Role of the Main Access Channel of Catalase-Peroxidase in Catalysis
2005; Elsevier BV; Volume: 280; Issue: 51 Linguagem: Inglês
10.1074/jbc.m508009200
ISSN1083-351X
AutoresChrista Jakopitsch, Enrica Droghetti, Florian Schmuckenschlager, Paul G. Furtmüller, Giulietta Smulevich, Christian Obinger,
Tópico(s)Dye analysis and toxicity
ResumoCatalase-peroxidases (KatG) are bifunctional heme peroxidases with an overwhelming catalatic activity. The structures show that the buried heme b is connected to the exterior of the enzyme by a main channel built up by KatG-specific loops named large loop LL1 and LL2, the former containing the highly conserved sequence Met-Gly-Leu-Ile-Tyr-Val-Asn-Pro-Glu-Gly. LL1 residues Ile248, Asn251, Pro252, and Glu253 of KatG from Synechocystis are the focus of this study because of their exposure to the solute matrix of the access channel. In particular, the I248F, N251L, P252A, E253Q, and E253D mutants have been analyzed by UV-visible and resonance Raman spectroscopies in combination with steady-state and presteady-state kinetic analyses. Exchange of these residues did not alter the kinetics of cyanide binding or the overall peroxidase activity. Moreover, the kinetics of compound I formation and reduction by one-electron donors was similar in the variants and the wild-type enzyme. However, the turnover numbers of the catalase activity of I248F, N251L, E253Q, and E253D were only 12.3, 32.6, 25, and 42% of the wild-type activity, respectively. These findings demonstrate that the oxidation reaction of hydrogen peroxide (not its reduction) was affected by these mutations. The altered kinetics allowed us to monitor the spectral features of the dominating redox intermediate of E253Q in the catalase cycle. Resonance Raman data and structural analysis demonstrated the existence of a very rigid and ordered structure built up by the interactions of these residues with distal side and also (via LL1) proximal side amino acids, with the heme itself, and with the solute matrix in the channel. The role of Glu253 and the other investigated channel residues in maintaining an ordered matrix of oriented water dipoles, which guides hydrogen peroxide to its site of oxidation, is discussed. Catalase-peroxidases (KatG) are bifunctional heme peroxidases with an overwhelming catalatic activity. The structures show that the buried heme b is connected to the exterior of the enzyme by a main channel built up by KatG-specific loops named large loop LL1 and LL2, the former containing the highly conserved sequence Met-Gly-Leu-Ile-Tyr-Val-Asn-Pro-Glu-Gly. LL1 residues Ile248, Asn251, Pro252, and Glu253 of KatG from Synechocystis are the focus of this study because of their exposure to the solute matrix of the access channel. In particular, the I248F, N251L, P252A, E253Q, and E253D mutants have been analyzed by UV-visible and resonance Raman spectroscopies in combination with steady-state and presteady-state kinetic analyses. Exchange of these residues did not alter the kinetics of cyanide binding or the overall peroxidase activity. Moreover, the kinetics of compound I formation and reduction by one-electron donors was similar in the variants and the wild-type enzyme. However, the turnover numbers of the catalase activity of I248F, N251L, E253Q, and E253D were only 12.3, 32.6, 25, and 42% of the wild-type activity, respectively. These findings demonstrate that the oxidation reaction of hydrogen peroxide (not its reduction) was affected by these mutations. The altered kinetics allowed us to monitor the spectral features of the dominating redox intermediate of E253Q in the catalase cycle. Resonance Raman data and structural analysis demonstrated the existence of a very rigid and ordered structure built up by the interactions