An Electrical Potential in the Access Channel of Catalases Enhances Catalysis

Substrate H2O2 must gain access to the deeply buried active site of catalases through channels of 30–50 Å in length. The most prominent or main channel approaches the active site perpendicular to the plane of the heme and contains a number of residues that are conserved in all catalases. Changes in Val169, 8 Å from the heme in catalase HPII from Escherichia coli, introducing smaller, larger or polar side chains reduces the catalase activity. Changes in Asp181, 12 Å from the heme, reduces activity by up to 90% if the negatively charged side chain is removed when Ala, Gln, Ser, Asn, or Ile are the substituted residues. Only the D181E variant retains wild type activity. Determination of the crystal structures of the Glu181, Ala181, Ser181, and Gln181 variants of HPII reveals lower water occupancy in the main channel of the less active variants, particularly at the position forming the sixth ligand to the heme iron and in the hydrophobic, constricted region adjacent to Val169. It is proposed that an electrical potential exists between the negatively charged aspartate (or glutamate) side chain at position 181 and the positively charged heme iron 12 Å distant. The potential field acts upon the electrical dipoles of water generating a common orientation that favors hydrogen bond formation and promotes interaction with the heme iron. Substrate hydrogen peroxide would be affected similarly and would enter the active site oriented optimally for interaction with active site residues. Substrate H2O2 must gain access to the deeply buried active site of catalases through channels of 30–50 Å in length. The most prominent or main channel approaches the active site perpendicular to the plane of the heme and contains a number of residues that are conserved in all catalases. Changes in Val169, 8 Å from the heme in catalase HPII from Escherichia coli, introducing smaller, larger or polar side chains reduces the catalase activity. Changes in Asp181, 12 Å from the heme, reduces activity by up to 90% if the negatively charged side chain is removed when Ala, Gln, Ser, Asn, or Ile are the substituted residues. Only the D181E variant retains wild type activity. Determination of the crystal structures of the Glu181, Ala181, Ser181, and Gln181 variants of HPII reveals lower water occupancy in the main channel of the less active variants, particularly at the position forming the sixth ligand to the heme iron and in the hydrophobic, constricted region adjacent to Val169. It is proposed that an electrical potential exists between the negatively charged aspartate (or glutamate) side chain at position 181 and the positively charged heme iron 12 Å distant. The potential field acts upon the electrical dipoles of water generating a common orientation that favors hydrogen bond formation and promotes interaction with the heme iron. Substrate hydrogen peroxide would be affected similarly and would enter the active site oriented optimally for interaction with active site residues. The monofunctional catalase (hydrogen peroxide:hydrogen peroxide oxidoreductase, EC 1.11.1.6) is a protective enzyme that degrades hydrogen peroxide to prevent damage from it or its more reactive degradation byproducts. The catalase reaction utilizes hydrogen peroxide as both an electron donor and an electron acceptor as summarized in the overall Reaction 1, which involves two distinct stages. In the first stage (Reaction 2) the resting state enzyme is oxidized by hydrogen peroxide to an oxy-ferryl intermediate, compound I, which in the second stage (Reaction 3) is reduced back to the resting state by a second hydrogen peroxide. 