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• W2078996434 abstract "Catalase-peroxidases have a predominant catalase activity but differ from monofunctional catalases in exhibiting a substantial peroxidase activity and in having different residues in the heme cavity. We present a kinetic study of the formation of the key intermediate compound I by probing the role of the conserved distal amino acid triad Arg-Trp-His of a recombinant catalase-peroxidase in its reaction with hydrogen peroxide, peroxoacetic acid, and m-chloroperbenzoic acid. Both the wild-type enzyme and six mutants (R119A, R119N, W122F, W122A, H123Q, H123E) have been investigated by steady-state and stopped-flow spectroscopy. The turnover number of catalase activity of R119A is 14.6%, R119N 0.5%, H123E 0.03%, and H123Q 0.02% of wild-type activity. Interestingly, W122F and W122A completely lost their catalase activity but retained their peroxidase activity. Bimolecular rate constants of compound I formation of the wild-type enzyme and the mutants have been determined. The Trp-122 mutants for the first time made it possible to follow the transition of the ferric enzyme to compound I by hydrogen peroxide spectroscopically underlining the important role of Trp-122 in catalase activity. The results demonstrate that the role of the distal His-Arg pair in catalase-peroxidases is important in the heterolytic cleavage of hydrogen peroxide (i.e. compound I formation), whereas the distal tryptophan is essential for compound I reduction by hydrogen peroxide. Catalase-peroxidases have a predominant catalase activity but differ from monofunctional catalases in exhibiting a substantial peroxidase activity and in having different residues in the heme cavity. We present a kinetic study of the formation of the key intermediate compound I by probing the role of the conserved distal amino acid triad Arg-Trp-His of a recombinant catalase-peroxidase in its reaction with hydrogen peroxide, peroxoacetic acid, and m-chloroperbenzoic acid. Both the wild-type enzyme and six mutants (R119A, R119N, W122F, W122A, H123Q, H123E) have been investigated by steady-state and stopped-flow spectroscopy. The turnover number of catalase activity of R119A is 14.6%, R119N 0.5%, H123E 0.03%, and H123Q 0.02% of wild-type activity. Interestingly, W122F and W122A completely lost their catalase activity but retained their peroxidase activity. Bimolecular rate constants of compound I formation of the wild-type enzyme and the mutants have been determined. The Trp-122 mutants for the first time made it possible to follow the transition of the ferric enzyme to compound I by hydrogen peroxide spectroscopically underlining the important role of Trp-122 in catalase activity. The results demonstrate that the role of the distal His-Arg pair in catalase-peroxidases is important in the heterolytic cleavage of hydrogen peroxide (i.e. compound I formation), whereas the distal tryptophan is essential for compound I reduction by hydrogen peroxide. catalase-peroxidase peroxoacetic acid m-chloroperbenzoic acid cytochrome cperoxidase ascorbate peroxidase polymerase chain reaction Catalase-peroxidases (KatGs)1 cover a growing group of enzyme. They are components of the oxidative defense system of bacterial (1Loewen P.C. Scandalios J.G. Oxidative Stress and the Molecular Biology of Antioxidant Defenses. Cold Spring Harbor Press, Cold Spring Harbor, NY1997: 273-308Google Scholar) and fungal (2Fraaije M.W. Roubroeks H.P. Hagen W.R. Van Berkel W.J.H. Eur. J. Biochem. 1996; 235: 192-198Crossref PubMed Scopus (70) Google Scholar, 3Levy E. Eyal Z. Hochman A. Arch. Biochem. Biophys. 