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• W2044264629 abstract "Catalase-peroxidase (KatG) is essential in Mycobacterium tuberculosis for oxidative stress management and activation of the antitubercular pro-drug isoniazid. The role of a unique distal side adduct found in KatG enzymes, involving linked side chains of residues Met255, Tyr229, and Trp107 (MYW), in the unusual catalase activity of KatG is addressed here and in our companion paper (Suarez, J., Ranguelova, K., Jarzecki, A. A., Manzerova, J., Krymov, V., Zhao, X., Yu, S., Metlitsky, L., Gerfen, G. J., and Magliozzo, R. S. (2009) J. Biol. Chem. 284, in press). The KatG[W107F] mutant exhibited severely reduced catalase activity yet normal peroxidase activity, and as isolated contains more abundant 6-coordinate heme in high spin and low spin forms compared with the wild-type enzyme. Most interestingly, oxyferrous heme is also found in the purified enzyme. Oxyferrous KatG[W107F] was prepared by photolysis in air of the carbonyl enzyme or was generated using hydrogen peroxide decayed with a t½ of 2 days compared with 6 min for wild-type protein. The stability of oxyenyzme was modestly enhanced in KatG[Y229F] but was not affected in KatG[M255A]. Optical stopped-flow experiments showed rapid formation of Compound I in KatG[W107F] and facile formation of oxyferrous heme in the presence of micromolar hydrogen peroxide. An analysis of the relationships between catalase activity, stability of oxyferrous enzyme, and a proposed MYW adduct radical is presented. The loss of catalase function is assigned to the loss of the MYW adduct radical and structural changes that lead to greatly enhanced stability of oxyenzyme, an intermediate of the catalase cycle of native enzyme. Catalase-peroxidase (KatG) is essential in Mycobacterium tuberculosis for oxidative stress management and activation of the antitubercular pro-drug isoniazid. The role of a unique distal side adduct found in KatG enzymes, involving linked side chains of residues Met255, Tyr229, and Trp107 (MYW), in the unusual catalase activity of KatG is addressed here and in our companion paper (Suarez, J., Ranguelova, K., Jarzecki, A. A., Manzerova, J., Krymov, V., Zhao, X., Yu, S., Metlitsky, L., Gerfen, G. J., and Magliozzo, R. S. (2009) J. Biol. Chem. 284, in press). The KatG[W107F] mutant exhibited severely reduced catalase activity yet normal peroxidase activity, and as isolated contains more abundant 6-coordinate heme in high spin and low spin forms compared with the wild-type enzyme. Most interestingly, oxyferrous heme is also found in the purified enzyme. Oxyferrous KatG[W107F] was prepared by photolysis in air of the carbonyl enzyme or was generated using hydrogen peroxide decayed with a t½ of 2 days compared with 6 min for wild-type protein. The stability of oxyenyzme was modestly enhanced in KatG[Y229F] but was not affected in KatG[M255A]. Optical stopped-flow experiments showed rapid formation of Compound I in KatG[W107F] and facile formation of oxyferrous heme in the presence of micromolar hydrogen peroxide. An analysis of the relationships between catalase activity, stability of oxyferrous enzyme, and a proposed MYW adduct radical is presented. The loss of catalase function is assigned to the loss of the MYW adduct radical and structural changes that lead to greatly enhanced stability of oxyenzyme, an intermediate of the catalase cycle of native enzyme. Catalase-peroxidase (KatG) 4The abbreviations used are: KatG, catalase-peroxidase; KatG[W107F], W107F mutant of KatG; KatG[Y229F], Y229F mutant of KatG; KatG[M255A], M255A mutant of KatG; INH, isoniazid (isonicotinic acid hydrazide); Cmpd, compound; PAA, peroxyacetic acid; 5-c, five coordinate; 6-c, six coordinate; LS, low spin; MYW, Met-Tyr-Trp; WT, wild type. is a dual function heme enzyme responsible in Mycobacterium tuberculosis for activation of the antitubercular pro-drug INH (1Zhang Y. Res. Microbiol. 