2H2O2→2H2O+O2Enz(Por-FeIII)+H2O2→CpdI(Por+·-FeIV=O)+H2OCpdI(Por+·-FeIV=O)+H2O2→Enz(Por-FeIII)+H2O+O2Reactions1-3(Eq. 1) The structures of heme-containing monofunctional catalases isolated from eight different sources have been reported including those from bovine liver (1Murthy M.R.N. Reid T.J. Sicignano A. Tanaka N. Rossmann M.G. J. Mol. Biol. 1981; 152: 465-499Crossref PubMed Scopus (375) Google Scholar, 2Fita I. Silva A.M. Murthy M.R.N. Rossmann M.G. Acta Crystallogr. Sect. B Struct. Crystallogr. Cryst. Chem. 1986; 42: 497-515Crossref Scopus (101) Google Scholar), human erythrocytes (3Ko T.-P. Safo M.K. Musayev F.N. Di Salvo M.L. Wang C. Wu S.-H. Abraham D.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 56: 241-245Crossref Scopus (52) Google Scholar, 4Putnam C.D. Arvai A.S. Bourne Y. Tainer J.A. J. Mol. Biol. 1999; 296: 295-309Crossref Scopus (339) Google Scholar), Penicillium vitale (5Vainshtein B.K. Melik-Adamyan W.R. Barynin V.V. Vagin A.A. Grebenko A.I. Nature. 1981; 293: 411-412Crossref PubMed Scopus (105) Google Scholar, 6Vainshtein B.K. Melik-Adamyan W.R. Barynin V.V. Vagin A.A. Grebenko A.I. Borisov V.V. Bartels K.S. Fita I. Rossmann M.G. J. Mol. Biol. 1986; 188: 49-61Crossref PubMed Scopus (128) Google Scholar), Saccharomyces cerevisiae (7Maté M.J. Zamocky M. Nykyri L.M. Herzog C. Alzari P.M. Betzel C. Koller F. Fita I. J. Mol. Biol. 1999; 286: 135-139Crossref PubMed Scopus (92) Google Scholar), Proteus mirabilis (8Gouet P. Jouve H.M. Dideberg O. J. Mol. Biol. 1995; 249: 933-954Crossref PubMed Scopus (118) Google Scholar), Micrococcus lysodeikticus (9Murshudov G.N. Melik-Adamyan W.R. Grebenko A.I. Barynin V.V. Vagin A.A. Vainshtein B.K. Dauter Z. Wilson K. FEBS Lett. 1982; 312: 127-131Crossref Scopus (86) Google Scholar), Escherichia coli (10Bravo J. Verdaguer N. Tormo J. Betzel C. Switala J. Loewen P.C. Fita I. Structure. 1995; 3: 491-502Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 11Bravo J. Maté M.J. Schneider T. Switala J. Wilson K. Loewen P.C. Fita I. Proteins. 1999; 34: 155-166Crossref PubMed Scopus (57) Google Scholar), and Pseudomonas syringae (12Carpena X. Soriano M. Klotz M.G. Duckworth H.W. Donald L.J. Melik-Adamyan W. Fita I. Loewen P.C. Proteins. 2003; 50: 423-436Crossref PubMed Scopus (46) Google Scholar), revealing a highly conserved β-barrel core structure in all enzymes. The active center, composed of a heme with a tyrosine as the fifth ligand to the iron, a histidine, and an asparagine, is deeply buried in this core structure. HPII from E. coli contains two post-translational modifications in the active center, including an oxidized, cis-spirolactone, heme d (13Murshudov G.N. Grebenko A.I. Barynin V. Dauter Z. Wilson K.S. Vainshtein B.K. Melik-Adamyan W. Bravo J. Ferrán J.M. Ferrer J.C. Switala J. Loewen P.C. Fita I. J. Biol. Chem. 1996; 271: 8863-8868Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar), and a covalent bond between the Nδ of His392 and the Cβ of Tyr415, the proximal side fifth ligand of the heme (14Bravo J. Fita I. Ferrer J.C. Ens W. Hillar A. Switala J. Loewen P.C. Protein Sci. 1997; 6: 1016-1023Crossref PubMed Scopus (49) Google Scholar). Both modifications are generated self-catalytically by the catalase and seem to require some degree of catalase activity (15Loewen P.C. Switala J. von Ossowski I. Hillar A. Christie A. Tattrie B. Nicholls P. Biochemistry. 