385, or (2) ferric enzyme reduces a hydroperoxide activator and the incipient ferryl-oxo heme oxidizes Tyr385. The participation of ferrous PGHS in cyclooxygenase activation was evaluated by determining the reduction potential of PGHS-2. Under all conditions tested, this potential (<-135 mV) was well below that required for reactions leading to cyclooxygenase activation. Substitution of the proximal heme ligand, His388, with tyrosine was used as a mechanistic probe of cyclooxygenase activation. His388Tyr PGHS-2, expressed in insect cells and purified to homogeneity, retained cyclooxygenase activity but its peroxidase activity was diminished more than 300-fold. Concordant with this poor peroxidase activity, an extensive lag in His388Tyr cyclooxygenase activity was observed. Addition of hydroperoxides resulted in a concentration-dependent decrease in lag time consistent with each peroxide's ability to act as a His388Tyr peroxidase substrate. However, hydroperoxide treatment had no effect on the maximal rate of arachidonate oxygenation. These data imply that the ferryl-oxo intermediates of peroxidase catalysis, but not the Fe(III)/Fe(II) couple of PGHS, are essential for cyclooxygenase activation. In addition, our findings are strongly supportive of a branched-chain mechanism of cyclooxygenase catalysis in which one activation event leads to many cyclooxygenase turnovers.     

Histidine 386 and its role in cyclooxygenase and peroxidase catalysis by prostaglandin-endoperoxide H synthases

Prostaglandin-endoperoxide H synthases (PGHSs) have a cyclooxygenase that forms prostaglandin (PG) G2 from arachidonic acid (AA) plus oxygen and a peroxidase that reduces the PGG2 to PGH2. The peroxidase activates the cyclooxygenase. This involves an initial oxidation of the peroxidase heme group by hydroperoxide, followed by oxidation of Tyr385 to a tyrosyl radical within the cyclooxygenase site. His386 of PGHS-1 is not formally part of either active site, but lies in an extended helix between Tyr385, which protrudes into the cyclooxygenase site, and His388, the proximal ligand of the peroxidase heme. When His386 was substituted with alanine in PGHS-1, the mutant retained <2.5% of the native peroxidase activity, but >20% of the native cyclooxygenase activity. However, peroxidase activity could be restored (10-30%) by treating H386A PGHS-1 with cyclooxygenase inhibitors or AA, but not with linoleic acid; in contrast, mere occupancy of the cyclooxygenase site of native PGHS-1 had no effect on peroxidase activity. Heme titrations indicated that H386A PGHS-1 binds heme less tightly than does native PGHS-1. The low peroxidase activity and decreased affinity for heme of H386A PGHS-1 imply that His386 helps optimize heme binding. Molecular dynamic simulations suggest that this is accomplished in part by a hydrogen bond between the heme D-ring propionate and the N-delta of Asn382 of the extended helix. The structure of the extended helix is, in turn, strongly supported by stable hydrogen bonding between the N-delta of His386 and the backbone carbonyl oxygens of Asn382 and Gln383. We speculate that the binding of cyclooxygenase inhibitors or AA to the cyclooxygenase site of ovine H386A PGHS-1 reopens the constriction in the cyclooxygenase site between the extended helix and a helix containing Gly526 and Ser530 and restores native-like structure to the extended helix. Being less bulky than AA, linoleic acid is apparently unable to reopen this constriction.     

Interactions between nitric oxide and peroxynitrite during prostaglandin endoperoxide H synthase-1 catalysis: a free radical mechanism of inactivation

