Detection of an oxyferryl porphyrin pi-cation-radical intermediate in the reaction between hydrogen peroxide and a mutant yeast cytochrome c peroxidase. Evidence for tryptophan-191 involvement in the radical site of compound I

Peroxide oxidation of a mutant cytochrome c peroxidase, in which Trp-191 has been replaced by Phe through site-directed mutagenesis, produces an oxidized intermediate whose stable UV/visible absorption spectrum is very similar to that of compound I of the native yeast enzyme. This spectrum is characteristic of an oxyferryl, Fe(IV), heme. Stopped-flow studies reveal that the reaction between the mutant enzyme and hydrogen peroxide is biphasic with the transient formation of an intermediate whose absorption spectrum is quite distinct from that of either the native ferric enzyme or the final product. Rapid spectral scanning of the intermediate provides a spectrum characteristic of an oxyferryl porphyrin pi-cation-radical species. At pH 6, 100 mM ionic strength, and 25 degrees C, the rate constant for formation of the oxyferryl pi-cation radical has a lower limit of 6 X 10(7) M-1 s-1 and the rate of conversion of the transient intermediate to the final oxidized product is 51 +/- 4 s-1. Evidence is presented indicating that Trp-191 either is the site of the radical in CcP compound I or is intimately involved in formation of the radical.     

A steady-state study on the formation of Compounds II and III of myeloperoxidase

The reaction between native myeloperoxidase and hydrogen peroxide, yielding Compound II, was investigated using the stopped-flow technique. The pH dependence of the apparent second-order rate constant showed the existence of a protonatable group on the enzyme with a pKa of 4.9. This group is ascribed to the distal histidine imidazole, which must be deprotonated to enable the reaction of Compound I with hydrogen peroxidase to take place. The rate constant for the formation of Compound II by hydrogen peroxide was 3.5.10(4) M-1.s-1. During the reaction of myeloperoxidase with H2O2, rapid reduction of added cytochrome c was observed. This reduction was inhibitable by superoxide dismutase, and this demonstrates that superoxide anion radicals are generated. When potassium ferrocyanide was used as an electron donor to generate Compound II from Compound I, the pH dependence of the apparent second-order rate constant indicated involvement of a group with a pKa of 4.5. However, with ferrocyanide as an electron donor, protonation of the group was necessary to enable the reaction to take place. The rate constant for the generation of Compound II by ferrocyanide was 1.6.10(7) M-1.s-1. We also investigated the reaction of Compound II with hydrogen peroxide, yielding Compound III. Formation of Compound III (k = 50 M-1.s-1) proceeded via two different pathways, one of which was inhibitable by tetranitromethane. We further investigated the stability of Compound II and Compound III as a function of pH, ionic strength and enzyme concentration. The half-life values of both Compound II and Compound III were independent of the enzyme concentration and ionic strength. The half-life value of Compound III was pH-dependent, showing a decreasing stability with increasing pH, whereas the stability of Compound II was independent of pH over the range 3-11.     

Laser flash photolysis studies of the reduction kinetics of NADPH:cytochrome P-450 reductase

The reduction kinetics of NADPH:cytochrome P-450 reductase have been investigated by the laser flash photolysis technique, using the semiquinone of 5-deazariboflavin (5-dRfH.) as the reductant. Transients observed at 470 nm at neutral pH indicated that the oxidized reductase was reduced via second-order kinetics with a rate constant of 6.8 X 10(7) M-1 s-1. The second-order rate constant corresponding to the formation of the protein-bound semiquinone (measured at 585 nm) was essentially the same as that obtained at 470 nm (7.1 X 10(7) M-1 s-1). Subsequent to this rapid formation of protein-bound semiquinone, a partial exponential decay was observed at 585 nm. The rate of this decay remained invariant with protein concentration between pH 5.0 and 7.0, and a first-order rate constant of 70 s-1 was obtained for this process. This is assigned to intramolecular electron transfer from FADH. to FMN. Prior reduction of the enzyme to the one-electron level led to a decrease in both the second-order rate constant for reduction (2 X 10(7) M-1 s-1) and the first-order intraflavin electron transfer rate constant (15 s-1). The protein-bound FAD moiety of FMN-depleted reductase was reduced by 5-dRfH. with a second-order rate constant that was identical with that observed with the native enzyme (6.9 X 10(7) M-1 s-1). However, with this species no significant decay of the FAD semiquinone was observed at 585 nm following its rapid formation, consistent with the above assignment of this kinetic process.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reaction of bovine-liver copper-zinc superoxide dismutase with hydrogen peroxide. Evidence for reaction with H2O2 and HO2- leading to loss of copper