1993; 32: 10159-10164Crossref PubMed Scopus (62) Google Scholar). Three channels, the main channel oriented perpendicular to the plane of the heme, the lateral channel approaching in the plane of the heme, and a channel leading to the central cavity, connect the active site to the exterior of the enzyme, providing routes for substrate ingress and product egress. A number of catalase HPII variants have been constructed (16Nicholls P. Fita I. Loewen P.C. Adv. Inorg. Chem. 2001; 51: 51-106Crossref Google Scholar) to study the roles of various residues in the enzyme, including the active site residues. Most recently, the characterization of inactive variants has allowed the identification of substrate H2O2 localized in the main or perpendicular channel (17Melik-Adamyan W. Bravo J. Carpena X. Switala J. Maté M.J. Fita I. Loewen P.C. Proteins. 2001; 44: 270-281Crossref PubMed Scopus (44) Google Scholar). The presence of H2O2 in the channel of HPII, the relatively direct route provided by the main channel to the heme in other catalases, and molecular dynamic studies (18Kalko S.G. Gelpi J.L. Fita I. Orozco M. J. Am. Chem. Soc. 2001; 123: 9665-9672Crossref PubMed Scopus (46) Google Scholar, 19Amara P. Andreoletti P. Jouve H.M. Field M.J. Protein Sci. 2001; 10: 1927-1935Crossref PubMed Scopus (45) Google Scholar) all suggest that the main channel is the primary route for substrate movement to the active site. On the other hand, evidence has been presented that the lateral channel in HPII does have a role (20Sevinc M.S. Mate M.J. Switala J. Fita I. Loewen P.C. Protein Sci. 1999; 8: 490-498Crossref PubMed Scopus (29) Google Scholar). A number of highly conserved residues are situated in the main channel. These include the essential histidine, a valine and an aspartate, (His128, Val169, and Asp181 in HPII) situated 4, 8, and 12 Å from the heme, respectively (Fig. 1). His128 is essential for catalysis in HPII (15Loewen P.C. Switala J. von Ossowski I. Hillar A. Christie A. Tattrie B. Nicholls P. Biochemistry. 1993; 32: 10159-10164Crossref PubMed Scopus (62) Google Scholar), and the importance of Val169 in constricting the narrowest, hydrophobic portion of the channel has been investigated in yeast CATA (7Maté M.J. Zamocky M. Nykyri L.M. Herzog C. Alzari P.M. Betzel C. Koller F. Fita I. J. Mol. Biol. 1999; 286: 135-139Crossref PubMed Scopus (92) Google Scholar) and HPII (21Mate M.J. Sevinc M.S. Hu B. Bujons J. Bravo J. Switala J. Ens W. Loewen P.C. Fita I. J. Biol. Chem. 1999; 274: 27717-27725Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar), although without a definitive conclusion. The importance of the other residues further up the channel has not been studied, and this paper focuses on a number of these residues in the main channel of HPII. The importance of the highly conserved aspartate, Asp181, in particular the negative charge on its side chain, is revealed. Materials—Standard chemicals and biochemicals were obtained from Sigma. Restriction nucleases, polynucleotide kinase, DNA ligase, and the Klenow fragment of DNA polymerase were obtained from Invitrogen. Strains and Plasmids—The plasmid pAMkatE72 (22von Ossowski I. Mulvey M.R. Leco P.A. Borys A. Loewen P.C. J. Bacteriol. 1991; 173: 514-520Crossref PubMed Scopus (126) Google Scholar) was used as the source for the katE gene. Phagemids pKS+ and pKS– from Stratagene Cloning Systems were used for mutagenesis, sequencing, and cloning. E. coli strains NM522 (supE thi (lac-proAB) hsd-5 [F′ proAB lacI q lacZ)15]) (23Mead D.A. Skorupa E.S. Kemper B. Nucleic Acids Res. 1985; 13: 1103-1118Crossref PubMed Scopus (81) Google Scholar), JM109 (recA1 supE44 endA1 hsdR17 gyrA96 relA1 thi) (lac-proAB) (24Yanisch-Perron C. Vieria C.J. Messing J. Gene (Amst.). 1985; 33: 103-119Crossref PubMed Scopus (11410) Google Scholar), and CJ236 (dut-1 ung-1 thi-1 relA1/pCJ105 F′) (25Kunkel T.A. Roberts J.D. Zakour R.A. Methods Enzymol. 1987; 154: 367-382Crossref PubMed Scopus (4540) Google Scholar) were used as hosts for the plasmids and for generation of single-strand phage DNA using helper phage R408. Strain UM255 (pro leu rpsL hsdM hsdR endI lacY katG2 katE12::Tn10 recA (26Mulvey M.R. Sorby P.A. Triggs-Raine B.L. Loewen P.C. Gene (Amst.). 