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An Electrical Potential in the Access Channel of Catalases Enhances Catalysis

2003; Elsevier BV; Volume: 278; Issue: 33 Linguagem: Inglês

10.1074/jbc.m304076200

1083-351X

Prashen Chelikani, X. Carpena, Ignacio Fita, P.C. Loewen,

Microfluidic and Capillary Electrophoresis Applications

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 strain UM255 transformed with plasmids pAMkatE72, pD181A, pD181S, pD181Q, pD181N, and pD181E, encoding HPII or the Ala181, Ser181, Gln181, Asn181, and Glu181 variants, respectively, were grown in Luria broth containing 10 g/liter tryptone, 5 g/liter yeast extract, and 5 g/liter NaCl. Growth of the mutant variants was for 16 h at 37 °C or 22 h at 28 °C and of the wild type HPII was for 16 h at 37 °C with shaking. The cells were harvested, and HPII was isolated as previously described (31Loewen P.C. Switala J. Biochem. Cell Biol. 1986; 64: 638-646Crossref PubMed Scopus (72) Google Scholar). Following resuspension of the ammonium sulfate fraction, the solution was heated at 50 °C for 15 min followed by centrifugation prior to chromatography on DEAE cellulose (Whatman). Crystallization, Data Collection, and Refinement—Crystals of the Asp181 variants of HPII were obtained at 22 °C using the hanging drop vapor diffusion method over a reservoir solution containing 15–17% PEG 3350 (Carbowax), 1.6–1.7 m LiCl (Baker), and 0.1 m Tris, pH 9.0. The crystals were monoclinic, space group P21 with one tetrameric molecule in the crystal asymmetric unit and had a solvent content of ∼40%. The diffraction data were obtained from crystals transferred to a solution containing 30% PEG 3350 and flash cooled with a nitrogen cryo-stream giving unit cell parameters listed in Table III. The diffraction data were autoindexed and integrated using programs DENZO and merged using SCALEPACK (32Otwinowski Z. Minor W. Methods Enzymol. 1996; 276: 307-326Crossref Scopus (38253) Google Scholar) (see Table III).Table IIIData collection and structural refinement statistics for the Asp181 variants of HPIID181AD181SD181QD181EData collection statisticsSpace groupP21P21P21P21Cella (Å)93.1193.3493.7693.38b (Å)132.50132.88133.13132.86c (Å)121.49121.45122.50122.04β0109.5109.4109.5109.5Resolution range (Å)29.8-2.4 (2.49-2.4)aValues in parentheses correspond to the highest resolution shell.29.8-2.2 (2.27-2.2)29.8-1.65 (1.71-1.65)29.9-1.8 (1.86-1.8)Unique reflections (F > 0)105,578 (7,994)138,759 (11,109)323,591 (23,749)251,911 (20,645)Completeness (%)96.8 (88.8)99.6 (99.5)96.0 (79.4)99.2 (98.0)I/σ(I)9.88.510.67.5R sym (%)bR sym = Σhkl Σj|Ihklj— 〈Ihkl 〉|/Σhkl〈Ihkl 〉.13.510.010.79.6Refinement statisticsWorking set100,246 (7,579)131,677 (10,519)307,191 (22,578)239,324 (19,579)Free reflections5,332 (415)6,965 (590)16,400 (1,171)12,587 (1,066)R cryst (%)cR cryst = Σ∥F obs| — |F calc∥/Σ|F obs|. R free is as for R cryst but calculated for a test set comprising reflections not used in the refinement (5%).14.4 (18.7)15.2 (17.4)17.4 (23.3)17.7 (23.2)R free (%)22.5 (27.8)21.6 (25.2)20.6 (26.7)21.8 (26.5)No. of non-hydrogen atomsProtein22,97222,97622,98822,988Water2,7672,7333,2213,072Heme172172172176Root mean square deviation from idealityBond lengths (Å)0.0120.0120.0080.011Bond angles (deg.)2.42.01.61.6Peptide planarity (Å)0.0240.0230.0200.022Aromatic planarity (Å)0.0120.0120.0070.010Est. coordinate error (Luzzati) (Å)0.200.180.170.18Averaged B factor (Å2)Main chain26.222.218.216.3Side chain26.222.719.517.1Water31.929.631.226.a Values in parentheses correspond to the highest resolution shell.b R sym = Σhkl Σj|Ihklj— 〈Ihkl 〉|/Σhkl〈Ihkl 〉.c R cryst = Σ∥F obs| — |F calc∥/Σ|F obs|. R free is as for R cryst but calculated for a test set comprising reflections not used in the refinement (5%). Open table in a new tab Structure determination was carried out with the program MOLREP using native HPII as the initial searching model. Refinements were completed using the program REFMAC (33Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13712) Google Scholar) with solvent molecules modeled with the program WATPEAK (34Collaborative Computational Project Number 4Acta Crystallogr. Sect. A. 1994; 50: 760-763Crossref PubMed Scopus (19668) Google Scholar) and manually with the graphics program O (35Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (12999) Google Scholar). Solvent molecules were only introduced when they corresponded to the strongest peaks in the difference Fourier maps that could make at least one hydrogen bond with atoms already in the model. In the final rounds of refinement the four subunits were treated independently with the bulk solvent correction applied and the whole resolution range available used for each variant. The analysis of solvent accessibility and molecular cavities was carried out with program VOIDOO (36Kleywegt G.J. Jones T.A. Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 178-185Crossref PubMed Scopus (970) Google Scholar) using a reduced atomic radius for polar atoms in accounting for possible hydrogen bonds (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). All of the figures were prepared using SETOR (37Evans S. J. Mol. Graphics. 1993; 11: 134-138Crossref PubMed Scopus (1249) Google Scholar). The structure factors and coordinates have been submitted to the Protein Data Bank under the accession numbers 1P7Y for D181A, 1P7Z for D181S, 1P80 for D181Q, and 1P81 for D181E. Effect of Changes in Val169 Situated 8 Å from the Heme—A conserved valine is situated in the main channel of all catalases approximately 8 Å from heme. Its side chain causes a constriction or narrowing of the channel to a diameter of approximately 3 Å that prevents any molecules much larger than H2O and H2O2 from gaining access to the active site heme. Changing this valine to alanine 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 increase in peroxidatic activity consistent with the concept that valine restricted access of larger molecules to the active site. Counterintuitively, the valine to alanine change in both CATA and HPII also caused a decrease in catalase activity, leading to the conclusion that the dimensions or volume of the channel were critical in determining the rate of H2O2 movement into the active site. Introducing the possibility for hydrogen bonding between water and protein in the V169S 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 decreased the efficiency of the catalase reaction. To expand the study of Val169, the effect of larger side chains at this location in HPII was investigated with the construction of the V169I, V169F, and V169W variants. Purification and characterization of variants V169I and V169F revealed activities that are 10–15% of wild type (Table II), even lower than those of the V169A and V169S, consistent with the larger side chains interfering with substrate access to the active site. Heme conversion from heme b to heme d was normal despite the lower catalytic rate. The V169W variant did not accumulate protein, presumably because protein folding was adversely affected by the larger side chain, and the nascent protein was proteolyzed.Table IISpecific activity of purified catalase variants and heme compositionVariantSpecific activityHemeunits/mgWild type19,100 ± 900dV169I3,730 ± 400dV169F1,470 ± 120dV169WNDaND, not determined because no protein accumulated.R180A11,200 ± 1,510dR180K22,700 ± 1,650dD181A810 ± 60bD181S2,550 ± 190bD181E21,900 ± 700dD181Q1,770 ± 50bD181N2,800 ± 400bD181I2,330 ± 350bD181WNDa ND, not determined because no protein accumulated. Open table in a new tab Effect of Changes to Asp181 Situated 12 Å from the Heme—A conserved aspartate is present in the main channel of all catalases approximately 12 Å from the heme. The role of this residue has not been investigated in any catalase, and katE was mutated to express the HPII variants D181A, D181S, D181Q, D181N, D181I, D181E, and D181W (Table I). Purified variants D181A, D181S, D181Q, D181N, and D181I all exhibited between 5 and 15% of wild type activity, indicating that the loss of activity was independent of side chain size or ability to form hydrogen bonds with adjacent waters. Significantly, the D181E variant retained wild type levels of activity, revealing that the presence of a negatively charged side chain at this location is critical for the catalytic process. As with the V169W variant, the D181W variant did not accumulate protein, presumably because the large side chain interfered with folding. The crystal structures of D181A, D181S, D181Q, and D181E were determined to provide insight into how Asp181 influenced catalysis (Table III). The structures of the four variants differed from the structure of native HPII only in the immediate vicinity of the changed residue and more distantly with changes in the number and location of solvent molecules in the main channel and active site (Fig. 2). In the active