The reaction of hydrogen peroxide with the copper-zinc bovine-liver superoxide dismutase at low molar ratios (0.2-20.0) of H2O2/active site between pH 7.3-10.0 leads to the loss of native enzyme as a distinct form monitored by electrophoresis. The pH dependence of the loss of native enzyme between 7.3 and 9.0 indicates the involvement of a conjugate base on the enzyme of pKa of 8.7 +/- 0.1. The rate of loss of the native enzyme is first order with respect to the concentration of both enzyme and hydrogen peroxide between pH 7.3 and 9.0 with no evidence for binding of peroxide. A second-order rate constant of 3.0 +/- 1.0 M-1 s-1 is obtained from these data. At pH 10.0 the reaction is first order with respect to enzyme concentration but saturable in H2O2. All data are consistent with the interpretation that H2O2 reacts with the enzyme at the lower pH where the reaction is dependent upon the conjugate base of a functional group on the enzyme. At the higher pH, the data are consistent with the reaction of HO2- and H2O2 with the dismutase. The dissociation constant for HO2- calculated from the kinetic data at pH 10.0 is between 25-50 microM and the rate constant for the breakdown of the HO2- dismutase complex is 1.10 + 0.05 x 10(-2) s-1. The change in the electrophoretic pattern at all pH values is accompanied by the loss of the ability of the enzyme to bind copper. Weakly bound or free copper can be detected using bathocuproine disulfonate. Furthermore copper-defficient forms of the enzyme can be detected by staining gels of the peroxide-treated dismutase with diethyldithiocarbamate.     

Cytochrome c peroxidase catalyzed oxidation of ferrocytochrome c by hydrogen peroxide: ionic strength dependence of the steady-state rate parameters

The steady-state kinetics of the cytochrome c peroxidase catalyzed oxidation of horse heart ferrocytochrome c by hydrogen peroxide have been studied at both pH 7.0 and pH 7.5 as a function of ionic strength. Plots of the initial velocity versus hydrogen peroxide concentration at fixed cytochrome c are hyperbolic. The limiting slope at low hydrogen peroxide give apparent bimolecular rate constants for the cytochrome c peroxidase-hydrogen peroxide reaction identical with those determined directly by stopped-flow techniques. Plots of the initial velocity versus cytochrome c concentration at saturating hydrogen peroxide (200 microM) are nonhyperbolic. The rate expression requires squared terms in cytochrome c concentration. The maximum turnover rate of the enzyme is independent of ionic strength, with values of 470 +/- 50 s-1 and 290 +/- 30 s-1 at pH 7.0 and 7.5, respectively. The limiting slope of velocity versus cytochrome c concentration plots provides a lower limit for the association rate constant between cytochrome c and the oxidized intermediates of cytochrome c peroxidase. The limiting slope varies from 10(6) M-1 s-1 at 300 mM ionic strength to 10(8) M-1 s-1 at 20 mM ionic strength and extrapolates to 5 x 10(8) M-1 s-1 at zero ionic strength. The data are discussed in terms of both a two-binding-site mechanism and a single-binding-site, multiple-pathway mechanism.     