1988; 73: 337-345Crossref PubMed Scopus (97) Google Scholar) was used for expression of the mutant katE constructs and isolation of the mutant HPII proteins. Oligonucleotide-directed Mutagenesis—Oligonucleotides were purchased from Invitrogen and are listed in Table I. The restriction nuclease fragments that were mutagenized following the Kunkel procedure (25Kunkel T.A. Roberts J.D. Zakour R.A. Methods Enzymol. 1987; 154: 367-382Crossref PubMed Scopus (4540) Google Scholar), sequenced, and subsequently reincorporated into pAMkatE72 to generate the plasmids encoding the mutagenized katE genes are also listed. Sequence confirmation of all sequences was by the Sanger method (27Sanger F.S. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52251) Google Scholar) on double-stranded plasmid DNA generated in JM109. Subsequent expression and purification were carried out as described previously (15Loewen P.C. Switala J. von Ossowski I. Hillar A. Christie A. Tattrie B. Nicholls P. Biochemistry. 1993; 32: 10159-10164Crossref PubMed Scopus (62) Google Scholar).Table IOligonucleotides and katE restriction fragments used in oligonucleotide-directed mutagenesis of katEMutantSequence changeOligonucleotideaThe sequence in bold type is the codon that has been modified.Restriction fragmentV169I(GTT → ATT)TTCTCTACCATTCAGGGTGGTHindIII-EcoRI (1246-1856)V169F(GTT → TTT)TTCTCTACCTTTCAGGGTGGTHindIII-EcoRI (1246-1856)V169W(GTT → TGG)TTCTCTACCTGGCAGGGTGGTHindIII-EcoRI (1246-1856)R180A(CGT → GCT)GATACCGTGGCTGATATCCGTHindIII-EcoRI (1246-1856)R180K(CGT → AAA)GATACCGTGAAAGATATCCGTHindIII-EcoRI (1246-1856)D181A(GAT → GCT)ACCGTGCGTGCTATCCGTGGCHindIII-EcoRI (1246-1856)D181S(GAT → TCT)ACCGTGCGTTCTATCCGTGGCHindIII-EcoRI (1246-1856)D181E(GAT → GAA)ACCGTGCGTGAAATCCGTGGCHindIII-EcoRI (1246-1856)D181Q(GAT → CAA)ACCGTGCGTCAAATCCGTGGCHindIII-EcoRI (1246-1856)D181N(GAT → AAT)ACCGTGCGTAATATCCGTGGCHindIII-EcoRI (1246-1856)D181I(GAT → ATT)ACCGTGCGTATTATCCGTGGCHindIII-EcoRI (1246-1856)D181W(GAT → TGG)ACCGTGCGTTGGATCCGTGGCHindIII-EcoRI (1246-1856)a The sequence in bold type is the codon that has been modified. Open table in a new tab Catalase, Protein, and Spectral Determination—Catalase activity was determined by the method of Rorth and Jensen (28Rorth H.M. Jensen P.K. Biochim. Biophys. Acta. 1967; 139: 171-173Crossref PubMed Scopus (112) Google Scholar) in a Gilson oxygraph equipped with a Clark electrode. One unit of catalase is defined as the amount that decomposes 1 μmol of H2O2 in 1 min in a 60 mm H2O2 solution at pH 7.0 at 37 °C. The initial rates of oxygen evolution were used to determine the turnover rates to minimize the inactivation caused by high [H2O2] (29Ogura Y. Arch. Biochem. Biophys. 1955; 57: 288-300Crossref PubMed Scopus (106) Google Scholar). Protein was estimated according to the methods outlined by Layne (30Layne E. Methods Enzymol. 1957; 3: 447-454Crossref Scopus (2718) Google Scholar). The absorption spectra were obtained using a Milton Roy MR3000 spectrophotometer. The samples were dissolved in 50 mm potassium phosphate, pH 7.0. Enzyme Purification—Cultures of E. coli UM255 with plasmids and encoding HPII or the Ala181, Ser181, and variants, were in yeast and of the mutant variants was for at 37 or at and of the wild type HPII was for at 37 with The were and HPII was isolated as previously described P.C. Switala J. Biochem. Biol. 1986; PubMed Scopus Google Scholar). of the the solution was at 50 for min by to on and of the Asp181 variants of HPII were obtained at using the method a solution and pH The were with in the crystal unit and a of The were obtained from to a solution and with a unit listed in Table The were and using and using Z. W. Methods Enzymol. 