site cavity, the feature common to all three inactive variants, D181A, D181S, and D181Q, is the absence of water 1, the sixth ligand of the heme, whereas nearby water 2 is present in the active sites of all variants. In addition, there are fewer waters in the channels of the less active variants generally (Table IV), even in positions over 20 Å away from the heme. By contrast, the native enzyme and the active variant D181E have water present at most positions in the channel. In particular, position 1, the sixth ligand to the heme, is occupied in three of four subunits of HPII and in all four subunits of D181E. Even the constricted, hydrophobic portion of the channel near Val169 is occupied at 3B in one subunit of HPII and at 3A and 3B in all four subunits of D181E.Table IVWater occupancy in the main or perpendicular channel of catalase HPII subunits listed as B factors (Å2)Water #Subunit ASubunit BSubunit CSubunit DHPII (1.9 Å; 1GGE) average B factor: 9.2 (protein), 17.7 (water)140.028.142.7215.317.416.312.93b32.3424.020.522.529.9530.741.229.027.9610.711.614.810.4728.031.133.324.187.710.918.66.697.19.412.04.71015.317.114.813.41116.718.823.710.9128.819.514.813.81319.335.517.51433.631.325.31530.130.1I3.77.14.86.2II5.16.15.07.1III4.08.96.48.1D181A (2.4 Å) average B factor: 26.2 (protein), 31.9 (water)216.720.022.121.646.18.115.111.5544.128.831.331.4625.434.328.032.5921.818.525.420.01037.025.022.439.01143.01248.934.133.435.01339.945.847.844.31538.0I19.321.213.313.5II17.819.918.812.2III15.515.517.117.2D181E (1.8 Å) average B factor: 16.7 (protein), 26.2 (water)135.143.444.246.1226.524.928.429.43a44.669.639.543.73b34.030.232.125.4435.047.542.341.7537.836.737.732.7616.017.020.116.5820.722.837.317.4912.615.818.114.91026.633.628.924.11124.828.233.720.411b30.135.81219.220.921.323.71327.530.91436.237.448.928.41534.448.9I10.410.39.714.1II9.011.514.07.9III11.713.610.713.3D181Q (1.65 Å) average B factor: 18.8 (protein), 31.2 (water)214.117.918.114.6418.020.924.420.6522.827.427.024.9618.121.218.918.2823.524.327.422.31037.135.339.139.61119.422.421.418.711b23.023.422.911c22.921.825.120.21219.625.924.024.512b30.322.927.431.21325.926.823.526.61434.431.61556.238.6I13.515.312.911.9II11.311.514.213.7II14.314.214.613.2D181S (2.2 Å) average B factor: 22.5 (protein), 29.6 (water)222.918.820.616.0413.914.221.313.94b25.123.226.625.6539.047.553.954.9625.327.233.919.7833.232.237.032.1916.513.716.110b44.840.838.41134.731.340.11244.137.31343.846.81444.1I12.013.613.812.5II10.716.415.511.2III11.710.711.513.5 Open table in a new tab As expected there are some changes in the positions of waters in the vicinity of the modified side chain at residue 181. These changes are most extensive in the D181Q variant where the nearby Gln233 side chain unexpectedly adopts a different conformation. The two waters that are adjacent to and interacting with the side chain of Asp181 at positions 4 and 5 are present in all variants, although they are shifted approximately 0.5 Å in D181A/S/Q, indicating that the interaction with Asp181 is not required for their presence. The three waters surrounding the heme, labeled I, II, and III, are conserved in all catalases for which structures have been determined and are included as controls. Effect of Changes to Arg180—Arg180 is a another highly conserved residue in catalases, and even though it is adjacent to Asp181, its side chain is oriented away from the channel (Fig. 1) and is situated almost 20 Å from the heme. To determine whether the influence of residues in this region of the enzyme was general or specific, katE was mutated to express variants R180A and R180K. Both presented wild type activities and heme d (Table II), indicating that other residues in the vicinity of Asp181 did not exert as great an influence on catalysis. Even with the wealth of knowledge accumulated from over 100 years of investigation, a clear understanding of how catalase maintains high selectivity for substrate hydrogen peroxide while at the same time exhibiting turnover rates in excess of 106/s remains elusive. The issue of selectivity can be explained in part by the active site heme being deeply buried within the β-barrel core of the subunit, necessitating passage of the substrate through 30–50 Å of narrow channels. Such narrow channels might conceivably hinder substrate movement to the active site and creates the conceptual problem of how the product water and oxygen find their way back to the surface of the