1993; 144: 143-149Crossref PubMed Scopus (17) Google Scholar, 2Zhang Y. Heym B. Allen B. Young D. Cole S. Nature. 1992; 358: 591-593Crossref PubMed Scopus (1097) Google Scholar, 3Rozwarski D.A. Grant G.A. Barton D.H.R. Jacobs Jr., W.R. Sacchettini J.C. Science. 1998; 279: 98-102Crossref PubMed Scopus (618) Google Scholar, 4Johnsson K. Schultz P.G. J. Am. Chem. Soc. 1994; 116: 7425-7426Crossref Scopus (271) Google Scholar), and it is the sole catalase in this pathogen. KatG enzymes are homologous to class I peroxidases such as cytochrome c peroxidase and ascorbate peroxidase (5Dunford H.B. Heme Peroxidases. Wiley-VCH, New York1999: 276-278Google Scholar), although certain features clearly distinguish the KatG enzymes. The most unusual of these is a three-amino acid distal side adduct (MYW) in which the side chains of Met255, Tyr229, and Trp107 are covalently linked. This adduct (Fig. 1) is found in all KatG enzymes (6Yamada Y. Fujiwara T. Sato T. Igarashi N. Tanaka N. Nat. Struct. Biol. 2002; 9: 691-695Crossref PubMed Scopus (140) Google Scholar, 7Bertrand 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 (160) Google Scholar, 8Carpena X. Loprasert S. Mongkolsuk S. Switala J. Loewen P.C. Fita I. J. Mol. Biol. 2003; 327: 475-489Crossref PubMed Scopus (123) Google Scholar) and is a key structural element required for catalase activity. The details of post-translational modification and the exact role of the MYW adduct in the catalase reaction mechanism remain under active investigation. Our companion paper (52Suarez J. Ranguelova K. Jarzecki A.A. Manzerova J. Krymov V. Zhao X. Yu S. Metlitsky L. Gerfen G.J. Magliozzo R.S. J. Biol. Chem. 2009; 284: 7017-7029Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar) provides kinetic and spectroscopic evidence that the adduct harbors a catalytically competent radical during catalase turnover. Disruption of the adduct by mutation of either Trp107 or Tyr229, or changing its structure by mutation of Met255, nearly eliminates catalase activity in M. tuberculosis KatG (9Ghiladi R.A. Knudsen G.M. Medzihradszky K.F. Ortiz de Montellano P.R. J. Biol. Chem. 2005; 280: 22651-22663Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 10Ghiladi R.A. Medzihradszky K.F. Ortiz de Montellano P.R. Biochemistry. 2005; 44: 15093-15105Crossref PubMed Scopus (42) Google Scholar) and in each mutant abolishes the unique radical described in the companion paper (52Suarez J. Ranguelova K. Jarzecki A.A. Manzerova J. Krymov V. Zhao X. Yu S. Metlitsky L. Gerfen G.J. Magliozzo R.S. J. Biol. Chem. 2009; 284: 7017-7029Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). These mutations are not damaging to the peroxidase mechanism with artificial substrates in reactions where turnover is initiated with alkyl peroxides (9Ghiladi R.A. Knudsen G.M. Medzihradszky K.F. Ortiz de Montellano P.R. J. Biol. Chem. 2005; 280: 22651-22663Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 10Ghiladi R.A. Medzihradszky K.F. Ortiz de Montellano P.R. Biochemistry. 2005; 44: 15093-15105Crossref PubMed Scopus (42) Google Scholar, 11Jakopitsch C. Auer M. Ivancich A. Ruker F. Furtmuller P.G. Obinger C. J. Biol. Chem. 2003; 278: 20185-20191Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 12Regelsberger 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 (72) Google Scholar, 13Regelsberger G. Jakopitsch C. Furtmuller P.G. Rueker F. Switala J. Loewen P.C. Obinger C. Biochem. Soc. Trans. 