Lignin peroxidase compound III. Mechanism of formation and decomposition

Lignin peroxidase compound III (LiPIII) was prepared via three procedures: (a) ferrous LiP + O2 (LiPIIIa), (b) ferric LiP + O2-. (LiPIIIb), and (c) LiP compound II + excess H2O2 followed by treatment with catalase (LiPIIIc). LiPIIIa, b, and c each have a Soret maximum at approximately 414 nm and visible bands at 543 and 578 nm. LiPIIIa, b, and c each slowly reverted to native ferric LiP, releasing stoichiometric amounts of O2-. in the process. Electronic absorption spectra of LiPIII reversion to the native enzyme displayed isosbestic points in the visible region at 470, 525, and 597 nm, suggesting a single-step reversion with no intermediates. The LiPIII reversion reactions obeyed first-order kinetics with rate constants of approximately 1.0 X 10(-3) s-1. In the presence of excess peroxide, at pH 3.0, native LiP, LiPII, and LiPIIIa, b, and c are all converted to a unique oxidized species (LiPIII*) with a spectrum displaying visible bands at 543 and 578 nm, but with a Soret maximum at 419 nm, red-shifted 5 nm from that of LiPIII. LiPIII* is bleached and inactivated in the presence of excess H2O2 via a biphasic process. The fast first phase of this bleaching reaction obeys second-order kinetics, with a rate constant of 1.7 X 10(1) M-1 s-1. Addition of veratryl alcohol to LiPIII* results in its rapid reversion to the native enzyme, via an apparent one-step reaction that obeys second-order kinetics with a rate constant of 3.5 X 10(1) M-1 s-1. Stoichiometric amounts of O2-. are released during this reaction. When this reaction was run under conditions that prevented further reactions, HPLC analysis of the products demonstrated that veratryl alcohol was not oxidized. These results suggest that the binding of veratryl alcohol to LiPIII* displaces O2-., thus returning the enzyme to its native state. In contrast, the addition of veratryl alcohol to LiPIII did not affect the rate of spontaneous reversion of LiPIII to the native enzyme.     

The reactions of Pseudomonas cytochrome c-551 oxidase with potassium cyanide.

The binding of cyanide to both oxidized and ascorbate-reduced forms of Pseudomonas cytochrome c-551 oxidase was investigated. Spectral studies on the oxidized enzyme and its apoprotein showed that the ligand can bind to both the c and d, haem components of the molecule, and kinetic observations indicated that both chromophores reacted, under a variety of conditions, with very similar rates. Cyanide combination velocities were dependent on ligand concentration, and increasing the pH also accelerated the reaction; the second-order rate constant was estimated as approx. 0.2M-1 . s-1 at pH 7.0. The binding of cyanide to the protein was observed to have a considerable influence on reduction of the enzyme by ascorbate. Spectral and kinetic observations have revealed that the species haem d13+-cyanide and any unbound haem c may react relatively rapidly with the reductant, but the behaviour of cyanide-bound haem c indicates that it may not be reduced without prior dissociation of the ligand, which occurs relatively slowly. The reaction of reduced Pseudomonas cytochrome oxidase with cyanide is radically different from that of the oxidized protein. In this case the ligand only binds to the haem d1 component and reacts much more rapidly. Stopped-flow kinetic measurements showed the binding to be biphasic in form. Both the rates of these processes were dependent on cyanide concentration, with the fast phase having a second-order rate constant of 9.3 X 10(5) M-1 . s-1 and the slow phase one of 2.3 X 10(5) M-1 . s-1. The relative proportions of the two phases also showed a dependency on cyanide concentration, the slower phase increasing as the cyanide concentration decreased. Computer simulations indicate that a reaction scheme originally proposed for the reaction of the enzyme with CO is capable of providing a reasonable explanation of the experimental results. Static-titration data of the reduced enzyme with with cyanide indicated that the binding was non-stoicheiometric, the ligand-binding curve being sigmoidal in shape. A Hill plot of the results yielded a Hill coefficient of 2.6.     

Characterization of vanadium bromoperoxidase from Macrocystis and Fucus: reactivity of vanadium bromoperoxidase toward acyl and alkyl peroxides and bromination of amines