of these residues with distal side and also (via LL1) proximal side amino acids, with the heme itself, and with the solute matrix in the channel. The role of Glu253 and the other investigated channel residues in maintaining an ordered matrix of oriented water dipoles, which guides hydrogen peroxide to its site of oxidation, is discussed. Catalase-peroxidases (KatG) are present in prokaryotes and fungi. These bifunctional enzymes have a predominant catalase activity together with a substantial peroxidatic activity with broad specificity. KatG enzymes are the only heme peroxidases that exhibit a catalatic activity comparable with that of the classical monofunctional heme catalases. On the basis of sequence similarity, KatG enzymes have been recognized as part of the class I superfamily of plant, fungal, and bacterial peroxidases (1Welinder K.G. Curr. Opin. Struct. Biol. 1992; 2: 388-393Crossref Scopus (761) Google Scholar). Moreover, the four available crystal structures of KatG enzymes from Haloarcula marismortui (Protein Data Bank code 1ITK), Burkholderia pseudomallei (code 1MWV), Mycobacterium tuberculosis (code 1SJ2), and Synechococcus PCC 7942 (code 1UB2) (2Yamada Y. Fujiwara T. Sato T. Igarashi N. Tanaka N. Nat. Struct. Biol. 2002; 9: 691-695Crossref PubMed Scopus (138) Google Scholar, 3Carpena X. Loprasert S. Mongkolsuk S. Switala J. Loewen P.C. Fita I. J. Mol. Biol. 2003; 327: 475-489Crossref PubMed Scopus (120) Google Scholar, 4Wada K. Tada T. Nakamura Y. Kinoshita T. Tamoi M. Shigeoka S. Nishimura K. Acta Crystallogr. Sect. D Biol. Crystallogr. 2002; 58: 157-159Crossref PubMed Scopus (40) Google Scholar, 5Bertrand T. Eady N.A.J Jones J.N. Jesmin Nagy J.M. Jamart-Gregoire B. Raven E.L. Brown K.A. J. Biol. Chem. 2004; 279: 38991-38999Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar) revealed that the heme pocket contains catalytic residues virtually identical to those of other peroxidases belonging to class I, such as cytochrome c peroxidase and ascorbate peroxidase. In particular, the distal and proximal heme pockets contains the amino acid triads His-Arg-Trp and His-Trp-Asp, respectively. Moreover, features unique to catalase-peroxidases were found. (a) Compared with cytochrome c peroxidase or ascorbate peroxidase, they are double in length, with the N-terminal half, which includes the heme b-binding site, being more conserved. This has been ascribed to gene duplication (6Welinder K.G. Biochim. Biophys. Acta. 1991; 1080: 215-220Crossref PubMed Scopus (150) Google Scholar). (b) They have an unusual covalent adduct consisting of the distal side tryptophan, tyrosine, and methionine (Trp122, Tyr249, and Met275 in Synechocystis numbering) (2Yamada Y. Fujiwara T. Sato T. Igarashi N. Tanaka N. Nat. Struct. Biol. 2002; 9: 691-695Crossref PubMed Scopus (138) Google Scholar, 3Carpena X. Loprasert S. Mongkolsuk S. Switala J. Loewen P.C. Fita I. J. Mol. Biol. 2003; 327: 475-489Crossref PubMed Scopus (120) Google Scholar, 4Wada K. Tada T. Nakamura Y. Kinoshita T. Tamoi M. Shigeoka S. Nishimura K. Acta Crystallogr. Sect. D Biol. Crystallogr. 2002; 58: 157-159Crossref PubMed Scopus (40) Google Scholar, 5Bertrand T. Eady N.A.J Jones J.N. Jesmin Nagy J.M. Jamart-Gregoire B. Raven E.L. Brown K.A. J. Biol. Chem. 2004; 279: 38991-38999Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). (c) They have one short stretch and three large loop (LL) 4The abbreviations used are: LLlarge loopRRresonance RamanMES2-2-(N-morpholino)ethanesulfonic acidWTwild-type5-cfive-coordinate6-csix-coordinateHShigh-spinLSlow-spinmWmilliwatt.4The abbreviations used are: LLlarge loopRRresonance RamanMES2-2-(N-morpholino)ethanesulfonic acidWTwild-type5-cfive-coordinate6-csix-coordinateHShigh-spinLSlow-spinmWmilliwatt. insertions (LL1-LL3) that are not found in either ascorbate peroxidase or cytochrome c peroxidase (2Yamada Y. Fujiwara T. Sato T. Igarashi N. Tanaka N. Nat. Struct. Biol. 2002; 9: 691-695Crossref PubMed Scopus (138) Google Scholar). (d) They have a more deeply buried active site, and the proposed access route for H2O2 is provided by a channel that is similar but longer and more constricted than that in the other heme peroxidases (3Carpena X. Loprasert S. Mongkolsuk S. Switala J. Loewen P.C. Fita I. J. Mol. Biol. 2003; 327: 475-489Crossref PubMed Scopus (120) Google Scholar). The constriction of the access channel results from the LL1 and LL2 insertions, as the former is positioned at one edge of the heme between helices D and E (see Fig. 1A). It also connects the distal and proximal catalytic domains. The LL1 insertion is of particular interest, as it contains a number of residues neighboring the covalently linked tyrosine (Tyr249 in Synechocystis numbering) that are highly conserved in KatG enzymes, viz. Ile248, Asn251, Pro252, and Glu253 (see Fig. 1B). This latter residue creates an acidic entrance to the channel, which is characterized by a pronounced funnel shape and a continuum of water (2Yamada Y. Fujiwara T. Sato T. Igarashi N. Tanaka N. Nat. Struct. Biol. 2002; 9: 691-695Crossref PubMed Scopus (138) Google Scholar, 3Carpena X. Loprasert S. Mongkolsuk S. Switala J. Loewen P.C. Fita I. J. Mol. Biol. 2003; 327: 475-489Crossref PubMed Scopus (120) Google Scholar, 4Wada K. Tada T. Nakamura Y. Kinoshita T. Tamoi M. Shigeoka S. Nishimura K. Acta Crystallogr. Sect. D Biol. Crystallogr. 2002; 58: 157-159Crossref PubMed Scopus (40) Google Scholar, 5Bertrand T. Eady N.A.J Jones J.N. Jesmin Nagy J.M. Jamart-Gregoire B. Raven E.L. Brown K.A. J. Biol. Chem. 2004; 279: 38991-38999Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). After passing the narrowest part of the channel, corresponding to the two KatG-specific residues Asp152 and Ser335, substrates come immediately into contact the with distal active-site residues Arg119, His123, and Trp122. Recently, it has been demonstrated that Asp152 is hydrogen-bonded to the LL1 residue Ile248 (7Santoni E. Jakopitsch C. Obinger C. Smulevich G. Biochemistry. 2004; 43: 5792-5802Crossref PubMed Scopus (26) Google Scholar). This hydrogen bond is important for the stability of the heme architecture, and its substitution markedly changes the proximal His-Asp hydrogen bond interaction (7Santoni E. Jakopitsch C. Obinger C. Smulevich G. Biochemistry. 2004; 43: 5792-5802Crossref PubMed Scopus (26) Google Scholar), underlining the role of the LL1 insertion in linking the distal and proximal heme sites. Moreover, Asp152 has been shown to be essential for the catalase (but not peroxidase) activity of KatG enzymes (8Jakopitsch C. Auer M. Regelsberger G. Jantschko W. Furtmuller P.G. Ruker F. Obinger C. Biochemistry. 2003; 42: 5292-5300Crossref PubMed Scopus (52) Google Scholar). large loop resonance Raman 2-2-(N-morpholino)ethanesulfonic acid wild-type five-coordinate six-coordinate high-spin low-spin milliwatt. large loop resonance Raman 2-2-(N-morpholino)ethanesulfonic acid wild-type five-coordinate six-coordinate high-spin low-spin milliwatt. To date, only two KatG-specific amino acids of the substrate channel have been studied in detail, viz. Asp152 of Synechocystis (8Jakopitsch C. Auer M. Regelsberger G. Jantschko W. Furtmuller P.G. Ruker F. Obinger C. Biochemistry. 2003; 42: 5292-5300Crossref PubMed Scopus (52) Google Scholar) and Ser315 of M. tuberculosis KatG (9Yu S. Girotto S. Lee C. Magliozzo R.S. J. Biol. Chem. 2003; 278: 14769-14775Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 10Wengenack N.L. Lane B.D. Hill P.J. Uhl J.R. Lukat-Rodgers G.S. Roberts G.D. Cockerill F.R. Brennan P.J. Rodgers K.R. Belisle J.T. Rusnak F. Protein Expression Purif. 