2001; 29: 99-105Crossref PubMed Google Scholar, 14Yu S. Girotto S. Zhao X. Magliozzo R.S. J. Biol. Chem. 2003; 278: 44121-44127Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). The origin of the changes in catalatic function in Synechocystis KatG and Burkholderia pseudomallei KatG distal Trp mutants had been assigned to faulty binding of the second molecule of hydrogen peroxide that should participate in reduction of Compound (Cmpd) I in a classical catalase cycle (12Regelsberger 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 (72) Google Scholar, 15Hillar A. Peters B. Pauls R. Loboda A. Zhang H. Mauk A.G. Loewen P.C. Biochemistry. 2000; 39: 5868-5875Crossref PubMed Scopus (95) Google Scholar), because the formation of Cmpd I was undamaged and in fact was very rapid in those cases. More recently, residue Trp111 together with His112 were described as key sites in Synechocystis KatG for the steering of Cmpd I reduction by hydrogen peroxide (16Deemagarn T. Wiseman B. Carpena X. Ivancich A. Fita I. Loewen P.C. Proteins. 2007; 66: 219-228Crossref PubMed Scopus (26) Google Scholar). Mutations in other residues surrounding the heme pocket have also been shown to interfere with catalase activity for reasons ascribed to disruption of hydrogen bonding networks (17Jakopitsch C. Droghetti E. Schmuckenschlager F. Furtmuller P.G. Smulevich G. Obinger C. J. Biol. Chem. 2005; 280: 42411-42422Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 18Jakopitsch C. Auer M. Regelsberger G. Jantschko W. Furtmuller G.P. Ruker F. Obinger C. Eur. J. Biochem. 2003; 270: 1006-1013Crossref PubMed Scopus (24) Google Scholar). Other issues such as stabilization of the oxyferrous heme intermediate formed in the presence of excess H2O2 was not presented in studies of KatG from other laboratories, but in the case of M. tuberculosis KatG[Y229F] (14Yu S. Girotto S. Zhao X. Magliozzo R.S. J. Biol. Chem. 2003; 278: 44121-44127Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar) and in KatG[W107F] as shown here, such stabilization is clearly a fundamental alteration in function that sets apart the behavior of these mutants. No prior work has defined a specific mechanistic role for the MYW adduct structure that could not also be fulfilled by the individual residues whether they were covalently linked or not. DFT calculations along with extensive analysis of EPR spectra allowed a reasonable assignment in the companion paper (52Suarez J. Ranguelova K. Jarzecki A.A. Manzerova J. Krymov V. Zhao X. Yu S. Metlitsky L. Gerfen G.J. Magliozzo R.S. J. Biol. Chem. 2009; 284: 7017-7029Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar) of a catalytically competent radical to this adduct in KatG. An intermediate exhibiting an optical spectrum typical of peroxidase Cmpd III (oxyperoxidase) is found when WT KatG reacts with a large excess of H2O2 (12Regelsberger 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 (72) Google Scholar, 15Hillar A. Peters B. Pauls R. Loboda A. Zhang H. Mauk A.G. Loewen P.C. Biochemistry. 2000; 39: 5868-5875Crossref PubMed Scopus (95) Google Scholar, 19Chouchane S. Lippai I. Magliozzo R.S. Biochemistry. 2000; 39: 9975-9983Crossref PubMed Scopus (101) Google Scholar, 20Jakopitsch C. Ivancich A. Schmuckenschlager F. Wanasinghe A. Poltl G. Furtmuller P.G. Ruker F. Obinger C. J. Biol. Chem. 2004; 279: 46082-46095Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar), yet catalase function proceeds under such conditions. The formation of this species occurs in KatG[Y229F], W107F, and M255A mutants in the presence of even a few molar equivalents of peroxide, but catalase function is severely reduced. The properties of the oxy intermediates in WT KatG and three distal side mutants and a proposed mechanism for the catalase reaction are discussed