Vanadium bromoperoxidase (V-BrPO) has been isolated and purified from the marine brown algae Fucus distichus and Macrocystis pyrifera. V-BrPO catalyzes the oxidation of bromide by hydrogen peroxide, resulting in the bromination of certain organic acceptors or the formation of dioxygen. V-BrPO from F. distichus and M. pyrifera have subunit molecular weights of 65,000 and 74,000, respectively, and specific activities of 1580 units/mg (pH 6.5) and 1730 units/mg (pH 6) for the bromination of monochlorodimedone, respectively. As isolated, the enzymes contain a substoichiometric vanadium/subunit ratio; the vanadium content and specific activity are increased by addition of vanadate. V-BrPO (F. distichus, M. pyrifera, and Ascophyllum nodosum) also catalyzes the oxidation of bromide using peracetic acid. In the absence of an organic acceptor, a mixture of oxidized bromine species (e.g., hypobromous acid, bromine, and tribromide) is formed. Bromamine derivatives are formed from the corresponding amines, while 5-bromocytosine is formed from cytosine. In all cases, the rate of the V-BrPO-catalyzed reaction is much faster than that of the uncatalyzed oxidation of bromide by peracetic acid, at pH 8.5, 1 mM bromide, and 2 mM peracetic acid. In contrast to hydrogen peroxide, V-BrPO does not catalyze formation of dioxygen from peracetic acid in either the presence or absence of bromide. V-BrPO also uses phenylperacetic acid, m-chloroperoxybenzoic acid, and p-nitroperoxybenzoic acid to catalyze the oxidation of bromide; dioxygen is not formed with these peracids. V-BrPO does not catalyze bromide oxidation or dioxygen formation with the alkyl peroxides ethyl hydroperoxide, tert-butyl hydroperoxide, and cuminyl hydroperoxide.     

Mechanism of dioxygen formation catalyzed by vanadium bromoperoxidase. Steady state kinetic analysis and comparison to the mechanism of bromination

The steady state kinetic mechanism of the bromide-assisted disproportionation of hydrogen peroxide, forming dioxygen, catalyzed by vanadium bromoperoxidase has been investigated and compared to the mechanism of monochlorodimedone (MCD) bromination under conditions of 0.0125-6 mM H2O2, 1-500 mM Br-, and pH 4.55-6.52. Under these conditions, 50 microM MCD was sufficient to inhibit at least 90% of the dioxygen formation during MCD bromination. The rate data is consistent with a substrate-inhibited Bi Bi Ping Pong mechanism, in which the substrate bromide, is also an inhibitor at pH 4.55 and 5.25, but not at pH 5.91 and 6.52. The kinetic parameter KmBr, KmH2O2, KisBr, and KiiBr determined for the reactions of bromide-assisted disproportionation fo hydrogen peroxide and MCD bromination are similar, indicating that the mechanisms of both reactions occur via the formation of a common intermediate, the formation of which is rate-limiting. Fluoride is a competitive inhibitor with respect to hydrogen peroxide in both reactions at pH 6.5. At high concentrations of hydrogen peroxide, the bromide-assisted disproportionation of hydrogen peroxide occurs during the bromination of MCD. The sum of the rates of MCD bromination and dioxygen formation during MCD bromination is equal to the rate of dioxygen formation in the absence of MCD. The apportionment of the reaction through the MCD bromination and dioxygen formation pathways depends on pH, with much lower hydrogen peroxide concentrations causing significant dioxygen formation at higher pH.     

Characterization of the oxidized states of bromoperoxidase

Bromoperoxidase Compound I has been formed in reactions between bromoperoxidase and organic peroxide substrates. The absorbance spectrum of bromoperoxidase Compound I closely resembles the Compound I spectra of other peroxidases. The pH dependence of the second order rate constant for the formation of Compound I with hydrogen peroxide demonstrates the presence of an ionizable group at the enzyme active site having a pKa of 5.3. Protonation of this acidic group inhibits the rate of Compound I formation. This pKa value is higher than that determined for other peroxidases but the overall pH rate profiles for Compound I formation are similar. The one-electron reduction of bromoperoxidase Compound I yields Compound II and a second reduction yields native enzyme. Bromoperoxidase Compound II readily forms Compound III in the presence of an excess of hydrogen peroxide. Compound III passes through an as yet uncharacterized intermediate (III) in its decay to native enzyme. Compound III is produced and accumulates in enzymatic bromination reactions to become the predominate steady state form of the enzyme. Since Compound III is inactive as catalyst for enzymatic bromination, its accumulation leads to an idling reaction pathway which displays an unusual kinetic pattern for the bromination of monochlorodimedone.     