Peroxynitrite (ONOO(-)) can serve either as a peroxide substrate or as an inactivator of prostaglandin endoperoxide H synthase-1 (PGHS-1). Herein, the mechanism of PGHS-1 inactivation by ONOO(-) and the modulatory role that nitric oxide (*NO) plays in this process were studied. PGHS-1 reacted with ONOO(-) with a second-order rate constant of 1.7 x 10(7) M(-1) s(-1) at pH 7.0 and 8 degrees C. In the absence of substrates, the enzyme was dose-dependently inactivated by ONOO(-) in parallel with 3-nitrotyrosine formation. However, when PGHS-1 was incubated with ONOO(-) in the presence of substrates, the direct reaction with ONOO(-) was less relevant and ONOO(-)-derived radicals became involved in enzyme inactivation. Bicarbonate at physiologically relevant concentrations enhanced PGHS-1 inactivation and nitration by ONOO(-), further supporting a free radical mechanism. Importantly, *NO (0.4-1.5 microM min(-1)) was able to spare the peroxidase activity of PGHS-1 but it enhanced ONOO(-)-mediated inactivation of cyclooxygenase. The observed differential effects of *NO on ONOO(-)-mediated PGHS-1 inactivation emphasize a novel aspect of the complex modulatory role that *NO plays during inflammatory processes. We conclude that ONOO(-)-derived radicals inactivate both peroxidase and cyclooxygenase activities of PGHS-1 during enzyme turnover. Finally, our results reconcile the proposed alternative effects of ONOO(-) on PGHS-1 (activation versus inactivation).     

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.     

Manganese peroxidase from the lignin-degrading basidiomycete Phanerochaete chrysosporium. Transient state kinetics and reaction mechanism

Stopped-flow techniques were used to investigate the kinetics of the formation of manganese peroxidase compound I (MnPI) and of the reactions of MnPI and manganese peroxidase compound II (MnPII) with p-cresol and MnII. All of the rate data were obtained from single turnover experiments under pseudo-first order conditions. In the presence of H2O2 the formation of MnPI is independent of pH over the range 3.12-8.29 with a second-order rate constant of (2.0 +/- 0.1) x 10(6) M-1 s-1. The activation energy for MnPI formation is 20 kJ mol-1. MnPI formation also occurs with organic peroxides such as peracetic acid, m-chloroperoxybenzoic acid, and p-nitroperoxybenzoic acid with second-order rate constants of 9.7 x 10(5), 9.5 x 10(4), and 5.9 x 10(4) M-1 s-1, respectively. The reactions of MnPI and MnPII with p-cresol strictly obeyed second-order kinetics. The second-order rate constant for the reaction of MnPII with p-cresol is extremely low, (9.5 +/- 0.5) M-1 s-1. Kinetic analysis of the reaction of MnII with MnPI and MnPII showed a binding interaction with the oxidized enzymes which led to saturation kinetics. The first-order dissociation rate constants for the reaction of MnII with MnPI and MnPII are (0.7 +/- 0.1) and (0.14 +/- 0.01) s-1, respectively, when the reaction is conducted in lactate buffer. Rate constants are considerably lower when the reactions are conducted in succinate buffer. Single turnover experiments confirmed that MnII serves as an obligatory substrate for MnPII and that both oxidized forms of the enzyme form productive complexes with MnII. Finally, these results suggest the alpha-hydroxy acids such as lactate facilitate the dissociation of MnIII from the enzyme.     

O2-mediated oxidation of hemopexin-heme(II)-NO

Hemopexin (HPX), serving as scavenger and transporter of toxic plasma heme, has been postulated to play a key role in the homeostasis of NO. Here, kinetics of HPX-heme(II) nitrosylation and O2-mediated oxidation of HPX-heme(II)-NO are reported. NO reacts reversibly with HPX-heme(II) yielding HPX-heme(II)-NO, according to the minimum reaction scheme: HPX-heme(II)+NO kon<-->koff HPX-heme(II)-NO values of kon, koff, and K (=kon/koff) are (6.3+/-0.3)x10(3)M-1s-1, (9.1+/-0.4)x10(-4)s-1, and (6.9+/-0.6)x10(6)M-1, respectively, at pH 7.0 and 10.0 degrees C. O2 reacts with HPX-heme(II)-NO yielding HPX-heme(III) and NO3-, by means of the ferric heme-bound peroxynitrite intermediate (HPX-heme(III)-N(O)OO), according to the minimum reaction scheme: HPX-heme(II)-NO+O2 hon<--> HPX-heme(III)-N(O)OO l-->HPX-heme(III)+NO3- the backward reaction rate is negligible. Values of hon and l are (2.4+/-0.3)x10(1)M-1s-1 and (1.4+/-0.2)x10(-3)s-1, respectively, at pH 7.0 and 10.0 degrees C. The decay of HPX-heme(III)-N(O)OO (i.e., l) is rate limiting. The HPX-heme(III)-N(O)OO intermediate has been characterized by optical absorption spectroscopy in the Soret region (lambdamax=409 nm and epsilon409=1.51x10(5)M-1cm-1). These results, representing the first kinetic evidence for HPX-heme(II) nitrosylation and O2-mediated oxidation of HPX-heme(II)-NO, might be predictive of transient (pseudo-enzymatic) function(s) of heme carriers.     