here. Chemicals and Reagents-INH, PAA, hydrogen peroxide, o-dianisidine, and all the other chemicals were purchased from Sigma and were of the highest purity available. INH was recrystallized from methanol and stored at 4 °C. PAA (32%) was diluted to 10 mm in potassium phosphate buffer and was incubated with 780 units/ml catalase (Roche Applied Science) for 4 h at 37 °C, followed by removal of the enzyme by ultrafiltration, to remove hydrogen peroxide that interfered with optical stopped-flow experiments. This preparation of PAA was stored at -80 °C in small aliquots. Construction, Expression, and Purification of Distal Mutants- Overexpression of recombinant WT KatG and KatG mutant enzymes and their purification were achieved as described in the companion paper (52Suarez J. Ranguelova K. Jarzecki A.A. Manzerova J. Krymov V. Zhao X. Yu S. Metlitsky L. Gerfen G.J. Magliozzo R.S. J. Biol. Chem. 2009; 284: 7017-7029Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar) and previous reports (14Yu S. Girotto S. Zhao X. Magliozzo R.S. J. Biol. Chem. 2003; 278: 44121-44127Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 21Ranguelova K. Girotto S. Gerfen G.J. Yu S. Suarez J. Metlitsky L. Magliozzo R.S. J. Biol. Chem. 2007; 282: 6255-6264Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Optical Measurements-All standard spectrophotometric measurements were performed using an NT14 UV-visible spectrophotometer (Aviv Biomedical, Lakewood, NJ). Protein concentration, expressed as heme concentration, was determined using a heme extinction coefficient at 407 nm equal to 100 mm-1 cm-1 (22Marcinkeviciene J.A. Magliozzo R.S. Blanchard J.S. J. Biol. Chem. 1995; 270: 22290-22295Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Catalase and peroxidase activities were determined spectrophotometrically in replicate analyses using initial rates of reactions with 25 mm hydrogen peroxide (catalase activity) or t-butyl hydroperoxide plus o-dianisidine (peroxidase activity) according to published methods (22Marcinkeviciene J.A. Magliozzo R.S. Blanchard J.S. J. Biol. Chem. 1995; 270: 22290-22295Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 23Saint-Joanis B. Souchon H. Wilming M. Johnsson K. Alzari P.M. Cole S.T. Biochem. J. 1999; 338: 753-760Crossref PubMed Scopus (110) Google Scholar). A rapid scanning diode array stopped-flow spectrophotometer (HiTech Scientific model SF-61DX2) was used for kinetics experiments. Data acquisition and analyses were performed using the Kinet-Asyst software package (HiTech Scientific). All reactions were followed at 25 °C using freshly prepared solutions of PAA or H2O2, all in 20 mm potassium phosphate buffer, pH 7.2. Preparation of Oxyferrous KatG[W107F]-Oxyferrous KatG[W107F] was generated through photolysis of the carbonyl enzyme dissolved in oxygen-containing buffer as described by Miller et al. (24Miller M.A. Bandyopadhyay D. Mauro J.M. Traylor T.G. Kraut J. Biochemistry. 1992; 31: 2789-2797Crossref PubMed Scopus (22) Google Scholar). Briefly, enzyme solutions were placed in a sealed cuvette and purged with argon for 30 min followed by carbon monoxide for 10 min. Addition of a small excess of sodium dithionite resulted in conversion to the enzyme-CO complex according to the optical features of the product (the wavelengths of the Soret, β, and α bands were 422, 540, 570 nm, respectively). The enzyme-CO complex was then separated in air from the reaction mixture using a small ion exchange column. After extensive rinsing of the bound enzyme-CO complex, it was eluted with potassium phosphate buffer containing 0.3 m NaCl. The chromatographic purification procedure was performed in a cold room with reduced light and completed within 10 min to minimize