Conformational Mobility in the Active Site of a Heme Peroxidase

Abstract

Conformational mobility of the distal histidine residue has been implicated for several different heme peroxidase enzymes, but unambiguous structural evidence is not available. In this work, we present mechanistic, spectroscopic, and structural evidence for peroxide- and ligand-induced conformational mobility of the distal histidine residue (His-42) in a site-directed variant of ascorbate peroxidase (W41A). In this variant, His-42 binds “on” to the heme in the oxidized form, duplicating the active site structure of the cytochromes b but, in contrast to the cytochromes b, is able to swing “off” the iron during catalysis. This conformational flexibility between the on and off forms is fully reversible and is used as a means to overcome the inherently unreactive nature of the on form toward peroxide, so that essentially complete catalytic activity is maintained. Contrary to the widely adopted view of heme enzyme catalysis, these data indicate that strong coordination of the distal histidine to the heme iron does not automatically undermine catalytic activity. The data add a new dimension to our wider appreciation of structure/activity correlations in other heme enzymes. Conformational mobility of the distal histidine residue has been implicated for several different heme peroxidase enzymes, but unambiguous structural evidence is not available. In this work, we present mechanistic, spectroscopic, and structural evidence for peroxide- and ligand-induced conformational mobility of the distal histidine residue (His-42) in a site-directed variant of ascorbate peroxidase (W41A). In this variant, His-42 binds “on” to the heme in the oxidized form, duplicating the active site structure of the cytochromes b but, in contrast to the cytochromes b, is able to swing “off” the iron during catalysis. This conformational flexibility between the on and off forms is fully reversible and is used as a means to overcome the inherently unreactive nature of the on form toward peroxide, so that essentially complete catalytic activity is maintained. Contrary to the widely adopted view of heme enzyme catalysis, these data indicate that strong coordination of the distal histidine to the heme iron does not automatically undermine catalytic activity. The data add a new dimension to our wider appreciation of structure/activity correlations in other heme enzymes. The heme peroxidase enzymes catalyze H2O2-dependent oxidation of a range of substrates through a mechanism that, in all cases, involves formation of an oxidized ferryl intermediate (known as Compound I; see Equation 1) that is subsequently reduced by substrate (Equations 2 and 3) (1Everse J. Everse K.E. Grishham M.B. Peroxidases in Chemistry and Biology. Vol. 1 and 2. CRC Press, Boca Raton, FL1991Google Scholar, 2Dunford H.B. Heme Peroxidases. John Wiley, Chichester, United Kingdom1999Google Scholar). In the majority of cases, reduction of Compound I occurs by two successive single-electron transfer steps, as follows (where P = peroxidase, HS = substrate, S· = 1-electron oxidized form of substrate). P+H2O2k1→Compound I+H2O(Eq. 1) Compound I+HSk2→Compound II+S⋅ (Eq. 2) Compound II+HSk3→P+S⋅+H2O(Eq. 3) Structural information is available for a number of heme peroxidase enzymes, and in all cases the heme iron is poised in a 5- or 6-coordinate environment with the sixth ligand provided by a weakly coordinated water molecule. This differs from the heme coordination geometry in other noncatalytic heme proteins that do not require binding of an exogenous ligand at the metal site. The classic example is the cytochromes, which typically have a strong endogenous protein ligand at the sixth site (usually His or Met) and no vacant coordination site for iron-catalyzed chemistry to occur. The prevailing view that has emerged, therefore, is that the catalytic enzymes (which include the heme peroxidases but also embraces other, more complex heme enzymes