1992; 296: 321-327Crossref PubMed Scopus (52) Google Scholar) cells and function primarily as catalases to remove hydrogen peroxide before it can damage cellular components. They are different from classical monofunctional catalases. On the basis of sequence similarities with fungal cytochromec peroxidase (CCP) and plant ascorbate peroxidases (APX), KatGs have been shown to be members of class I of the superfamily of plant, fungal, and bacterial peroxidases (4Welinder K.G. Curr. Opin. Struct. Biol. 1992; 2: 388-393Crossref Scopus (767) Google Scholar). Despite striking sequence homologies between class I enzymes, there are dramatic differences in the catalytic activity and substrate specificity (5Obinger C. Regelsberger G. Strasser G. Burner U. Peschek G.A. Biochem. Biophys. Res. Commun. 1997; 235: 545-552Crossref PubMed Scopus (59) Google Scholar, 6Regelsberger G. Obinger C. Zoder R. Altmann F. Peschek G.A. FEMS Microbiol. Lett. 1999; 170: 1-12Crossref PubMed Google Scholar, 7Regelsberger G. Jakopitsch C. Engleder M. Rüker F. Peschek G.A. Obinger C. Biochemistry. 1999; 38: 10480-10488Crossref PubMed Scopus (45) Google Scholar). The most interesting feature of bifunctional catalase-peroxidases is the overwhelming catalase activity with overall rate constants comparable with those of monofunctional catalases. In both CCP and APX, the catalase activity can be neglected. KatGs also function as broad specificity peroxidases, oxidizing various electron donors, including NAD(P)H (8Marcinkeviciene J.A. Magliozzo R.S. Blanchard J.S. J. Biol. Chem. 1995; 270: 22290-22295Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 9Nagy J.M. Cass A.E.G. Brown K.A. J. Biol. Chem. 1997; 272: 31265-31271Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 10Johnsson K. Froland W.A. Schultz P.G. J. Biol. Chem. 1997; 272: 2834-2840Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar), phenols, and anilines (7Regelsberger G. Jakopitsch C. Engleder M. Rüker F. Peschek G.A. Obinger C. Biochemistry. 1999; 38: 10480-10488Crossref PubMed Scopus (45) Google Scholar), whereas typical substrates for APX and CCP (ascorbate and cytochrome c, respectively) are extremely poor electron donors for KatGs (5Obinger C. Regelsberger G. Strasser G. Burner U. Peschek G.A. Biochem. Biophys. Res. Commun. 1997; 235: 545-552Crossref PubMed Scopus (59) Google Scholar, 6Regelsberger G. Obinger C. Zoder R. Altmann F. Peschek G.A. FEMS Microbiol. Lett. 1999; 170: 1-12Crossref PubMed Google Scholar, 7Regelsberger G. Jakopitsch C. Engleder M. Rüker F. Peschek G.A. Obinger C. Biochemistry. 1999; 38: 10480-10488Crossref PubMed Scopus (45) Google Scholar, 11Obinger C. Regelsberger G. Furtmüller P.G. Jakopitsch C. Rüker F. Pircher A. Peschek G.A. Free Radical Res. 1999; 31: S243-S249Crossref PubMed Scopus (12) Google Scholar). From both CCP and APX, the three-dimensional structures are known (12Finzel B.C. Poulos T.L. Kraut J. J. Biol. Chem. 1984; 259: 13027-13036Abstract Full Text PDF PubMed Google Scholar,13Patterson W.R. Poulos T.L. Biochemistry. 1995; 34: 4331-4341Crossref PubMed Scopus (252) Google Scholar) and exhibit highly conserved amino acid residues at the active site. They indicate the presence of a proximal histidine as well as the triad Arg-Trp-His at the distal side. Both physical characterization as well as sequence analysis suggest the presence of these residues also in catalase-peroxidases (6Regelsberger G. Obinger C. Zoder R. Altmann F. Peschek G.A. FEMS Microbiol. Lett. 1999; 170: 1-12Crossref PubMed Google Scholar, 14Jakopitsch C. Rüker F. Regelsberger G. Dockal M. Peschek G.A. Obinger C. Biol. Chem. 1999; 380: 1987Crossref Scopus (42) Google Scholar). At the moment there is no structural basis to understand the catalytic features of catalase-peroxidases. Thus, in developing ideas how protein structure