protein without interfering with substrate ingress. Molecular dynamic simulations support the concept of hydrogen peroxide entering the enzyme through the main channel but do not agree on the route of product exhaust (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). Furthermore, classical molecular interaction potential calculations carried out on CATA suggest that the substrate arrives in the active site properly oriented for interaction with the heme iron and side chains of the catalytic His and Asn 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 demonstration that a negatively charged side chain in the main channel of HPII enhances water occupancy in the access channel, particularly at the sixth ligand position and coincidently enhances enzyme activity provides a valuable insight into the mechanism of the catalase reaction. Both observations can be explained in terms of an electrical potential between the negatively charged carboxylate and the positively charged heme iron, which will influence the orientation of any molecule with an electrical dipole passing through the channel. Furthermore, given the location of the sixth ligand water in roughly the same position as substrate H2O2, on a direct line between the carboxylate and heme irons (Fig. 3), the electrical potential may influence transition state formation. The hydrogens in H2O2 are separated by an angle of approximately 110° when viewed along the O–O axis resulting in an asymmetric or skewed structure. Orbital interactions present an energy barrier to rotation around the O–O bond of approximately 2.5 kcal/mol, limiting H2O2 to one predominant conformation (38Schumb W.C. Satterfield C.N. Wentworth R.L. Hydrogen Peroxide. Reinhold, New York1955: 310-353Google Scholar). The structure gives rise to an electrical dipole of 2.3 Debye, just slightly larger than the 1.9 Debye dipole of water. Consequently, water and hydrogen peroxide passing Asp181 in the main channel will be affected by the electrical potential and be forced into an orientation with the oxygens directed toward the heme iron (Fig. 4). When oxygen O-1 of H2O2 becomes associated with the heme iron, spatial constraints in the active site fix H-1 within hydrogen bond distance of the imidazole ring of the active site histidine and O-2 within hydrogen bonding distance of the NH2 of the active site asparagine. Thus, orientation of the dipole of H2O2 in the potential field presents a simple mechanism to explain the prediction arising from molecular dynamic studies that substrate molecules enter the active site in a preferred orientation. Similarly, the greater occupancy of water in the channel of the native enzyme and the D181E variant, compared with the other less active Asp181 variants, can be attributed to the electrical potential acting on the dipoles of the solvent to create a population of waters with common orientation, thereby favoring the formation of a hydrogen bonded matrix. In the hydrophobic portion of the channel between Asp181 and His128, waters have only other waters to hydrogen bond with, and the bond lengths separating waters 2, 3a, 3b, and 4 are longer than optimal for strong hydrogen bonds (Fig. 3). The favorable orientation of the molecules induced by the electrical potential may be critical in stabilizing the solute matrix in the channel. What cannot be satisfactorily explained is the difference in occupancies between the channels of D181E and the native enzyme. One possibility is that it is simply an artifact of the refinement process, but the resolutions of the two data sets are approximately equivalent, and there are few other differences between the two variants. At this juncture, it seems reasonable to conclude that water occupancy in the hydrophobic portion of the channel is not a prerequisite for catalase activity, but that water can occupy the channel if certain subtle conditions, which we do not yet fully understand, are fulfilled. Certainly a complete explanation for waters in the channel and their impact on reactivity and solute selectivity involves more than simply the length of the hydrophobic region (4Putnam C.D. Arvai A.S. Bourne Y. Tainer J.A. J. Mol. Biol. 1999; 296: 295-309Crossref Scopus (339) Google Scholar). At a minimum, the volume and shape of the hydrophobic region must work in concert with electrostatic influences for optimum substrate access, and electrostatic effects have an additional key role in catalysis.