such as heme oxygenase, the cytochrome P450s, and cytochrome c oxidase) usually contain 5-coordinate or weakly 6-coordinate heme groups that allow facile reaction with substrate, whereas the electron transfer proteins, for example the cytochromes, have no vacant site at the metal ion for catalysis to occur. In line with the above considerations, there are no known examples of a genuine heme peroxidase with bis-histidine ligation, but there are a few examples in the literature of different heme peroxidases, or site-directed variants thereof, in which coordination of the distal histidine residue has been proposed on the basis of spectroscopic studies (3Cheek J. Mandelman D. Poulos T.L. Dawson J.H. J. Biol. Inorg. Chem. 1999; 4: 64-72Crossref PubMed Scopus (35) Google Scholar, 4Sutherland G.R.J. Zapanta S. Tien M. Aust S.D. Biochemistry. 1997; 36: 3654-3662Crossref PubMed Scopus (69) Google Scholar, 5Smulevich G. Miller M.A. Kraut J. Spiro T.G. Biochemistry. 1991; 30: 9546Crossref PubMed Scopus (130) Google Scholar, 6Vitello L.B. Erman J.E. Miller M.A. Mauro J.M. Kraut J. Biochemistry. 1992; 31: 11524-11535Crossref PubMed Scopus (74) Google Scholar, 7Turano P. Ferrer J.C. Cheesman M.R. Thomson A.J. Banci L. Bertini I. Mauk A.G. Biochemistry. 1995; 34: 13895-13905Crossref PubMed Scopus (31) Google Scholar, 8Youngs H.L. Moenne-Loccoz P. Loehr T.M. Gold M.H. Biochemistry. 2000; 39: 9994-10000Crossref PubMed Scopus (18) Google Scholar). These examples include: the W51A (7Turano P. Ferrer J.C. Cheesman M.R. Thomson A.J. Banci L. Bertini I. Mauk A.G. Biochemistry. 1995; 34: 13895-13905Crossref PubMed Scopus (31) Google Scholar) and D235N (6Vitello L.B. Erman J.E. Miller M.A. Mauro J.M. Kraut J. Biochemistry. 1992; 31: 11524-11535Crossref PubMed Scopus (74) Google Scholar) variants of cytochrome c peroxidase; thermally inactivated manganese peroxidase (4Sutherland G.R.J. Zapanta S. Tien M. Aust S.D. Biochemistry. 1997; 36: 3654-3662Crossref PubMed Scopus (69) Google Scholar); and manganese peroxidase at alkaline pH (8Youngs H.L. Moenne-Loccoz P. Loehr T.M. Gold M.H. Biochemistry. 2000; 39: 9994-10000Crossref PubMed Scopus (18) Google Scholar). Removal of the ligands coordinating to the bound K+-site in ascorbate peroxidase also leads to formation of a low-spin species (3Cheek J. Mandelman D. Poulos T.L. Dawson J.H. J. Biol. Inorg. Chem. 1999; 4: 64-72Crossref PubMed Scopus (35) Google Scholar). In none of these cases has unambiguous structural information been obtained, however. In this work, we present the first crystallographically defined example of a functional peroxidase enzyme with bis-histidine ligation in the W41A variant of ascorbate peroxidase. This variant duplicates the heme coordination geometry of the cytochromes b in the oxidized form but remains fully competent for formation of the catalytic Compound I and Compound II intermediates, as well as for substrate oxidation, by means of a reaction mechanism in which a conformationally mobile ligand (His-42) binds “on” and then swings “off” the iron during catalysis. This switch between the on and off forms is triggered by reaction with hydrogen peroxide (or other ligands) and, under catalytic conditions, is fully reversible, allowing essentially complete activity to be maintained. These data indicate that strong coordination of the distal histidine residue to the heme iron does not automatically undermine peroxidase activity. Materials—l-Ascorbic acid (Aldrich), guaiacol (Sigma), and the chemicals used for buffers (Fisher) were of the highest analytical grade (more than 99% pure) and were used without further purification. Hydrogen peroxide solutions were freshly prepared by dilution of a 30% (v/v) solution (BDH Chemicals); exact concentrations were determined using the published absorption coefficient (ϵ240 = 39.4 m1- cm-1 (9Nelson D.P. Kiesow L.A. Anal. Biochem. 