2004; 36: 232-243Crossref PubMed Scopus (17) Google Scholar), which constitute the narrowest part of the access channel. Exchange of Asp152 significantly decreases the catalatic activity (8Jakopitsch C. Auer M. Regelsberger G. Jantschko W. Furtmuller P.G. Ruker F. Obinger C. Biochemistry. 2003; 42: 5292-5300Crossref PubMed Scopus (52) Google Scholar), whereas exchange of Ser315 in M. tuberculosis KatG only moderately reduces the rate of oxygen release from H2O2 (9Yu S. Girotto S. Lee C. Magliozzo R.S. J. Biol. Chem. 2003; 278: 14769-14775Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 10Wengenack N.L. Lane B.D. Hill P.J. Uhl J.R. Lukat-Rodgers G.S. Roberts G.D. Cockerill F.R. Brennan P.J. Rodgers K.R. Belisle J.T. Rusnak F. Protein Expression Purif. 2004; 36: 232-243Crossref PubMed Scopus (17) Google Scholar). Recent studies have unequivocally demonstrated that the catalase (but not peroxidase) activity is very susceptible to exchange of amino acids at both the distal and proximal heme sites (8Jakopitsch C. Auer M. Regelsberger G. Jantschko W. Furtmuller P.G. Ruker F. Obinger C. Biochemistry. 2003; 42: 5292-5300Crossref PubMed Scopus (52) Google Scholar, 11Regelsberger G. Jakopitsch C. Furtmüller P.G. Rüker F. Switala J. Loewen P.C. Obinger C. Biochem. Soc. Trans. 2001; 29: 99-105Crossref PubMed Google Scholar, 12Jakopitsch C. Regelsberger G. Furtmüller P.G. Rüker F. Peschek G.A. Obinger C. J. Inorg. Biochem. 2002; 91: 78-86Crossref PubMed Scopus (21) Google Scholar, 13Jakopitsch C. Auer M. Ivancich A. Ruker F. Furtmüller P.G. Obinger C. J. Biol. Chem. 2003; 278: 20185-20191Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 14Jakopitsch C. Ivancich A. Schmuckenschlager F. Wanasinghe A. Pöltl G. Furtmüller P.G. Rüker F. Obinger C. J. Biol. Chem. 2004; 279: 46082-46095Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 15Ivancich A. Jakopitsch C. Auer M. Un S. Obinger C. J. Am. Chem. Soc. 2003; 125: 14093-14102Crossref PubMed Scopus (102) Google Scholar). These findings suggest a correlation between the loss of catalase activity and the disruption of the particularly extensive hydrogen-bonding network typical of KatG (7Santoni E. Jakopitsch C. Obinger C. Smulevich G. Biochemistry. 2004; 43: 5792-5802Crossref PubMed Scopus (26) Google Scholar, 15Ivancich A. Jakopitsch C. Auer M. Un S. Obinger C. J. Am. Chem. Soc. 2003; 125: 14093-14102Crossref PubMed Scopus (102) Google Scholar) as well as the formation and stability of protein radicals (14Jakopitsch C. Ivancich A. Schmuckenschlager F. Wanasinghe A. Pöltl G. Furtmüller P.G. Rüker F. Obinger C. J. Biol. Chem. 2004; 279: 46082-46095Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 15Ivancich A. Jakopitsch C. Auer M. Un S. Obinger C. J. Am. Chem. Soc. 2003; 125: 14093-14102Crossref PubMed Scopus (102) Google Scholar). Therefore, to establish the role of the main access channel in KatG catalysis, we have extended the study to four conserved amino acids belonging to LL1 of Synechocystis KatG, viz. Ile248, Pro252, Asn251, and Glu253, the latter three being on the surface of this channel (Fig. 1). In particular, the I248F, N251L, P252A, E253Q, and E253D variants were investigated by combining pre-steady-state and steady-state kinetics with electronic absorption and resonance Raman (RR) spectroscopies. Reagents—Standard chemicals and biochemicals were obtained from Sigma at the highest grade available. Mutagenesis—A pET-3a expression vector containing the cloned catalase-peroxidase gene from the cyanobacterium Synechocystis PCC 6803 (16Jakopitsch C. Rüker F. Regelsberger G. Dockal M. Peschek G.A. Obinger C. Biol. Chem. 1999; 380: 1087-1096Crossref PubMed Scopus (42) Google Scholar) was