Kinetic parameters for the elimination reaction catalyzed by triosephosphate isomerase and an estimation of the reaction's physiological significance

Kinetic parameters for triosephosphate isomerase catalysis of the elimination reaction of an equilibrium mixture of dihydroxyacetone phosphate (DHAP) and D-glyceraldehyde-3-phosphate (DGAP) to form methylglyoxal and phosphate ion are reported for the enzyme from rabbit muscle. Pseudo-first-order rate constants for the disappearance of substrate (kelim) were determined for reactions at [Enzyme] much greater than [Substrate]. The second-order rate constant kEnz = 10.1 M-1 s-1 was determined from a plot of kelim against enzyme concentration. The kinetic parameters, determined from a steady-state kinetic analysis at [Substrate] much greater than [Enzyme], are kcat = 0.011 s-1, Km = 0.76 mM, and kcat/Km = 14 M-1 s-1. The estimated rate-constant ratio for partitioning of the enzyme-bound intermediate between protonation at carbon 2 and elimination, 1,000,000, is much larger than the ratio of 6.5 determined for the reaction of the enediolate phosphate in a loose complex with quinuclidinonium cation, a small buffer catalyst. There is a 10(5)-10(8)-fold decrease in the rate constant for the elimination reaction of the enediolate phosphate when this species binds to triosephosphate isomerase. The kinetic parameters for the elimination reaction catalyzed by the native triosephosphate isomerase and for the reaction catalyzed by a mutant form of the enzyme, which is missing a segment that forms hydrogen bonds with the phosphate group of substrate [Pompliano, D. L., Peyman, A., & Knowles, J. R. (1990) Biochemistry 29, 3186-3194] are similar.(ABSTRACT TRUNCATED AT 250 WORDS)     

The reaction of superoxide radical with catalase. Mechanism of the inhibition of catalase by superoxide radical

We have studied the time course of the absorption of bovine liver catalase after pulse radiolysis with oxygen saturation in the presence and absence of superoxide dismutase. In the absence of superoxide dismutase, catalase produced Compound I and another species. The formation of Compound I is due to the reaction of ferric catalase with hydrogen peroxide, which is generated by the disproportionation of the superoxide anion (O-2). The kinetic difference spectrum showed that the other species was neither Compound I nor II. In the presence of superoxide dismutase, the formation of this species was found to be inhibited, whereas that of Compound I was little affected. This suggests that this species is formed by the reaction of ferric catalase with O-2 and is probably the oxy form of this enzyme (Compound III). The rate constant for the reaction of O-2 and ferric catalase increased with a decrease in pH (cf. 4.5 X 10(4) M-1 s-1 at pH 9 and 4.6 X 10(6) M-1 s-1 at pH 5.). The pH dependence of the rate constant can be explained by assuming that HO2 reacts with this enzyme more rapidly than O-2.     

The reaction of hydrogen peroxide with the dimethyl ester heme derivative of cytochrome c peroxidase

A modified cytochrome c peroxidase was prepared by reconstituting apocytochrome c peroxidase with protoheme in which both heme propionic acid groups were converted to the methyl ester derivatives. The modified enzyme reacted with hydrogen peroxide with a rate constant of (1.3 +/- 0.2) x 10(7) M-1 s-1, which is 28% that of the native enzyme. The reaction between the modified enzyme and hydrogen peroxide was pH-dependent with an apparent pK of 5.1 +/- 0.1 compared to a value of 5.4 +/- 0.1 for the native enzyme. These observations support the conclusion that the apparent ionization near pH 5.4, which influences the hydrogen peroxide-cytochrome c peroxidase reaction is not due to the ionization of the propionate side chains of the heme group in the native enzyme. A second apparent ionization, with pK of 6.1 +/- 0.1, influences the spectrum of the modified enzyme which changes from a high spin type at low pH to a low spin type at high pH.     

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.     

The oxidation of Pseudomonas cytochrome c-551 oxidase by potassium ferricyanide.