dissociation of CO by photolysis. The enzyme-CO complex was then subjected to a 10–15-s illumination by a 75-watt xenon lamp, and the resulting protein was stored at 4 °C. For comparison, similar experiments were performed using WT KatG. In some cases, the oxyenzyme complexes were also prepared by treating the ferric enzymes with excess H2O2 followed by rapid removal of excess peroxide through ion exchange chromatography of the enzyme. EPR/Resonance Raman Spectroscopy-X-band EPR spectra were recorded using a Bruker E500 EPR spectrometer with data acquisition and manipulation performed using XeprView and WinEPR software (Bruker). Low temperature spectra were recorded using an Oxford Spectrostat continuous flow cryostat and ITC503 temperature controller. The spectra of KatG[W107F] (100 μm) were recorded at 10 K in 20 mm potassium phosphate buffer, pH 7.2 (at room temperature). The EPR spectrum of commercial ferric horse heart Mb in 50 mm glycine/NaOH buffer at pH 10 was used as a standard to estimate the low spin concentration in samples of KatG[W107F]. Final heme concentration in the standard was based on the Soret peak at 418 nm, assuming complete conversion to the low spin alkaline form of myoglobin (∈ = 95 mm-1·cm-1) (25Antonini E. Brunori M. Neuberger A. Tatum E.L. Hemoglobin and Myoglobin in Their Reactions with Ligands. North-Holland Publishing Co., Amsterdam1971: 44Google Scholar, 26Ikeda-Saito M. Hori H. Andersson L.A. Prince R.C. Pickering I.J. George G.N. Sanders II, C.R. Lutz R.S. McKelvey E.J. Mattera R. J. Biol. Chem. 1992; 267: 22843-22852Abstract Full Text PDF PubMed Google Scholar). Resonance Raman spectra were obtained using instrumentation described previously (27Kapetanaki S. Chouchane S. Girotto S. Yu S. Magliozzo R.S. Schelvis J.P. Biochemistry. 2003; 42: 3835-3845Crossref PubMed Scopus (24) Google Scholar). Enzyme samples (40 μm) were maintained at 6 ± 2 °C and were excited at 406.7 nm with a Kr+ laser (Coherent, I-302). The laser power at the sample was 10 milliwatts. Toluene was used to calibrate the spectra, and the spectra were corrected for a sloping base line with a polynomial function. The goal of this work was to provide characterization of resting KatG[W107F] and new mechanistic insights into the loss of catalase activity in this and two other distal side mutants to shed light on the mechanism in the WT enzyme. Spectroscopic examination of the ferric enzyme helps explain the major alteration in functional properties of the W107F mutant, which include stabilization of 6-c heme species. Prior reports have described certain properties of this mutant in the M. tuberculosis enzyme (21Ranguelova K. Girotto S. Gerfen G.J. Yu S. Suarez J. Metlitsky L. Magliozzo R.S. J. Biol. Chem. 2007; 282: 6255-6264Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar) and the KatG from other organisms (28Carpena X. Wiseman B. Deemagarn T. Herguedas B. Ivancich A. Singh R. Loewen P.C. Fita I. Biochemistry. 2006; 45: 5171-5179Crossref PubMed Scopus (38) Google Scholar, 29Jakopitsch C. Regelsberger G. Furtmuller P.G. Ruker F. Peschek G.A. Obinger C. Biochem. Biophys. Res. Commun. 2001; 287: 682-687Crossref PubMed Scopus (52) Google Scholar, 30Vlasits J. Jakopitsch C. Schwanninger M. Holubar P. Obinger C. FEBS Lett. 2007; 581: 320-324Crossref PubMed Scopus (38) Google Scholar). Here, changes in mechanism and the properties of heme and enzyme intermediates are described in the context of the unique mechanistic features afforded by the distal side MYW adduct presented in the companion paper (52Suarez J. Ranguelova K. Jarzecki A.A. Manzerova J. Krymov V. Zhao X. Yu S. Metlitsky L. Gerfen G.J. Magliozzo R.S. J. Biol. Chem. 2009; 284: 