used as the template for PCR. Oligonucleotide site-directed mutagenesis was performed using PCR-mediated introduction of silent mutations as described previously (17Regelsberger G. Jakopitsch C. Ruker F. Krois D. Peschek G.A. Obinger C. J. Biol. Chem. 2000; 275: 22854-22861Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Unique restriction sites flanking the region to be mutated were selected. The flanking primers were 5′-AAT GAT CAG GTA CCG GCC AGT AAA TG-3′ (containing a KpnI restriction site) and 5′-AGT GCA GAC TAG TTC GGA AAC G-3′ (containing an SpeI restriction site). The internal 5′-primer was 5′-GAG GGG GTC GAC GGG CAC CCG GAT C-3′ and possessed an SalI restriction site. The following mutant primers with the desired mutation and a silent mutation introducing the SalI restriction site were constructed (with point mutations in italics and restriction sites underlined): 5′-GCC CGT CGA CCC CCT CAG CGT TAA CGT AAA TTA ATC-3′ changed Pro252 to Ala, 5′-GCC CGT CGA CCC CCT CAG GGT TAA CGT AAA ATA ATC CCA TTT G-3′ changed Ile248 to Phe, 5′-GCC CGT CGA CCC CCT CAG GGA GAA CGT AAA TTA ATC CCA TTT G-3′ changed Asn251 to Leu, and 5′-GGT GCC CGT CGA CCC CCT GAG GGT TAA CG-3′ changed Glu253 to Gln. To substitute Glu253 with Asp, the following internal primers containing an AccIII restriction site were used: 5′-TAA TCC GGA CGG GGT GGA TGG-3′ and 5′-CGT CCG (G/A)T TAA CGT AAA TTA ATC CC-3′. The fragment defined by the KpnI and SpeI restriction sites was replaced with the new construct containing the point mutation. All constructs were sequenced to verify DNA changes by thermal cycle sequencing. Electronic Absorption—Absorption spectra were measured with a Cary 5 spectrophotometer at room temperature. Absorption spectra were measured prior to and after Raman experiments. No degradation was observed under the experimental conditions used. Kinetic measurements were performed using a Zeiss Specord S10 diode array spectrophotometer and a Hitachi U-3000 spectrophotometer equipped with a thermostatted cell holder. Circular Dichroism—CD studies were carried out using a Jasco J-600 spectropolarimeter. Far-UV (190-260 nm) experiments were carried out using a protein concentration of 1.5 μm and a cuvette with a 1-mm path length. A good signal-to-noise ratio in the CD spectra was obtained by averaging 12 scans (resolution, 1 nm; bandwidth, 1 nm; response, 2 s; and scan speed, 10 nm/min). The protein concentration was calculated from the known amino acid composition and absorption at 280 nm according to Gill and Hippel (18Gill C. Hippel P.H. Anal. Biochem. 1989; 182: 319-326Crossref PubMed Scopus (5010) Google Scholar). Resonance Raman Spectroscopy—The RR spectra of the ferric and ferrous forms were obtained with excitation from the 406.7-nm line of a Kr+ laser (Coherent Innova 302) and the 441.6-nm line of a helium/cadmium laser (Kimmon IK4121R-G), respectively. To minimize the local heating of the sample, a gentle flux of N2 passed through liquid N2 was used. The sample was cooled to a temperature of ∼15 °C. The backscattered light from a slowly rotating NMR tube was collected and focused into a computer-controlled triple spectrometer (consisting of two Acton Research SpectraPro 2300i models and one SpectraPro 2500i model in the final stage with a grating of 3600 grooves/mm) working in the subtractive mode and equipped with a liquid nitrogen-cooled CCD detector (Roper Scientific, Inc.). The polarized RR spectra were obtained by collecting and focusing the backscattered light from a slowly rotating NMR tube into a computer-controlled double monochromator (Jobin-Yvon HG2S) equipped with a cooled photomultiplier (RCA C31034A). Polarized spectra were obtained by inserting a Polaroid analyzer between the sample and the entrance