1972; 49: 474-478Crossref PubMed Scopus (826) Google Scholar)). Aqueous solutions were prepared using water purified through an Elgastat Option 2 water purifier, which itself was fed with deionized water. All pH measurements were made using a Russell pH-electrode attached to a digital pH-meter (Radiometer Copenhagen, model PHM 93). Mutagenesis and Protein Purification—Site-directed mutagenesis on recombinant soybean cytosolic APX (rsAPX) 2The abbreviations used are: APX, ascorbate peroxidase; rsAPX, recombinant soybean cytosolic ascorbate peroxidase; sh, shoulder; MALDI, matrix-assisted laser desorption ionization; TOF, time-of-flight. was performed according to the QuikChange™ protocol (Stratagene Ltd., Cambridge, UK). Two oligonucleotides encoding the desired mutation were synthesized and purified (Invitrogen). For W41A, the primers were: 5′-GCTCCGTTTGGCAGCGCACTCTGCTGGAACC-3′ (forward primer) and 3′-GGTTCCAGCAGAGTGCGCTGCCAAACGGAGC-5′ (reverse primer). DNA sequencing of the entire coding region using an Applied Biosystems 3730 DNA analyzer was used to confirm the desired mutation and the absence of spurious mutations. Bacterial fermentation of cells and purification of rsAPX and W41A was carried out according to published procedures (10Metcalfe C.L. Ott M. Patel N. Singh K. Mistry S.C. Goff H.M. Raven E.L. J. Am. Chem. Soc. 2004; 126: 16242-16248Crossref PubMed Scopus (40) Google Scholar, 11Lad L. Mewies M. Raven E.L. Biochemistry. 2002; 41: 13774-13781Crossref PubMed Scopus (72) Google Scholar). Enzyme purity was assessed by examination of the Asoret/A280 value; in all cases an Asoret/A280 value of >1.7 for rsAPX and W41A was considered pure. Enzyme purity was also assessed using SDS-PAGE, and the preparations were judged to be homogeneous by the observation of a single band on a Coomassie Blue-stained reducing SDS-polyacrylamide gel. Enzyme concentrations for the W41A mutant were determined using the pyridine hemochromagen method (12Antonini M. Brunori E. Hemoglobin and Myoglobin and Their Reactions with Ligands. North Holland Publishers, Amsterdam1971Google Scholar); an absorption coefficient of ϵ405 = 125 mm-1 cm-1 was determined for W41A. Enzyme concentrations of rsAPX were determined using the ϵ407 = 107 mm-1 cm-1 (13Jones D.K. Dalton D.A. Rosell F.I. Lloyd Raven E. Arch. Biochem. Biophys. 1998; 360: 173-178Crossref PubMed Scopus (40) Google Scholar). Mass Spectrometry—The integrity of the tryptophan mutant was confirmed by MALDI-TOF mass spectrometry. A 10 pmol/μl stock of the W41A mutant was made up in water and 0.5 μl of the 1:1 protein/matrix mixture (sinapinic acid (5 mg/ml) in 1:1 acetonitrile/water, 0.1% trifluoroacetic acid) was spotted onto a MALDI target plate using the drying droplet method. The MALDI-TOF mass spectrometer (Applied Biosystems) was calibrated in the range of 5000 to 35,000 Da with a protein mass standard kit (Sequazyme, Applied Biosystems). Spectra were accumulated in the same mass range using an average of at least 250 laser shots. The spectra were analyzed using Data Explorer software (Applied Biosystems). The MALDI-TOF mass spectrum of W41A gives a mass of 28,209.36 Da mass for W41A = that no has measurements pH = = were carried out according to published 1991; PubMed Scopus Google Scholar). of ascorbate was at = mm-1 K. Scopus Google Scholar) and were by a of 2 to for the of the to ascorbate and E. Google Scholar). For oxidation of stock solutions were prepared in 30% M. J. Biochem. PubMed Scopus Google and oxidation to was at = mm-1 cm-1 M. J. Biochem. PubMed Scopus Google Scholar)). for were by the of activity by the of the for were determined by a of the data to the using a All are the of measurements were performed using an (Applied with a water of are an average of at least were in all All data were analyzed using least on an (Applied using All was performed using the software for the formation of Compound I were by a in to the formation of Compound at for rsAPX and for W41A in single by enzyme with concentrations of absorption studies were carried out using a and software (Applied was performed by and using