1996; Scopus Google Scholar) Table and for the Asp181 variants of in to the is as for for a not used in the of from B in to the is as for for a not used in the Open table in a new tab was carried out with the using HPII as the initial were using the G.N. Vagin A.A. Acta Crystallogr. Sect. D Biol. Crystallogr. 1997; PubMed Scopus Google Scholar) with with the Crystallogr. Sect. A. 50: PubMed Scopus Google Scholar) and with the T.A. M. Acta Crystallogr. Sect. A. 1991; PubMed Scopus Google Scholar). were when to the in the that at hydrogen bond with in the In the of the were with the and the used for The of and molecular was carried out with T.A. Acta Crystallogr. Sect. D Biol. Crystallogr. 50: PubMed Scopus Google Scholar) using a reduced for polar in for hydrogen (21Mate M.J. Sevinc M.S. Hu B. Bujons J. Bravo J. Switala J. Ens W. Loewen P.C. Fita I. J. Biol. Chem. 1999; 274: 27717-27725Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). of the were using S. J. Mol. 1993; PubMed Scopus Google Scholar). The structure and have been to the Protein the for for for and for of Changes in Val169 8 Å from the conserved valine is situated in the main channel of all catalases 8 Å from side chain a or of the channel to a of Å that larger and H2O2 from access to the active site this valine to in yeast catalase CATA (7Maté M.J. Zamocky M. Nykyri L.M. Herzog C. Alzari P.M. Betzel C. Koller F. Fita I. J. Mol. Biol. 1999; 286: 135-139Crossref PubMed Scopus (92) Google Scholar) allowed an in activity with the that valine access of larger to the active site. the valine to in both CATA and HPII also caused a in catalase leading to the that the or of the channel were in the of H2O2 movement into the active site. the for hydrogen between water and in the variant (12Carpena X. Soriano M. Klotz M.G. Duckworth H.W. Donald L.J. Melik-Adamyan W. Fita I. Loewen P.C. Proteins. 2003; 50: 423-436Crossref PubMed Scopus (46) Google Scholar) also the of the catalase the study of Val169, the of larger side chains at this in HPII was investigated with the of the and and characterization of variants and that are of wild type lower those of the and with the larger side chains with substrate access to the active site. from heme to heme d was the lower The variant not was affected by the larger side chain, and the was activity of catalase variants and heme not determined not determined Open table in a new tab of Changes to Asp181 12 Å from the conserved aspartate is in the main channel of all catalases 12 Å from the The role of this has not been investigated in and katE was to the HPII variants and variants and all between and of wild type that the of activity was of side chain or to hydrogen with adjacent the D181E variant wild type of revealing that the presence of a negatively charged side chain at this is for the with the the variant not the side chain with The crystal structures of and D181E were determined to into Asp181 catalysis The structures of the variants from the structure of HPII in the of the and more with in the number and of in the main channel and active site (Fig. In the active site cavity, the common to all inactive variants, and is the of water 1, the sixth ligand of the heme, water is in the active of all In are in the channels of the less active variants in Å from the the enzyme and the active variant D181E have water at most in the channel. In position 1, the sixth ligand to the heme, is in of of HPII and in all of the hydrophobic portion of the channel Val169 is at in of HPII and at and in all of occupancy in the main or perpendicular channel of catalase HPII listed as B B B B B B Open table in a new