Stopped-flow kinetics were made of the reaction between ascorbate-reduced Pseudomonas cytochrome oxidase and potassium ferricyanide under both N2 and CO atmospheres. Under N2 three kinetic processes were observed, two being dependent on ferricyanide concentration, with second-order rate constants of 9.6 X 10(4)M-1.s-1 and 1.5 X 10(4)M-1.s-1, whereas the other was concentration-independent, with a first-order rate constant of 0.17 +/- 0.03s-1. Measurements of their kinetic difference spectra have allowed the fastest and second-fastest phases of the reaction to be assigned to direct bimolecular reactions of ferricyanide with the haem c and haem d, moieties of the enzyme respectively. Under CO, the second-order rate constant for the reaction of the haem c was, at 1.3 X 10(5)M-1.s-1, slightly enhanced over the rate in a N2 atmosphere, but the reaction velocity of the haem d1 component was greatly decreased, being apparently limited to that of the rates of CO dissociation from the molecule (0.15s-1 and 0.03s-1). The results are compared with those obtained during a previous study of the reaction of reduced Pseudomonas cytochrome oxidase with oxidized azurin.     

Compound I formation is a partially rate-limiting process in chloroperoxidase-catalyzed bromination reactions

The kinetics of chloroperoxidase-catalyzed bromination and chlorination reactions were studied at various halide and hydrogen peroxide concentrations. At very high concentrations, both chloride (KI = 370 mM) and bromide (KI = 150 mM) are competitive substrate inhibitors versus hydrogen peroxide. Results at subinhibitory halide concentrations for bromination reactions (kcat = 4 ms-1, kcat/KPeroxide = 1.6 microM-1 x s-1 and kcat/KBr = 4.0 microM-1 x s-1) and chlorination reactions (kcat = 1.5 ms-1, kcat/Kperoxide = 2.3 microM-1 x s-1, and kcat/KBr = 0.32 microM-1 x s-1) indicate that halide oxidation is rate-limiting in chlorination reactions. However, in bromination reactions, both compound I formation and bromide oxidation are partially rate-limiting. This is the first documented case where compound I formation participates in determining the overall rate of a peroxidase reaction.     

The kinetics of electron transfer between pseudomonas aeruginosa cytochrome c-551 and its oxidase.

The redox reaction between cytochrome c-551 and its oxidase from the respiratory chain of pseudomonas aeruginosa was studied by rapid-mixing techniques at both pH7 and 9.1. The electron transfer in the direction of cytochrome c-551 reduction, starting with the oxidase in the reduced and CO-bound form, is monophasic, and the governing bimolecular rate constants are 1.3(+/- 0.2) x 10(7) M-1 . s-1 at pH 9.1 and 4 (+/- 1) x 10(6) M-1 . s-1 at pH 7.0. In the opposite direction, i.e. mixing the oxidized oxidase with the reduced cytochrome c-551 in the absence of O2, both a lower absorbance change and a more complex kinetic pattern were observed. With oxidized azurin instead of oxidized cytochrome c-551 the oxidation of the c haem in the CO-bound oxidase is also monophasic, and the second-order rate constant is 2 (+/- 0.7) x 10(6) M-1 . s-1 at pH 9.1. The redox potential of the c haem in the oxidase, as obtained from kinetic titrations of the completely oxidized enzyme with reduced azurin as the variable substrate, is 288 mV at pH 7.0 and 255 mV at pH 9.1. This is in contrast with the very high affinity observed in similar titrations performed with both oxidized azurin and oxidized cytochrome c-551 starting from the CO derivative of the reduced oxidase. It is concluded that: (i) azurin and cytochrome c-551 are not equally efficient in vitro as reducing substrates of the oxidase in the respiratory chain of Pseudomonas aeruginosa; (ii) CO ligation to the d1 haem in the oxidase induces a large decrease (at least 80 mV) in the redox potential of the c-haem moiety.     