software (Applied spectra = were in = and spectra were on a and to a and with an from and a from and of rsAPX and the W41A mutant were using the published procedures Mewies M. Raven E.L. Biol. PubMed Scopus Google Scholar). of the forms of rsAPX and the W41A mutant were by a few of in 10 of pH and of the heme were by in in the by the of of which with to D. K. M.A. J. Biol. Chem. 2000; PubMed Scopus Google Scholar). For of W41A, were in for and then in The heme coordination geometry in cytochrome c peroxidase is known to be to the of see for 6Vitello L.B. Erman J.E. Miller M.A. Mauro J.M. Kraut J. Biochemistry. 1992; 31: 11524-11535Crossref PubMed Scopus (74) Google Scholar, 7Turano P. Ferrer J.C. Cheesman M.R. Thomson A.J. Banci L. Bertini I. Mauk A.G. Biochemistry. 1995; 34: 13895-13905Crossref PubMed Scopus (31) Google Scholar, and J.C. P. Banci L. I. M. Mauk A.G. Biochemistry. PubMed Scopus Google Scholar, L.B. M. Erman J.E. Biochemistry. PubMed Scopus Google Scholar, L.B. Erman J.E. Miller M.A. J. Kraut J. Biochemistry. PubMed Scopus Google Scholar, J.E. L.B. Miller M.A. Kraut J. Biochemistry. PubMed Scopus Google Scholar). These were not used in the also was not used in the data for W41A and the and forms were at using an whereas data for the was at using an all at the Data for the and were at at using a All data were at K. Data at the was and using on Protein 1992; Google Scholar) and Biol. PubMed Scopus Google and data at was and using the and 1997; Scholar). Data and are in of the data were for the of and from The were from a model from the complex Data by the of bound ligand and water of using Biol. 1997; PubMed Scopus Google Scholar) from the Biol. PubMed Scopus Google Scholar) and of the protein model using P. K. Biol. 2004; PubMed Scopus Google Scholar) by the of water were carried out the and In and data have been with the Protein Data and The of all are in and Protein Data for are in in a new Heme spectrum of the of W41A is in The spectrum cm-1 = differs from that of the enzyme = L. Mewies M. Raven E.L. Biochemistry. 2002; 41: 13774-13781Crossref PubMed Scopus (72) Google Scholar); 1) and a in the region that is with the of low-spin W41A variant in cytosolic APX was also not in this variant are but the band is to The at in the enzyme is in the variant evidence for the formation of heme was at alkaline pH not the low-spin species was as from coordination of an protein of W41A with noncatalytic ligands is to the proposed protein ligand is or bound to the of to the of W41A leads to a spectrum in which complete formation of low-spin heme is = This that of a strong exogenous ligand leads to of the the structure of the of W41A this we have that binds to the reduced heme = the structure of the also that His-42 is further evidence in of a low-spin heme species The spectrum of W41A is by a low-spin species with of and is to be A with of and is with an The low-spin species is the majority species at 10 and is with bis-histidine the are with an in which the are not to species in the spectrum of the which has a species = and and a of low-spin heme = and (13Jones D.K. Dalton D.A. Rosell F.I. Lloyd Raven E. Arch. Biochem. Biophys. 1998; 360: 173-178Crossref PubMed Scopus (40) Google Scholar, Thomson A.J. J. Am. Chem. Soc. Scopus Google Scholar). A low-spin species = as a on the = and the of = in W41A are to from a heme in which the are to other that there is also evidence for of the histidine in the structure of the form of see of W41A and and structure of the of W41A and 1) the made in In this structure which onto that for the form of rsAPX the iron is by the ligand and the of the bound ligand is to of His-42 in For the the electron for the residue is with two of this has to as for rsAPX the other has to the of in In the structure a single of is the data are to with and a of the histidine not be of the of the heme electron The structure of the of W41A also confirm that His-42 is In this and in contrast to of the hydrogen to means that the