tab are some in the of in the of the side chain at These are most in the variant the side chain a different The two that are adjacent to and with the side chain of Asp181 at and are in all variants, although are Å in that the interaction with Asp181 is not for The the heme, I, and are conserved in all catalases for which structures have been determined and are as of Changes to is a highly conserved in catalases, and it is adjacent to Asp181, its side chain is oriented from the channel (Fig. 1) and is situated Å from the determine the of residues in this region of the enzyme was or katE was to variants and Both presented wild type and heme d that other residues in the of Asp181 not as an on with the of from of a of catalase high for substrate hydrogen peroxide at the turnover rates in of The of be in by the active site heme deeply buried the β-barrel core of the of the substrate through 30–50 Å of channels substrate movement to the active site and the of the product water and oxygen back to the of the without with substrate dynamic the of hydrogen peroxide the enzyme through the main channel not on the route of product (18Kalko S.G. Gelpi J.L. Fita I. Orozco M. J. Am. Chem. Soc. 2001; 123: 9665-9672Crossref PubMed Scopus (46) Google Scholar, 19Amara P. Andreoletti P. Jouve H.M. Field M.J. Protein Sci. 2001; 10: 1927-1935Crossref PubMed Scopus (45) Google Scholar). molecular interaction potential carried out on CATA suggest that the substrate in the active site oriented for interaction with the heme iron and side chains of the and side chains (18Kalko S.G. Gelpi J.L. Fita I. Orozco M. J. Am. Chem. Soc. 2001; 123: 9665-9672Crossref PubMed Scopus (46) Google Scholar). The that a negatively charged side chain in the main channel of HPII water occupancy in the access particularly at the sixth ligand position and enzyme activity a into the of the catalase Both be in of an electrical potential between the negatively charged and the positively charged heme iron, which the orientation of with an electrical through the channel. the of the sixth ligand water in the position as substrate on a direct between the and heme (Fig. the electrical potential state The in H2O2 are by an of when the in an or structure. an to the bond of H2O2 to Scholar). The structure to an electrical of larger the of water and hydrogen peroxide Asp181 in the main channel be affected by the electrical potential and be into an orientation with the the heme iron (Fig. oxygen of H2O2 with the heme iron, in the active site hydrogen bond of the of the active site and hydrogen of the of the active site orientation of the of H2O2 in the potential field a to the from molecular dynamic studies that substrate enter the active site in a the occupancy of water in the channel of the enzyme and the D181E with the other less active Asp181 variants, be to the electrical potential on the dipoles of the to a of with common the formation of a hydrogen In the hydrophobic portion of the channel between Asp181 and have other to hydrogen bond and the bond and are for hydrogen (Fig. The orientation of the by the electrical potential be in the in the channel. be is the in between the channels of D181E and the One is that it is an of the the of the two are and are other between the two this it to that water occupancy in the hydrophobic portion of the channel is not a for catalase that water the channel if which not are a for in the channel and on and involves more the of the hydrophobic region C.D. Arvai A.S. Bourne Y. Tainer J.A. J. Mol. Biol. 1999; 296: 295-309Crossref Scopus (339) Google Scholar). a the and of the hydrophobic region must in with for substrate and have an role in

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