modifies heme reactivity, class I peroxidases are an extremely exciting field of research, with KatGs being the least understood type of peroxidase. The initial step in the catalytic mechanism of a peroxidase or catalase is heterolysis of the oxygen-oxygen bond of hydrogen peroxide. This reaction causes the release of one water molecule (15Schonbaum G.R. Lo S. J. Biol. Chem. 1972; 247: 3353-3360Abstract Full Text PDF PubMed Google Scholar) and coordination of the second oxygen atom to the iron center (16Hager L.P. Doubek D.L. Silverstein R.M. Harges J.H. Martin J.C. J. Am. Chem. Soc. 1972; 94: 4364-4366Crossref PubMed Scopus (90) Google Scholar). Two electrons are transferred from the enzyme to the coordinated oxygen atom, one from the iron and one from a second donor. In pea cytosolic APX, the porphyrin serves as the second donor (17Patterson W.R. Poulos T.L. Goodin D.B. Biochemistry. 1995; 34: 4342-4345Crossref PubMed Scopus (162) Google Scholar), whereas, in CCP, the second donor is Trp-191 (18Erman J.E. Vitello L.B. Mauro J.M. Kraut J. Biochemistry. 1989; 28: 7992-7995Crossref PubMed Scopus (178) Google Scholar, 19Scholes C.P. Liu Y. Fishel L.A. Farnum M.A. Mauro J.M. Kraut J. Isr. J. Chem. 1989; 29: 85-92Crossref Scopus (69) Google Scholar, 20Sivaraja M. Goodin D.B. Mauk A.G. Smith M. Hoffman B.A. Science. 1989; 245: 738-740Crossref PubMed Scopus (472) Google Scholar). Recently, we have shown that the spectrum of KatG compound I is reminiscent to APX compound I (7Regelsberger G. Jakopitsch C. Engleder M. Rüker F. Peschek G.A. Obinger C. Biochemistry. 1999; 38: 10480-10488Crossref PubMed Scopus (45) Google Scholar) with the important difference (and this is caused by the overwhelming catalase activity) that compound I formation of KatG could never be monitored with hydrogen peroxide. Instead of H2O2, it was necessary to use peroxoacetic acid (7Regelsberger G. Jakopitsch C. Engleder M. Rüker F. Peschek G.A. Obinger C. Biochemistry. 1999; 38: 10480-10488Crossref PubMed Scopus (45) Google Scholar). The triad Arg-Trp-His is located near the peroxide binding site. Histidine is suggested to function as a general acid/base catalyst that assists in deprotonating the hydroperoxide and protonating the departing water (18Erman J.E. Vitello L.B. Mauro J.M. Kraut J. Biochemistry. 1989; 28: 7992-7995Crossref PubMed Scopus (178) Google Scholar), whereas distal arginine is proposed to stabilize the transition state for compound I formation by interacting with the developing negative charge on the oxygen atom being reduced during the heterolytic cleavage of the peroxide bond and to stabilize the resulting oxy-ferryl center of compound I (21Vitello L.B. Erman J.E. Miller M.A. Wang J. Kraut J. Biochemistry. 1993; 32: 9807-9818Crossref PubMed Scopus (142) Google Scholar). The role of distal tryptophan is least clear. In CCP the mutant W51F has been shown to be hyperactive (22Roe J.A. Goodin D.B. J. Biol. Chem. 1993; 268: 20037-20045Abstract Full Text PDF PubMed Google Scholar). In the present work we undertook a detailed analysis of compound I formation of recombinant catalase-peroxidase from the cyanobacteriumSynechocystis PCC 6803. Our goal was to elucidate the role of the distal triad Arg-Trp-His in peroxide binding and cleavage of KatG and to ascertain similarities or differences to both APX and CCP. The wild-type enzyme and six mutants (R119A, R119N, W122F, W122A, H123Q, H123E) have been investigated by both steady-state and presteady-state kinetic analysis. The most exciting finding of this work is the role of tryptophan in catalase activity of KatG. The Trp-122 mutants lost their catalase activity completely but retained their peroxidase activity. The consequence was that, for the first