Horseradish peroxidase monitored by infrared spectroscopy:e¡ect of temperature, substrate and calcium

Andras D. Kaposi a;*, Judit Fidy a, Eric S. Manas b, Jane M. Vanderkooi b,Wayne W. Wright b

a Institute of Biophysics and Radiation Biology, Semmelweis University of Medicine, Puskin u. 9, Budapest H-1088, Hungaryb Johnson Research Foundation, Department of Biochemistry and Biophysics, School of Medicine, University of Pennsylvania,

Philadelphia, PA 19104, USA

Received 22 June 1999; received in revised form 17 September 1999; accepted 20 September 1999

Abstract

Horseradish peroxidase was examined as a function of Ca and substrate binding using infrared spectroscopy in thetemperature range of 10^300 K. The Ca complex could be identified by the carboxylate stretches. The amide peak positionsindicate that the protein remains stable from room temperature to 10 K. Shifts in these peaks are consistent with increasedhydrogen bonding as temperature decreases, but the protein conformation is maintained at cryogenic temperatures. Thesubstrate, benzohydroxamic acid, produced no detectable change in the infrared spectrum, consistent with X-raycrystallography results. With removal of Ca, the protein maintained its overall helicity. ß 1999 Elsevier Science B.V. Allrights reserved.

Keywords: Hydrogen bonding; Benzohydroxamic acid; Alpha helix

1. Introduction

Proteins have been examined by many spectro-scopic techniques, and the information one wishesto obtain includes structure, bonding strengths, ki-netics of conformational changes, characterizationof binding sites and so on. In this study we are ex-amining the conformation of horseradish peroxidase(HRP) by infrared (IR) spectroscopy. IR spectro-scopy allows one to detect changes in protein con-formation, while at the same time monitoring the

solvent conditions. IR has long been used to examineH bonding in amide groups [1] and peak positionshelp to identify K and L structures in proteins [2^5].As presently conventionally applied, IR spectroscopyis useful for detecting large changes within the pro-tein.

HRP is an enzyme that catalyzes the H2O2-de-pendent oxidation of many aromatic electron donormolecules. Recent crystallographic work has eluci-dated the structure of HRP isoenzyme C [6] andthe structure of its substrate complex with benzohy-droxamic acid (BHA) [7]. The protein contains asingle heme group, and two Ca binding domains.In the absence of Ca, its activity is reduced [8,9].Since the Ca does not directly participate in the re-action, its role is likely to be structural. In this work,

0167-4838 / 99 / $ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved.PII: S 0 1 6 7 - 4 8 3 8 ( 9 9 ) 0 0 2 0 6 - X

Abbreviations: BHA, benzohydroxamic acid; HRP, horserad-ish peroxidase; IR, infrared

* Corresponding author. Fax: +36-1-266-6656;E-mail : kaposi@puskin.sote.hu

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www.elsevier.com/locate/bba

we examine whether there are changes in the proteinin the presence of Ca, and the substrate. We examinethe spectra over a range in temperature from roomtemperature to 10 K. The temperature excursion is ofinterest in protein studies because it is well knownthat H-bonding increases as temperature decreases[10,11], and therefore temperature shifts in the spec-tra can help to identify vibrational modes arisingfrom H-bonded groups that are free to changebond strength with temperature. Furthermore,heme proteins, including HRP, have been extensivelystudied at low temperature because spectroscopy ofthe optical centers often becomes more resolved atlow temperature [12^14]. In particular, our work onmetal substituted HRP has relied on the resolutiona¡orded at low temperature to monitor conforma-tional distributions and substrate interactions [15^18]. For such studies, IR measurements provide in-dependent evidence to indicate whether the cyrogenictemperature required alters the structure of the pro-tein, information important to ascertain the rele-vance of low temperature studies.

2. Materials and methods

2.1. Materials

Water was deionized and then glass distilled. Per-deuterated glycerol and benzohydroxamic acid wereobtained from Sigma (St. Louis, MO). Other materi-als were the highest purity commercially available.

HRP (RZ = 3.0, where this value, Reinheitzahl, isthe ratio of absorbances at 402 and 280 nm) waspurchased from Sigma. To purify the basic form(isoenzyme C) of this protein, column chromatogra-phy was used essentially as described by Paul [19].The protein (300 mg) was applied to a CM Sephar-ose (Pharmacia) in a column equilibrated with0.005 M Tris bu¡er at pH 7.4 and eluted with 0.01M NaCl. The RZ values of the fractions werechecked by an absorption spectrophotometer (Cary4E, Varian) and the fraction of RZ = 3.4 was usedfor investigations.