slit of the monochromator. The depolarization ratios (ρ = I⊥/I∥) of the bands at 314 and 460 cm-1 of CCl4 were measured to check the reliability of the polarization measurements. The values obtained (0.73 and 0.00, respectively) compare well with the theoretical values of 0.75 and 0.00, respectively. The RR spectra were calibrated with indene and CCl4 as standards to an accuracy of 1 cm-1 for intense isolated bands. In the figures, the relative intensities of the high- and low-frequency RR bands of the ferric form were normalized on the ν4 and ν7 bands at 1373 and 679 cm-1, respectively. In the case of the ferrous form, the low-frequency region was normalized on the ν8 band at 347 cm-1. Peak intensities and positions of the ν(C=C) vinyl stretches in the 1600-1650 cm-1 frequency region of the RR spectra of ferric KatG enzymes and mutants were determined by a curve-fitting program (Lab Calc, Galactic Industries Corp.) (TABLE ONE).TABLE ONERR frequencies and mode assignments for WT KatG and mutants 1248F and N251L at pH 7.0WTI248FN251Lcm−16-c LSν10 (depolarized)1639163916395-c HSν10 (depolarized)163116311631ν(C=C) (polarized)162916291626ν(C=C) (polarized)1624162416226-c HSν10 (depolarized)16151615 Open table in a new tab The following buffers were used: 100 mm MES and 100 mm citric acid at pH 6.0, 100 mm KH2PO4 and 100 mm citric acid at pH 6.5, 100 mm sodium phosphate at pH 7.0 and 7.5, and 100 mm Tris-HCl at pH 8.0. The ferrous form of the protein was obtained by adding a small volume (2-5 μl) of fresh sodium dithionite solution (10-20 g/liter) to a deoxygenated protein solution. The sample concentrations were ∼80-150 μm for electronic absorption spectra and ∼30-80 μm for RR experiments. Steady-state Kinetics—Catalase activity was determined polarographically in 50 mm phosphate buffer using a Clark-type electrode (YSI 5331 oxygen probe) inserted into a stirred thermostatted water bath (YSI 5301B). All reactions were performed at 30 °C and started by addition of KatG. One unit of catalase is defined as the amount that decomposes 1 μmol of H2O2/min at pH 7 and 30 °C. To cover the pH range 4.0-9.0, 50 mm citrate/phosphate or 50 mm Tris-HCl buffer was used. Peroxidase activity was monitored spectrophotometrically using 1 mm H2O2 and 5 mm guaiacol (ϵ470 = 26.6 mm-1 cm-1) or 1 mm o-dianisidine (ϵ460 = 11.3 mm-1 cm-1). Alternatively, 1 mm peroxoacetic acid and 5 mm guaiacol were used. One unit of peroxidase is defined as the amount that oxidizes 1 μmol of electron donor/min at pH 7 and 30 °C. Transient-state Kinetics—Transient-state measurements were made using a Model SX.18MV stopped-flow spectrophotometer and a PiStar-180 circular dichroism spectrometer (Applied Photophysics Ltd.) equipped with a 1-cm observation cell thermostatted at 15 °C. Calculation of pseudo first-order rate constants (kobs) from experimental traces at the Soret maximum was performed with a SpectraKinetic work station (Version 4.38) interfaced to the instrument. The substrate concentrations were at least five times that of the enzyme to allow determination of pseudo first-order rate constants. Second-order rate constants were calculated from the slope of the linear plot of the pseudo first-order rate constants versus substrate concentration. To follow spectral transitions, a Model PD.1 photodiode array accessory (Applied Photophysics Ltd.) connected to the stopped-flow machine together with XScan diode array scanning software (Version 1.07) were utilized. The kinetics of oxidation of ferric catalase-peroxidase to compound I by peroxoacetic acid or hydrogen peroxide as well as the kinetics of cyanide binding to ferric KatG were followed in the single mixing mode. Catalase-peroxidase and the peroxides or cyanide were mixed to give a final