time, in these two proteins compound I formation could be followed spectroscopically also with hydrogen peroxide. Mutation of distal arginine and histidine gave 10- to 102-fold and 104-fold decreases in catalase activity, indicating a role in O-O heterolysis of hydrogen peroxide. In contrast, their role in compound I formation with peroxy acids was much less pronounced. Materials were from the following sources: phenylmethylsulfonyl fluoride, hemin, pepstatin A, leupeptin, chloramphenicol, and ampicillin from Sigma; GFX PCR DNA and gel band purification kit, chelating Sepharose Fast Flow, and HiPrep® Sephacryl S-300 HR 16/60 gels from Amersham Pharmacia Biotech; the Centriprep-30 concentrators from Amicon; KpnI from Stratagene;BamHI and PstI restriction enzymes, T4 DNA ligase, and alkaline phosphatase from Roche Molecular Biochemicals; and Pfu polymerases from Promega and Stratagene, respectively. All other chemicals were of the highest purity grade available. Oligonucleotide site-directed mutagenesis was performed using PCR-mediated introduction of silent mutations as described (23Kohli R.M. BioTechniques. 1998; 25: 184-188Crossref PubMed Scopus (8) Google Scholar). A pET-3a expression vector that contained the cloned catalase-peroxidase gene from the cyanobacteriumSynechocystis PCC 6803 (7Regelsberger G. Jakopitsch C. Engleder M. Rüker F. Peschek G.A. Obinger C. Biochemistry. 1999; 38: 10480-10488Crossref PubMed Scopus (45) Google Scholar, 14Jakopitsch C. Rüker F. Regelsberger G. Dockal M. Peschek G.A. Obinger C. Biol. Chem. 1999; 380: 1987Crossref Scopus (42) Google Scholar) was used as the template for PCR. At first unique restriction sites were selected flanking the region to be mutated. The flanking primers were 5′-AAT GAT CAG GTA CCG GCC AGT AAA TG-3′ containing a KpnI restriction site and 5′-TGC ATA AAG GAT CCG GGT GC-3′ containing a BamHI restriction site. The internal 5′-primer was 5′-GCA CGC TGC AGG CAC TTA TCG CAT TG-3′ and possessed a PstI restriction site. The following mutant primers with the desired mutation and a silent mutation introducing a restriction site were constructed (point mutations italicized and restriction sites underlined): 5′-AGT GCC TGCAGC GTG CCA GGC CAT AGC AAT CAT TAA TC-3′ changed Arg-119 to Ala, 5′-AGT GCC TGCAGC GTG CCA GGC CAT GTT GAT CAG CCC ACC ATA ATG ACC-3′ changed Arg-119 to Asn, 5′-AGT GCC TGCAGC GTG GAA GGC CAT AC-3′ changed Trp-122 to Phe, 5′-AGT GCC TGCAGC GTG CGC GGC CAT AC-3′ changed Trp-122 to Ala, 5′-AGT GCC TGCAGC CTCCCA GGC CAT AC-3′ changed His-123 to Glu, and 5′-AGT GCC TGCAGC CTG CCA GGC CAT AC-3′ changed His-123 to Gln. The fragment defined by the KpnI and BamHI restriction sites was replaced by the new construct containing the point mutation. All constructs were sequenced to verify DNA changes using thermal cycle sequencing. The mutant recombinant catalase-peroxidases were expressed in Escherichia coliBL21(DE3)pLysS and purified as described previously by Regelsbergeret al. (7Regelsberger G. Jakopitsch C. Engleder M. Rüker F. Peschek G.A. Obinger C. Biochemistry. 1999; 38: 10480-10488Crossref PubMed Scopus (45) Google Scholar) and Jakopitsch et al. (14Jakopitsch C. Rüker F. Regelsberger G. Dockal M. Peschek G.A. Obinger C. Biol. Chem. 1999; 380: 1987Crossref Scopus (42) Google Scholar) using the same conditions as for the wild-type enzyme. Catalase activity was determined in 50 mm citrate/phosphate or phosphate buffer adjusted to the corresponding pH in the range 3–9, both polarographically using a Clark-type electrode (YSI 5331 Oxygen Probe) inserted into a stirred water bath (YSI 5301B) at 25 °C and spectrophotometrically (24Beers R.F. Sizer I.W. J. Biol. Chem. 1952; 195: 133-140Abstract Full Text PDF PubMed Google Scholar) using an extinction coefficient for hydrogen peroxide at 240 nm of 39.4m−1 cm−1(25Nelson D.P. Kiesow L.A. Anal. Biochem. 