ligand two the nature of the low-spin heme in W41A, data to were and W41A is in structure to the the between for this structure and rsAPX is using Scopus Google Scholar)). The structure of W41A that by the is by two water 1 and 2 in the structure and of the active site structure is to rsAPX, there are in protein His-42 the of His-42 toward the heme in W41A such that of His-42 is with in of the to this as the “on” A further water in is in the region that was by The is essentially in W41A and rsAPX and This new histidine ligand a water that is to the iron in the protein This of the of His-42 toward the heme in the on form and the in heme geometry are the of the of the which the His-42 to to the of the electron for W41A His-42 that there is above His-42 and also to the of His-42 This with the of His-42 in rsAPX and is with the of a heme species in which His-42 is not are two of this the spectroscopic data above are also with heme in the in this the electron from a of and low-spin iron in the we used enzyme in our we that reduction of heme is during data J. 2002; PubMed Scopus Google Scholar). The spectrum of W41A = that are to for rsAPX = and are not with bis-histidine ligation, that of His-42 occurs on In this the electron from reduction of the heme during data were not able to data for the W41A the of the the is that the data indicate that His-42 is and more than toward data above that, the heme is 6-coordinate and low-spin in W41A two strong histidine His-42 is not bound to the heme and under to this form as the “off” this is the then reaction with and of substrate be there to our no unambiguous example of a crystallographically defined peroxidase in the was of to strong ligation, to that for example in the cytochromes b, reaction with for Compound I formation in W41A were determined under = The W41A variant was to be competent for formation of Compound I under these conditions, at a as by a in at These in for rsAPX L. Mewies M. Raven E.L. Biochemistry. 2002; 41: 13774-13781Crossref PubMed Scopus (72) Google Scholar). for this a on the of hydrogen peroxide This is in contrast to the data for rsAPX, in which a on is in the range = L. Mewies M. Raven E.L. Biochemistry. 2002; 41: 13774-13781Crossref PubMed Scopus (72) Google Scholar)). The is with a mechanism that a conformational of the proposed to be between the on and off to reaction with as in and = Compound In the of the be as follows A of these data for W41A to Equation for the of and the of In the reaction of W41A with was using Data a of were to a model as for rsAPX L. Mewies M. Raven E.L. Biochemistry. 2002; 41: 13774-13781Crossref PubMed Scopus (72) Google A is W41A, is Compound and is Compound Spectra for the Compound I = and Compound II = are in with for rsAPX = and for Compound I and = and this and 11Lad L. Mewies M. Raven E.L. Biochemistry. 2002; 41: 13774-13781Crossref PubMed Scopus (72) Google Scholar). of Compound I A for W41A occurs with an which is than for rsAPX = of Compound II occurs with for W41A and rsAPX = and These data indicate that reaction of W41A with leads to the formation of genuine Compound I and Compound II intermediates, as for rsAPX, and that conformational of the protein is during catalysis. toward W41A variant was also to be competent for oxidation of two of In to substrate APX is known to other substrates that are of the II and peroxidases E.L. PubMed Scopus Google Scholar). The binding site for these two of substrate ascorbate binds to the heme the Mewies M. Raven E.L. Biol. PubMed Scopus Google and substrates are to to the Raven E.L. Biochemistry. 2004; PubMed Scopus Google Scholar, Poulos T.L. Biochemistry. 2000; 39: PubMed Scopus Google Scholar). For ascorbate oxidation by W41A, data and the were = = = The for rsAPX were: = = = L. Mewies M. Raven E.L. Biochemistry. 2002; 41: 13774-13781Crossref PubMed Scopus (72) Google Scholar) rsAPX, data for oxidation of ascorbate not and these data were to the to for and L. Mewies M. Raven E.L. Biochemistry. 