7017-7029Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Enzymatic and Spectroscopic Characterization of KatG[W107F]- The yield of KatG[W107F] upon overexpression and purification from Escherichia coli is similar to that of WT KatG. The catalase activity of KatG[W107F] is greatly reduced compared with WT KatG (0.5 ± 0.2 versus 3800 ± 300 units/mg for WT KatG) under assay conditions using millimolar concentrations of H2O2, whereas its peroxidase activity (with t-butyl peroxide and o-dianisidine) is only moderately reduced (0.6 ± 0.2 versus 0.9 ± 0.1 unit/mg for WT KatG). Here, it was considered important to describe the characteristics of the resting (ferric) enzyme to help explain the behavior of the oxyenzyme presented below. The optical spectrum of KatG[W107F] has a Soret peak at 407 and sharp CT2 and CT1 bands around 504 and 628 nm, respectively (Fig. 2A), characteristic of enzyme containing a high level of 6-c heme. In freshly isolated WT KatG, which contains 5-c heme as the majority species, the CT1 band is found close to 640 nm, and in “aged” WT enzyme, which contains more 6-c heme, it occurs at 629 nm (31Chouchane S. Girotto S. Kapetanaki S. Schelvis J.P. Yu S. Magliozzo R.S. J. Biol. Chem. 2003; 278: 8154-8162Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Interestingly, the W107F mutant also exhibits optical features at 541 and 578 nm characteristic of the β and α bands of 6-c low spin (6-c LS) ferric heme and/or oxyferrous heme, both of which are confirmed here. These low spin features decay upon storage of the enzyme, consistent with loss of an exchangeable heme ligand. EPR and resonance Raman spectra also provided insight into coordination number in the W107F mutant. EPR spectra of freshly purified KatG[W107F] contain a broad axial signal (g⊥ = 5.80 and g ∼2.0) and some low intensity signals because of 6-c low spin ferric heme (g1 = 3.23, g2 = 2.05 and unresolved g3) estimated to be less than 10% of heme content in the fresh enzyme (Fig. 2B, spectrum 1). WT KatG under the same conditions instead exhibits signals assigned to 5-c heme and a rhombic signal also assigned to a 6-c heme species different from the one in the mutant (g1,2,3 = 5.94, 5.49, ∼2) (31Chouchane S. Girotto S. Kapetanaki S. Schelvis J.P. Yu S. Magliozzo R.S. J. Biol. Chem. 2003; 278: 8154-8162Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). The 6-c complexes other than the low spin species are assigned to enzyme containing water as a sixth ligand associated with heme iron. Certain similar findings were reported earlier for the analogous mutant of Synechocystis and B. pseudomallei KatG (28Carpena X. Wiseman B. Deemagarn T. Herguedas B. Ivancich A. Singh R. Loewen P.C. Fita I. Biochemistry. 2006; 45: 5171-5179Crossref PubMed Scopus (38) Google Scholar, 32Ivancich A. Jakopitsch C. Auer M. Un S. Obinger C. J. Am. Chem. Soc. 2003; 125: 14093-14102Crossref PubMed Scopus (102) Google Scholar). An endogenous strong field ligand is likely responsible for the 6-c LS species because the gmax value (3.21) is larger than that usually observed for O-type (oxygen and nitrogen as the strong field axial ligands) heme (33Peisach J. Blumberg W.E. Arends T. Bemski G. Nagel R.L. Proceedings of the 1st Inter-American Symposium on Hemoglobins, Caracas, 1969. Karger, Basel1971: 199Google Scholar, 34Svistunenko D.A. Sharpe M.A. Nicholls P. Blenkinsop C. Davies N.A. Dunne J. Wilson M.T. Cooper C.E. Biochem. J. 2000; 351: 595-605Crossref PubMed Scopus (45) Google Scholar). The hydroxy-form of horseradish peroxidase, for example, exhibits an EPR signal with gmax = 2.9 (35Blumberg W.E. Peisach J. Wittenberg B.A. Wittenberg J.B. J. Biol. Chem. 1968; 243: 1854-1862Abstract Full Text PDF PubMed Google Scholar), and a similar signal is reported