1972; 49: 474-478Crossref PubMed Scopus (820) Google Scholar). One unit of catalase is defined as the amount that decomposes 1 μmol of H2O2/min at pH 7 and 25 °C. Peroxidase activity was monitored spectrophotometrically using 1 mmH2O2 and either 1 mm o-dianisidine (ε460 = 11.3 mm−1cm−1) or 20 mm pyrogallol (ε430 = 2.47 mm−1cm−1) following the oxidation rate in 67 mm phosphate buffer, pH. 7.0. One unit of peroxidase is defined as the amount that decomposes 1 μmol of electron donor/min at pH 7 and 25 °C. CD spectra were recorded on a Jobin Yvon CD6 dichrograph equipped with a thermostated cell holder, and data were recorded on-line using a personal computer. Spectra are averages of four accumulated scans with subtraction of the base line. The quartz cuvette used had a path length of 1 mm. All samples were measured at 20 °C in 50 mm phosphate buffer, pH 7.0. Protein concentrations were 1 μm in all experiments. Transient-state measurements were performed using an Applied Photophysics instrument (Model SX-18MV) equipped with a 1-cm observation cell thermostated at 15 °C. Rate constants from experimental traces were calculated by the SpectraKinetic workstation version 4.38 interfaced to the apparatus. Conventional stopped-flow analysis was used to investigate the oxidation of ferric enzyme by peroxides and formation of compound I. At least three determinations of the pseudo-first-order rate constants,k obs, were measured for each substrate concentration, and the mean value was used to calculate the second-order rate constants. To allow calculation of pseudo-first-order rates, the concentrations of substrates were at least 5 times that of the enzyme. The slope of the linear plot of the pseudo-first-order rate constant versus substrate concentration was used to obtain the second-order rate constant for the reaction. All stopped-flow experiments were performed in 50 mm phosphate buffer, pH 7.0, with the exception of pH dependence studies on reaction rates for which the experimental traces were recorded in 50 mmcitrate/phosphate buffers at different pH values between 4.0 and 8.0. Reactions were also studied with a diode-array detector (model PD.1 from Applied Photophysics) as part of the stopped-flow machine, as well as in conventional steady-state spectroscopy using a Zeiss Specord S-10 diode-array spectrophotometer. Recently, we reported a high level expression in E. coli of a recombinant form of a homodimeric catalase-peroxidase from the cyanobacterium Synechocystis PCC 6803. Both physical and preliminary kinetic characterization revealed its identity with the wild-type protein (7Regelsberger G. Jakopitsch C. Engleder M. Rüker F. Peschek G.A. Obinger C. Biochemistry. 1999; 38: 10480-10488Crossref PubMed Scopus (45) Google Scholar, 14Jakopitsch C. Rüker F. Regelsberger G. Dockal M. Peschek G.A. Obinger C. Biol. Chem. 1999; 380: 1987Crossref Scopus (42) Google Scholar). Fig. 1depicts CD spectra of ferric native recombinant KatG and of all six mutants investigated in this study. The far-UV CD spectra give a measure of the protein secondary structure. The signal observed for catalase-peroxidase was characteristic of a protein composed primarily of α helices. Very little difference was observed between the CD spectra of wild-type and the six mutant proteins, indicating that there was no large scale conformational change in the structure. If conformational changes did occur, they must have been very localized and minimal and thus went undetected by CD. Since the α-helical content seemed to be insensitive to the amino acid substitution at the distal Arg, Trp, or