concentration of 1 μm enzyme and 5-250 μm peroxide or 20-500 μm cyanide. The first data point was recorded 1.5 ms after mixing, and 2000 data points were accumulated. Sequential mixing stopped-flow analysis was used to measure compound I reduction by one-electron donors. In the first step, the enzyme was mixed with peroxoacetic acid, and after a defined delay time, the formed compound I was mixed with the electron donor aniline, ascorbate, or o-dianisidine. All stopped-flow determinations were made in 50 mm phosphate buffer at pH 7.0 and 15 °C, and at least three determinations were performed per substrate concentration. Electronic Absorption and Resonance Raman Spectra of Ferric KatG Variants—The far-UV (190-250 nm) CD spectrum is a sensitive probe of protein secondary structure. The CD spectra of all variants (data not shown) were similar to that of wild-type KatG and showed the typical features of α-helical protein structure with the 222 and 208 nm dichroic bands. Therefore, mutations did not induce changes in the overall secondary structure, and if conformational changes did occur, they must be very localized and minimal. Fig. 2 (A and B) compares the UV-visible and RR spectra of ferric KatG and its variants at pH 7.0. The UV-visible absorption spectra of recombinant wild-type KatG and the four variants exhibited the typical bands of heme b-containing peroxidases in the visible and near-UV region. It was recently shown that, in the UV-visible spectrum of wildtype (WT) KatG, a Soret band at 407 nm, Q-bands at 502 and 542 nm, and a CT1 band (long wavelength (>600 nm) porphyrin-to-metal charge transfer band) at 637 nm are indicative of a five-coordinate (5-c) high-spin (HS) heme coexisting with a six-coordinate (6-c) HS heme and a 6-c low-spin (LS) heme (19Heering H.A. Indiani C. Regelsberger G. Jakopitsch C. Obinger C. Smulevich G. Biochemistry. 2002; 41: 9237-9247Crossref PubMed Scopus (32) Google Scholar). The RR spectrum in the high-frequency region confirmed that the predominant form is a 5-c HS heme (ν3 at 1493 cm-1, ν11 at 1551 cm-1, ν2 at 1571 cm-1, ν37 at 1594 cm-1, and ν10 at 1631 cm-1), coexisting with a 6-c LS heme (ν3 at 1501 cm-1 and ν10 at 1639 cm-1) and a 6-c HS heme (ν3 at 1485 cm-1, ν11 at 1545 cm-1, and ν2 at 1566 cm-1). Furthermore, by means of polarized RR spectra, two distinct vinyl stretches have been identified at 1624 and 1629 cm-1 (19Heering H.A. Indiani C. Regelsberger G. Jakopitsch C. Obinger C. Smulevich G. Biochemistry. 2002; 41: 9237-9247Crossref PubMed Scopus (32) Google Scholar). The different frequencies correspond to different torsion angles (τ; i.e. the relative orientation of the vinyl Ca=Cb π-bond with respect to one of the two pyrrole Cα=Cβ π-bonds) of the 2-vinyl group with respect to the 4-vinyl group (20Marzocchi M. Smulevich G. J. Raman Spectrosc. 2003; 34: 725-736Crossref Scopus (65) Google Scholar) resulting from interaction with the protein matrix. The UV-visible and RR spectra of E253Q and E253D were identical to those of the WT protein (data not shown). The replacement of Glu253 affected neither the spin state of the iron atom nor the vinyl stretches. On the contrary, mutation of Pro252, Asn251, and Ile248 led to the conversion of the 6-c HS form to both 5-c HS and 6-c LS hemes. The replacement had minor effects in the P252A mutant, but changes were progressively more evident in the order P252A, N251L, and I248F. The substitution of Pro252 with Ala only slightly affected the coordination of the iron atom. The maximum of the Soret band did not change with respect to WT KatG. However, the amount of the LS form increased, as indicated by the red shift in the Q-band to 507 nm and by the intensity increase of the band at 542 nm. Moreover, the increase of the shoulder