for the distal histidine (H123Q) mutant of Synechocystis KatG (32Ivancich A. Jakopitsch C. Auer M. Un S. Obinger C. J. Am. Chem. Soc. 2003; 125: 14093-14102Crossref PubMed Scopus (102) Google Scholar) arguing in favor of the hydroxide form in that histidine mutant and assignment of the 6-c LS species here to an endogenous ligand complex very likely from coordination of the distal imidazole (residue His108). Resonance Raman spectra of KatG[W107F] contained multiple ν3 bands, including a 6-c heme band at 1488 cm-1 (along with a small contribution from a band at 1483 cm-1 also assigned to 6-c heme), and a band at 1507 cm-1 typical of 6-c LS heme (Fig. 2C), consistent with the optical and EPR spectra. The bands usually associated with out-of-plane modes in the low frequency region of the resonance Raman spectra have lower intensities in the mutant compared with WT M. tuberculosis KatG, suggesting a more planar geometry of heme. This observation is consistent with the axial EPR signal arising from the majority species at low temperature. An oxyenzyme component was not detectable most likely because of photolysis of the ligand. In KatG[W107F], and in KatG[Y229F] as previously reported (14Yu S. Girotto S. Zhao X. Magliozzo R.S. J. Biol. Chem. 2003; 278: 44121-44127Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar), elimination of the MYW adduct apparently alters the hydrogen bonding arrangements on the distal side to favor and stabilize 6-c heme. Conversely, water coordination in WT KatG could be considered to be disfavored by the presence of the MYW adduct. The heme iron-associated water molecule in the crystal structure of WT M. tuberculosis KatG from this laboratory (36Zhao X. Yu H. Yu S. Wang F. Sacchettini J.C. Magliozzo R.S. Biochemistry. 2006; 45: 4131-4140Crossref PubMed Scopus (132) Google Scholar) is hydrogen bonded to the indole nitrogen of Trp107 (and is also hydrogen bonded to the distal histidyl imidazole but at a longer distance). The hydrogen bond to the indole nitrogen could disfavor optimal interaction between the water ligand and iron; in fact the water iron bond length is 2.8 Å in this structure (36Zhao X. Yu H. Yu S. Wang F. Sacchettini J.C. Magliozzo R.S. Biochemistry. 2006; 45: 4131-4140Crossref PubMed Scopus (132) Google Scholar). The 6-c form(s) of M. tuberculosis KatG enzyme in solution displays unique spectroscopic and functional properties (37Zhao X. Yu S. Magliozzo R.S. Biochemistry. 2007; 46: 3161-3170Crossref PubMed Scopus (35) Google Scholar, 38Ranguelova K. Suarez J. Metlitsky L. Yu S. Brejt S.Z. Zhao L. Schelvis J.P. Magliozzo R.S. Biochemistry. 2008; 47: 12583-12592Crossref PubMed Scopus (9) Google Scholar). Because both the 107 and the 229 mutants favor 6-c heme species, the distal sides must have something in common. The phenolic oxygen of Tyr229 in the MYW adduct of WT KatG faces away from heme iron (36Zhao X. Yu H. Yu S. Wang F. Sacchettini J.C. Magliozzo R.S. Biochemistry. 2006; 45: 4131-4140Crossref PubMed Scopus (132) Google Scholar), whereas the indole nitrogen of Trp107 faces toward the sixth ligand position, yet mutation of either residue has a similar effect on coordination number that could arise due to removal of the participation of the indole group. The position of the indole is likely to be altered in the Y229F mutant because of the missing covalent bond to Trp107. Thus, disruption of the indole-water interactions in these two distal side mutants can favor water coordination to iron. There may also be a change in the position of His108 because the adjacent Trp side chain is not incorporated into the MYW adduct in the Y229F mutant, and the link between Tyr229 and Trp107 is absent in the W107F mutant, such that this region