His, we assumed that the mutants were folded properly, as is the wild-type enzyme. The absorption spectrum of the recombinant enzyme in the resting state exhibited the typical bands of a heme b-containing ferric peroxidase in the visible and near ultraviolet region. Fig.2 A shows the Soret peak to be at 406 nm and the two charge-transfer bands at 500 (CT2) and 631 nm (CT1). Both the Soret and the CT1 band suggested the presence of a five-coordinate high spin heme coexisting with a six-coordinate high spin heme. The A 406/A 280ratio (i.e. Reinheitszahl) varied between 0.63 and 0.65 and was similar to that of wild-type KatG (6Regelsberger G. Obinger C. Zoder R. Altmann F. Peschek G.A. FEMS Microbiol. Lett. 1999; 170: 1-12Crossref PubMed Google Scholar). The spectral parameters of the mutants are summarized in Table I. Mutations of Arg, Trp, and His caused shifts of 1–4 nm at the Soret region and more pronounced shifts at both CT1 and CT2. Mutation of distal histidine led to a marked red-shift observed for the CT1 band, which (together with an increase of the shoulder at 380 nm) indicated an increase of the five-coordinate high spin heme at the expense of six-coordinate high spin heme. A similar but less pronounced transition was observed for R119A and R119N.Table IAbsorption maxima (ASoret, Aβ, Aα), Reinheitszahl (ASoret/A280 nm), apparent Km, kcat, and kcat/Km values (with H2O2) for wild-type (WT) and mutants (R119A, R119N, W122F, W122A, H123Q, H123E) of recombinant catalase-peroxidase from Synechocystis PCC 6803WTR119AR119NW122FW122AH123QH123EA Soret(nm)406410408409409404409Aβ(nm)500527505511521508515Aα(nm)631639637634628645640RZ (ASoret/A280 nm)0.640.680.630.600.640.460.48Km(mm)4.97.160NDND15.56.2k cat(s−1)350051317.3NDND0.831.17k cat/Km (s−1m−1)7.1 × 1057.2 × 1042.9 × 102NDND5.4 × 1011.9 × 102ND, not detectable; RZ, Reinheitzahl. Open table in a new tab ND, not detectable; RZ, Reinheitzahl. With the exception of His-123 mutants, both the protein yield (40–60 mg of recombinant KatG from 1 liter of E. coli culture) and the Reinheitszahl of the mutants were comparable with those of the wild-type enzyme. The lower heme content of H123Q and H123E could reflect that heme binding in these variants was disrupted. Recombinant KatG exhibited an overwhelming catalase activity. The polarographically measured specific catalase activity in the presence of 1 mm hydrogen peroxide was 553–624 units/mg of protein. The presence of classical peroxidase substrates influenced the oxygen release from hydrogen peroxide only to a small extent. With 1 mmH2O2 and either 5 mm o-dianisidine or 20 mm pyrogallol, the peroxidase activity was determined to be 2.0–2.8 units/mg and 5.4–6.1 units/mg, respectively. Although it is incorrect to treat catalase kinetics in a Michaelis-Menten way (and therefore the definition of Km and k cat should be avoided), we have calculated apparent Km and k cat values as has been done in the literature in recent years (5Obinger C. Regelsberger G. Strasser G. Burner U. Peschek G.A. Biochem. Biophys. Res. Commun. 1997; 235: 545-552Crossref PubMed Scopus (59) Google Scholar, 6Regelsberger G. Obinger C. Zoder R. Altmann F. Peschek G.A. FEMS Microbiol. Lett. 1999; 170: 1-12Crossref PubMed Google Scholar, 7Regelsberger G. Jakopitsch C. Engleder M. Rüker F. Peschek G.A. Obinger C. Biochemistry. 