2002; 41: 13774-13781Crossref PubMed Scopus (72) Google Scholar). For oxidation of guaiacol by W41A of the data to the gives = 2 = and = mm-1 = = = mm-1 L. Mewies M. Raven E.L. Biochemistry. 2002; 41: 13774-13781Crossref PubMed Scopus (72) Google Scholar)). ascorbate oxidation is by the but for guaiacol an in of the enzyme for the substrate was This from the of a that has been by of the that has also been implicated in binding of the substrate acid to the Raven E.L. Biochemistry. 2004; PubMed Scopus Google and these data further evidence for binding of substrates at this site. Structural for of His-42 during spectroscopic, and data above allow to be Removal of leads to conformational mobility the active site. This His-42 more mobile such that to the heme in the of W41A on The flexibility of His-42 means that from the heme under off for example in the of a strong exogenous ligand This switch between the on and off forms is also used during catalysis through a mechanism that involves of His-42 on reaction with and oxidation of substrate through the catalytic more information on the conformational mobility of His-42 during catalysis. we an in which of W41A were in for and then in The structure of W41A, of the active site is to W41A between is and to rsAPX but in with the W41A structure there are in His-42 His-42 swings off the iron to a that is to that in the rsAPX electron is above the iron in a region that for rsAPX Mewies M. Raven E.L. Biol. PubMed Scopus Google as well as for other peroxidases, has been as a water we have also this as from a water The between the iron and this distal water is to that in rsAPX of heme geometry in the structure of W41A, in which His-42 swings off the is different from the majority of low-spin species at or or from that in the structure of the For this we carried out solution on a to that of the to the nature of the heme species W41A was with from and the spectrum This spectrum evidence for formation of a Compound II as for W41A in the This Compound II species for for reduction by ascorbate a spectrum that was essentially to the low-spin heme species bis-histidine in the absence of ascorbate not the spectrum of Compound II to a species that the low-spin form but that a in the region of heme heme was This is as evidence for between the on and off forms and is with the data evidence for the of majority heme was under heme is rsAPX has a spectroscopic that is not for W41A. These data evidence for reversible between the on and off forms and are in with the mechanism proposed above and with the In the of these and no evidence for a heme was the data in are as from a Compound The at is than for other Compound II as M. Poulos T.L. Biochemistry. PubMed Scopus Google J. Biol. Inorg. Chem. 2002; PubMed Scopus Google Dawson J.H. H.B. 2004; PubMed Scopus Google J. 2002; PubMed Scopus Google which the that toward the on form, as has the spectroscopic, mechanistic, and data that of in ascorbate peroxidase leads to mobility of His-42 and indicate that this residue to the heme in the oxidized The are in Contrary to strong coordination of His-42 to the iron to form a heme does not reaction with His-42 is conformationally mobile and between on and off forms to allow reaction with peroxide to In this W41A is able to as a peroxidase, substrate binding at the and the is in peroxidase structure that mobility of through of the hydrogen between His-42 and or by of in the heme to cytochrome of on the distal by of with a more have the same by His-42 toward the These a new on the widely view of heme enzyme catalysis in which strong ligation, as in the cytochromes, is to reaction with ligands at the heme that reversible coordination of the distal histidine is also in 2002; Scholar) and Chem. 39: PubMed Scopus Google that a mechanism for activity be used more widely in other, more complex heme enzymes. Singh for are to Thomson and Cheesman for the of and to for of mass and are also for with MALDI data also and for data and for with