of the distal pocket can be more flexible in both cases. The histidyl imidazole of residue 108 may then become the more important hydrogen-bonding site for stabilizing sixth ligands to iron. The W107F mutant also exhibits more 6-c LS heme because of coordination of an endogenous ligand likely to be the distal imidazole, which constitutes specific evidence for enhanced flexibility of the distal side in the heme pocket. Most importantly, the observation of enhanced stability of oxyferrous heme in these distal side mutants is consistent with the idea that the histidyl imidazole can better participate in hydrogen bonding to stabilize ligands when the indole of Trp107 is missing. Oxyferrous KatG[W107F]-The presence of some oxyferrous enzyme in samples of purified KatG[W107F] was suspected because of the broad shape of the features around 540 and 580 nm in the optical spectrum of the enzyme as isolated. However, oxyferrous heme has not been reported for purified KatG or other “native” peroxidases, either because there is no route to its formation in situ or because this intermediate may be formed but is unstable. Thus, the presence of oxyferrous heme in preparations of KatG[W107F] would indicate first that it can be formed during cell growth and that it is stable enough to persist during purification and storage. To help confirm these ideas, authentic oxyferrous KatG[W107F] was prepared by photolysis in aerated buffer of the carbonyl complex formed from reduced (ferrous) enzyme and CO. The prepared oxy-KatG[W107F] shows characteristic absorption bands at 413, 542, and 578 nm. Like oxy-horseradish peroxidase (39Wittenberg B.A. Antonini E. Brunori M. Noble R.W. Wittenberg J.B. Wymann J. Biochemistry. 1967; 6: 1970-1974Crossref PubMed Scopus (18) Google Scholar), the oxyenzyme undergoes auto-oxidation to ferric enzyme with an isosbestic point consistent with direct conversion to the ferric enzyme (Fig. 3A). Oxyferrous KatG[W107F] decays very slowly, and the reaction follows single exponential kinetics. The t½ is estimated to be at least 2 days at 4 °C, as compared with 6 min for the similarly prepared oxy-WT KatG (Fig. 3B). In similar experiments, the half-lives of oxyferrous Y229F and M255A mutants were found to be 25 and 7 min, respectively. The oxy-WT KatG and KatG[Y229F] prepared here are more stable than the corresponding enzyme forms from Synechocystis (40Jakopitsch C. Wanasinghe A. Jantschko W. Furtmuller P.G. Obinger C. J. Biol. Chem. 2005; 280: 9037-9042Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Most interestingly, the stability of oxyferrous M. tuberculosis KatG was hardly affected by the Met255 mutation, was improved by the Tyr299 mutation, and was greatly enhanced by Trp107 mutation. The Met255 mutation still allows formation of a Tyr-Trp cross-link on the distal side (10Ghiladi R.A. Medzihradszky K.F. Ortiz de Montellano P.R. Biochemistry. 2005; 44: 15093-15105Crossref PubMed Scopus (42) Google Scholar). These observations lead to the conclusion that the MYW adduct in WT KatG disfavors ligand binding to the sixth coordination position of heme iron in confirmation of the observations on the ferric enzyme above. The very slow rate of decay" @default.
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• W2044264629 date "2009-03-01" @default.
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• W2044264629 title "Role of the Oxyferrous Heme Intermediate and Distal Side Adduct Radical in the Catalase Activity of Mycobacterium tuberculosis KatG Revealed by the W107F Mutant" @default.
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• W2044264629 doi "https://doi.org/10.1074/jbc.m808107200" @default.
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