1999; 38: 10480-10488Crossref PubMed Scopus (45) Google Scholar, 11Obinger C. Regelsberger G. Furtmüller P.G. Jakopitsch C. Rüker F. Pircher A. Peschek G.A. Free Radical Res. 1999; 31: S243-S249Crossref PubMed Scopus (12) Google Scholar, 14Jakopitsch C. Rüker F. Regelsberger G. Dockal M. Peschek G.A. Obinger C. Biol. Chem. 1999; 380: 1987Crossref Scopus (42) Google Scholar). With wild-type KatG (1 nm heme), there was a linear correlation in a Lineweaver-Burk plot between 50 μm and 10 mmhydrogen peroxide allowing determination of both apparentKm and v max. At H2O2 concentrations higher than 10 mm, a progressive inhibition of enzyme activity was seen. For the wild-type protein, the corresponding apparentk cat value with H2O2 as the sole substrate was determined to be 3500 s−1 and the apparent Km was 4.9 mm. The k cat/Km ratio was 7.1 × 105m−1s−1. Table I contains some of the kinetic constants for KatG and the investigated mutants. It shows how the mutations decrease the affinity for hydrogen peroxide as well as the turnover rates and thus confirms that the changed residues are involved in catalase activity of KatG. Interestingly, both Trp-122 mutants lost their catalase activity completely. Mutation of Arg-119 and His-123 gave partially active forms. Mutation of histidine decreased the k catvalue by a factor of 3000–4000, whereas the k cat value of catalase activity was 14.6% for R119A and 0.5% for R119N compared with the wild-type protein. The catalase activity of the variants was measured polarographically and was proportional to hydrogen peroxide concentrations in the range 2–20 mm. Corresponding to the effect of mutation on catalytic activity, the concentration of the mutants in the assays had to be 30–300 nm (per heme). At higher peroxide levels (>20 mm), the reaction rate again lost proportionality to H2O2 concentration. Generally, inactivation of mutants at high H2O2 concentration was more dramatic than inactivation of the wild-type protein. Both the wild-type protein and the Arg-119 and His-123 variants exhibited only a small decrease in absorbance (hypochromicity) of ∼0–2% at the Soret region. Assuming that compound I would give a Soret absorbance ∼60% that of the ferric enzyme (7Regelsberger G. Jakopitsch C. Engleder M. Rüker F. Peschek G.A. Obinger C. Biochemistry. 1999; 38: 10480-10488Crossref PubMed Scopus (45) Google Scholar), these hypochromicities fitted a steady-state compound I concentration between 0% and 4%. This calculation was based on a hypothetical catalase reaction mechanism involving Reactions 1 (a and b) and 2 (a and b) in Fig. 6. During catalase activity the ratio of free enzyme to compound I is a constant determined by the rate constants of compound I formation,k 1(app), and compound I reduction,k 2(app). From [KatG]/[compound I] =k 2(app)/k 1(app) follows that during catalase activity k 2(app) ≫k 1(app). Our experiments demonstrated that mutation of distal Arg and His did not alter this ratio. On the contrary, mutation of distal Trp dramatically shifted this ratio. Addition of hydrogen peroxide to both catalatically inactive mutants W122F and W122A led to compound I accumulation. Apparently, in the Trp-122 variants, Reaction 2 (a and b) was blocked. The peroxidatic activity of the Arg-119 and His-123 mutants was reduced proportionally to the catalase activity. In contrast, in both Trp-122 variants, the peroxidatic to catalatic ratio dramatically increased. Although having lost the catalase activity, the specific peroxidase activity of W122F was still 0.5 units/mg (1 mmH2O2, 5 mm o-dianisidine) and 0.83 units/mg (1 mmH2O2, 20 mm pyrogallol). The initial event in the catalytic mechanism of a peroxidase or catalase is a two-electron oxidation of the enzyme by hydrogen peroxide to an intermediate called compound I (Reaction 1, a and b). Compound I of peroxidases normally exhibits the same Soret band maximum as the corresponding native peroxidase but a hypochromicity of about 40–50%. We have recently demonstrated that bifunctional catalase-peroxidases exhibit similar spectral changes upon addition of peroxides (7Regelsberger G. Jakopitsch C. Engleder M. Rüker F. Peschek G.A. Obinger C. Biochemistry. 