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.     

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.     

Role of the low-affinity binding site in electron transfer from cytochrome C to cytochrome C peroxidase

The interaction of yeast iso-1-cytochrome c (yCc) with the high- and low-affinity binding sites on cytochrome c peroxidase compound I (CMPI) was studied by stopped-flow spectroscopy. When 3 microM reduced yCc(II) was mixed with 0.5 microM CMPI at 10 mM ionic strength, the Trp-191 radical cation was reduced from the high-affinity site with an apparent rate constant >3000 s(-1), followed by slow reduction of the oxyferryl heme with a rate constant of only 10 s(-1). In contrast, mixing 3 microM reduced yCc(II) with 0.5 microM preformed CMPI *yCc(III) complex led to reduction of the radical cation with a rate constant of 10 s(-1), followed by reduction of the oxyferryl heme in compound II with the same rate constant. The rate constants for reduction of the radical cation and the oxyferryl heme both increased with increasing concentrations of yCc(II) and remained equal to each other. These results are consistent with a mechanism in which both the Trp-191 radical cation and the oxyferryl heme are reduced by yCc(II) in the high-affinity binding site, and the reaction is rate-limited by product dissociation of yCc(III) from the high-affinity site with apparent rate constant k(d). Binding yCc(II) to the low-affinity site is proposed to increase the rate constant for dissociation of yCc(III) from the high-affinity site in a substrate-assisted product dissociation mechanism. The value of k(d) is <5 s(-1) for the 1:1 complex and >2000 s(-1) for the 2:1 complex at 10 mM ionic strength. The reaction of horse Cc(II) with CMPI was greatly inhibited by binding 1 equiv of yCc(III) to the high-affinity site, providing evidence that reduction of the oxyferryl heme involves electron transfer from the high-affinity binding site rather than the low-affinity site. The effects of CcP surface mutations on the dissociation rate constant indicate that the high-affinity binding site used for the reaction in solution is the same as the one identified in the yCc*CcP crystal structure.     

Reduction of prostaglandin H synthase compound II by phenol and hydroquinone, and the effect of indomethacin.

The reduction of prostaglandin H synthase compound II to native enzyme by phenol and by hydroquinone, in the presence of diethyldithiocarbamate as a stabilizing agent, was studied by rapid scan spectrometry and transient state kinetics at 4.0 +/- 0.5 degrees C in 0.1 M phosphate buffer, pH 8.0. The plot of pseudo-first-order rate constants for the conversion of prostaglandin H synthase compound II to native enzyme versus phenol concentration was linear with a non-zero intercept. The second-order rate constant was determined from the slope to be (5.3 +/- 0.3) x 10(5) M-1 s-1. For the reduction by hydroquinone, the second-order rate constant was determined from pointwise measurements of the pseudo-first-order rate constant to be (2.1 +/- 0.4) x 10(6) M-1 s-1. Rapid scan spectrum results also showed the reduction of compound I to compound II by both phenol and hydroquinone. Thus reduction of both compound I and compound II is one electron process. Our results suggest that the tyrosyl radical, detected in the presence of oxidizing agents, is formed by intramolecular electron transfer from the tyrosyl residue to the porphyrin pi-cation radical, and this reaction tends to disappear in the presence of sufficient reducing substrate. These in vitro results support speculation that there is a role of the peroxidase component of prostaglandin H synthase in benzene-induced toxicity. In the present work, the effect of indomethacin on the reduction of prostaglandin H synthase compound II by diethyldithiocarbamate, phenol, and hydroquinone was also investigated. Results revealed, for the first time, that indomethacin is an inhibitor of the peroxidase activity of prostaglandin H synthase, although not as effectively as in its well-known inhibition of cyclooxygenase activity.