at 380 nm and the red shift of the CT1 band to 643 nm indicate that the mutation increased the 5-c HS form at the expense of the aquo 6-c species, as confirmed by the intensity decrease of the ν3 band at 1485 cm-1 in the corresponding RR spectrum (Fig. 2B). The replacement of Asn251 with Leu gave rise to similar effects. However, the amount of the 6-c LS form was even larger, as shown by the red shift of the Q-band to 512 nm and the intensity increases of the 542 nm band (Fig. 2A) and of the RR bands at 1501 (ν3) and 1639 (ν10) cm-1 (Fig. 2B). Finally, the substitution of Ile248 with Phe led to the disappearance of the 6-c HS heme with the concomitant increase of the 5-c HS heme. In fact, in the UV-visible spectrum, the shoulder at 380 nm markedly increased, and the CT1 band shifted to 643 nm. Accordingly, the RR spectrum showed core size marker bands of 5-c HS and 6-c LS forms only. Because we observed an intensity variation in the 1600-1650 cm-1 region in the N251L and I248F RR spectra compared with the WT protein spectrum (Fig. 2B), we collected polarized RR spectra (Fig. 3) to determine if and how the frequencies of the vinyl stretches differ from that of the WT protein. In fact, in this region, upon Soret excitation, the polarized bands due the ν(C=C) vinyl stretching modes overlap with the ν10 depolarized bands due to the non-totally symmetric modes (B1g). The curve-fitting analysis (supported by measurement of the depolarization ratios, ρ = I⊥/I∥) showed that the two ν(C=C) vinyl stretching modes of the N251L mutant downshifted by 2-3 cm-1 with respect to the WT protein (TABLE ONE). Therefore, it appears that, compared with the WT protein, the N251L mutation changed the vinyl group orientation with respect to the heme plane. The conclusions drawn from the analysis of the UV-visible and RR core size marker bands were confirmed by the RR spectra in the low-frequency region (Fig. 4). The low-frequency region spectra of the E253Q and E253D mutants were identical to that of WT KatG. The spectra were analyzed following the previous assignment proposed for WT KatG (19Heering H.A. Indiani C. Regelsberger G. Jakopitsch C. Obinger C. Smulevich G. Biochemistry. 2002; 41: 9237-9247Crossref PubMed Scopus (32) Google Scholar) and the assignment of myoglobin by Hu et al. (30Hu S. Smith K.M. Spiro T.G. J. Am. Chem. Soc. 1996; 118: 12638-12646Crossref Scopus (463) Google Scholar), which was based on isotopically labeled hemes. The band at 336 cm-1 (assigned to the out-of-plane γ6 mode) is active only in the 5-c HS form and is thus present in the spectra of all mutants. The band at 349 cm-1 (ν8) in the spectrum of WT KatG was shifted to 347 cm-1 in the spectra of P252A, N251L, and I248F. The band at 376 cm-1 (assigned to the porphyrin propionate bending mode δ(CβCcCd)) was relatively less intense in the spectra of P252A and N251L compared with that of WT KatG. The variation of the intensity of this band may be related to conformational changes of the propionate substituents. The band observed at 430 cm-1 in the WT protein (assigned to the δ(CβCaCb) bending modes of the vinyl substituents) downshifted to 421 cm-1 in the N251L spectrum, reflecting the variations of the vinyl stretching modes observed in the high-frequency region. Moreover, in the I248F mutant, a remarkable intensity increase was observed for the band at 754 cm-1 (assigned to the ν15 mode; pyrrole breathing). Electronic Absorption and Resonance Raman Spectra of Ferrous KatG Variants—Further information on structural changes resulting from the mutation could be obtained from the ferrous forms of the proteins. The UV-visible spectrum of the ferrous form of the WT protein at pH 6.5 is typical of a 5-c HS
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