1999; 38: 10480-10488Crossref PubMed Scopus (45) Google Scholar). In contrast to its homologous members of class I peroxidases, APX and CCP, monitoring of compound I in KatGs is impossible because of its high catalase activity (k 2(app) ≫ k 1(app)). Consequently, peroxy acids had to be used to form a stable KatG compound I. From the single-mixing stopped-flow experiments on rates of compound I formation using excess peroxy acids, the following results were obtained. Reaction of wild-type KatG with both peroxoacetic acid (PA) and m-chloroperbenzoic acid (CPB) exhibited single exponential curves, indicating pseudo-first-order kinetics (Fig.2 B, inset). Plots of the first-order rate constants, k obs, versus peroxide concentration were linear with very small intercepts (<0.5 s−1) (Fig. 2 B). These small intercepts fitted well with the observation that wild-type compound I was stable for more than 30 s, even in the presence of excess PA. With PA and CPB the rate constants for compound I formation,k 1(app), were (3.9 ± 0.4) × 104m−1s−1 and (5.3 ± 0.2) × 104m−1s−1, respectively (pH 7.0, 15 °C). Fig.2 A shows the spectrum of KatG compound I produced by mixing 20 μm recombinant enzyme with 200 μmperoxoacetic acid. Its spectrum was distinguished from the resting state by a 40% hypochromicity at 406 nm and two distinct peaks at 604 and 643 nm. Isosbestic points between compound I and the resting enzyme were determined to be at 357, 430, and 516 nm. When PA or CPB was added to R119A and R119N, a similar exponential decrease of absorbance at the Soret maximum was observed. However, the exponential phase (representing ∼90% of total hypochromicity) was followed by a relatively slow decrease in absorbance of this mutant, and this decrease could be attributed to a decay of compound I. The pseudo-first-order rate constants, k obs, were calculated from the first exponential phase. The k 1(app) values determined from plots of these first-order rate constants, k obs,versus peroxide concentration are summarized in TableII. There was a distinct influence of mutations of Arg-119 on the reaction with PA, whereas the reactivity toward CPB seemed to be unaffected. With PA the intercepts were relatively small (0.2 s−1 with R119A and 1.1 s−1 with R119N), whereas with CPB the corresponding values were 3.6 and 4.5 s−1, respectively. This fitted well with the observation that CPB always caused a faster decrease in absorbance during the second (linear) phase than PA.Table IIApparent bimolecular rate constants, kapp, of the reaction of ferric catalase-peroxidase with various hydroperoxideskappWTR119AR119NW122FW122AH123QH123Em−1 s−1Hydrogen peroxideNDNDND8.2 × 1048.8 × 104NDNDPeroxoacetic acid3.9 × 1041.9 × 1034.5 × 1021.8 × 1052.1 × 105ND1.1 × 105m-Chloroperbenzoic acid5.3 × 1045.0 × 1041.4 × 1047.3 × 1049.0 × 1067.3 × 1047.5 × 106Wild-type (WT) and six mutants (R119A, R119N, W122F, W122A, H123Q, H123E) of recombinant enzyme from Synechocystis PCC 6803 were measured at 15 °C in 50 mm phosphate buffer, pH 7.0. ND, not detectable. Open table in a new tab Wild-type (WT) and six mutants (R119A, R119N, W122F, W122A, H123Q, H123E) of recombinant enzyme from Synechocystis PCC 6803 were measured at 15 °C in 50 mm phosphate buffer, pH 7.0. ND, not detectable. Mutation of His-123 and Trp-122 resulted also in a biphasic reactivity toward both PA and CPB and calculation of k 1(app) was performed by fitting the first exponential phase of reaction. Replacement of the distal His with Gln led to a complete depression of reaction with PA, whereas reaction with CPB gave a k 1(app) comparable with the wild-" @default.
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