Reactions of lignin peroxidase compounds I and II with veratryl alcohol. Transient-state kinetic characterization

Stopped-flow techniques were utilized to investigate the kinetics of the reaction of lignin peroxidase compounds I and II (LiPI and LiPII) with veratryl alcohol (VA). All rate data were collected from single turnover experiments under pseudo first-order conditions. The reaction of LiPI with VA strictly obeys second-order kinetics over the pH range 2.72-5.25 as demonstrated by linear plots of the pseudo first-order rate constants versus concentrations of VA. The second-order rate constants are strongly dependent on pH and range from 2.62 x 10(6) M-1 s-1 (pH 2.72) to 1.45 x 10(4) M-1 s-1 (pH 5.25). The reaction of LiPII and VA exhibits saturation behavior when the observed pseudo first-order rate constants are plotted against VA concentrations. The saturation phenomenon is quantitatively explained by the formation of a 1:1 LiPII-substrate complex. Results of kinetic and rapid scan spectral analyses exclude the formation of a LiPII-VA cation radical complex. The first-order dissociation rate constant and the equilibrium dissociation constant for the LiPII reaction are also pH dependent. Binding of VA to LiPII is controlled by a heme-linked ionizable group of pKa approximately 4.2. The pH profiles of the second-order rate constants for the LiPI reaction and of the first-order dissociation constants for the LiPII reaction both demonstrate two pKa values at approximately 3.0 and approximately 4.2. Protonated oxidized enzyme intermediates are most active, suggesting that only electron transfer, not proton uptake from the reducing substrate, occurs at the enzyme active site. These results are consistent with the one-electron oxidation of VA to an aryl cation radical by LiPI and LiPII.     

The reaction of Pseudomonas aeruginosa cytochrome c-551 oxidase with oxygen.

The reaction of ascorbate-reduced Pseudomonas cytochrome oxidase with oxygen was studied by using stopped-flow techniques at pH 7.0 and 25 degrees C. The observed time courses were complex, the reaction consisting of three phases. Of these, only the fastest process, with a second-order rate constant of 3.3 X 10(4) M-1.S-1, was dependent on oxygen concentration. The two slower processes were first-order reactions with rates of 1.0 +/- 0.4s-1 and 0.1 +/- 0.03s-1. A kinetic titration experiment revealed that the enzyme had a relatively low affinity constant for oxygen, approx. 10(4)M-1. Kinetic difference spectra were determined for all three reaction phases, showing each to have different characteristics. The fast-phase difference spectrum showed that changes occurred at both the haem c and haem d1 components of the enzyme during this process. These changes were consistent with the haem c becoming oxidized, but with the haem d1 assuming a form that did not correspond to the normal oxidized state, a situation that was not restored even after the second kinetic phase, which reflected further changes in the haem d1 component. The results are discussed in terms of a kinetic scheme.     

Laser flash photolysis study of intermolecular and intramolecular electron transfer in trimethylamine dehydrogenase

Laser flash photolysis has been used to investigate the kinetics of reduction of trimethylamine dehydrogenase by substoichiometric amounts of 5-deazariboflavin semiquinone, and the subsequent intramolecular electron transfer from the FMN cofactor to the Fe4S4 center. The initial reduction event followed second-order kinetics (k = 1.0 x 10(8) M-1 s-1 at pH 7.0 and 6.4 x 10(7) M-1 s-1 at pH 8.5) and resulted in the formation of the neutral FMN semiquinone and the reduced iron-sulfur cluster (in a ratio of approximately 1:3). Following this, a slower, protein concentration independent (and thus intramolecular) electron transfer was observed corresponding to FMN semiquinone oxidation and iron-sulfur cluster reduction (k = 62 s-1 at pH 7.0 and 30 s-1 at pH 8.5). The addition of the inhibitor tetramethylammonium chloride to the reaction mixture had no effect on these kinetic properties, suggesting that this compound exerts its effect on the reduced form of the enzyme. Treatment of the enzyme with phenylhydrazine, which introduces a phenyl group at the 4a-position of the FMN cofactor, decreased both the rate constant for reduction of the protein and the extent of FMN semiquinone production, while increasing the amount of iron-sulfur center reduction, consistent with the results obtained with the native enzyme. Experiments in which the kinetics of reduction of the enzyme were determined during various stages of partial reduction were also consistent with these results, and further indicated that the FMN semiquinone form of the enzyme is more reactive toward the deazariboflavin reductant than is the oxidized FMN.(ABSTRACT TRUNCATED AT 250 WORDS)