2.2. Methods

IR spectra were obtained with a Bruker IFS 66

Fourier transform IR instrument (Bruker, Brookline,MA) as previously described [20]. The sample com-partment was purged with nitrogen to reduce thecontribution from water vapor. The spectral resolu-tion was 0.5 cm31 ; following data acquisition andsubtraction of the background, the data were con-volved to give 2 cm31 resolution. In some cases,the spectra were further smoothed using a 9-pointsmoothing function. As indicated in the ¢gurelegends, either the attenuated total internal re£ec-tance (ATR) technique or the transmission modewas used. The cell holders were obtained from Gra-seby Specac (Smyrna GA 30082). The ATR top platewas ¢tted with a 45³ ZnSe 6 re£ection crystal. TheATR holder allowed additions to be made withoutremoving the sample. This holder was used for pDand EDTA titrations. The pD of the sample wasmeasured in the ATR cell using a model MI-414bmicroelectrode (Microelectrodes, Londonderry,NH). The observed pH value was changed to pDby adding 0.4.

The transmission cell holder was used when tem-perature was varied. Temperature regulation between5 and 20³C was achieved by circulating waterthrough the holder. For low temperatures, an APDclosed cycle Helitran cryostat (Advanced ResearchSystems, Allentown PA) was used. The temperaturewas measured by a silicon diode near the sample, andthe temperature was controlled using a digital tem-perature controller (Scienti¢c Instruments). Spectrawere taken on cooling with a cooling rate of100 K/h. The outer cryostat windows were made ofCaF2. The inner cryostat windows were made ofZnSe.

To study proteins in aqueous solution at cryogenictemperatures a cosolvent must be used to prevent icecrystals from forming [21]. To see what happenswhen a cosolvent is not used, the spectra of HRPwas examined above and below the temperature offreezing of D2O. The results, given in Fig. 1, showthat there is essentially no di¡erence between thespectra of the protein between 20 and 5³C, but at4³C, the freezing point, the amide IP region (1650cm31) broadens and intensi¢es. The OH stretch re-gion of D2O shows a characteristic double band inthe ice crystal [22]. These spectra served as a refer-ence to ascertain that crystals did not form in theprotein samples studied with glycerol as a cosolvent.

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In separate experiments on proteins, the IR spectraof other proteins in D2O and D2O/glycerol werecompared. There was no signi¢cant e¡ect in theamide IP region (W.W. Wright, unpublished observa-tions).

3. Results

3.1. Infrared spectra of HRP

The spectrum of HRP in D2O is shown in Fig. 2.Several peaks can be identi¢ed. The largest peaks, at1645 and 1455 cm31, are the amide IP and IIP peaks,respectively. The small peak at 1516 cm31 is attrib-uted to the Tyr ring stretch [22]. The small peaksaround 1400 cm31 are in the range of the symmetriccarboxylate stretching mode (XsOCO). The peak at1582 cm31, marked `F', and a peak at V1545cm31 marked `B' in the ¢gure are in the range ofanti-symmetric stretching modes (XaOCO) for carbox-ylate groups on the surface of proteins, and/or in-volved in metal binding [20,23,24]. To see whetherremoval of Ca a¡ects the spectrum in this region,

the Ca-chelator EDTA was added. With increasingEDTA concentration, the peak of the B form de-creased (Fig. 2, inset), relative to the F form. Thepeak at 1413 cm31 is also gaining intensity. In acontrol experiment, the spectrum of EDTA andthat of Ca-EDTA were taken. These are also shownin the ¢gure for comparison. EDTA has a contribu-tion in the range of the symmetric stretch region (i.e.1413 cm31), but does not interfere with the anti-sym-metric stretch region.

Another means to identify the spectral contribu-tion of the carboxylates is to do a pD titration. AspD is decreased, the peak intensity from both B andF is decreased (Fig. 3), consistent with the protona-tion of the carboxylates. In addition to changes ofthe surface charges, it is known that lowering the pH(below pH 2.5) causes the heme to be removed [25]and this may lead to the denaturation of the protein.The appearance of a shoulder at 1620 cm31 on theamide IP band, is characteristic of anti-parallel inter-molecular L-sheet, which indicates aggregation of theprotein at the pD values of 1.7 and 2.6. A similarfeature was seen for thermally denatured peroxidases[26].

Fig. 1. Spectra of HRP in D2O. Prior to measurement the protein was incubated in 100 mM NaCl and 10 mM phosphate bu¡er pD7 for V18 h at room temperature. The protein concentration was 2.5 mM. Temperatures were: 20³C (thin line), 5³C (thick line) and4³C (thin line). The measurements were made in a transmission cell with a 50-Wm Te£on spacer.

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3.2. Temperature dependence of the IR spectralfeatures of HRP as a function of substrateand Ca

A comparison of the IR spectra of resting enzyme,the substrate-bound enzyme and Ca-free enzyme wasmade from room temperature to 10 K. Representa-tive spectra are shown under the three conditions(Fig. 4). It can be seen that the amide IP peak gen-erally widens and shifts to lower frequency with low-ering of temperature. The Tyr ring stretch at1516 cm31 is invariant with temperature and proteincondition. Within experimental error of detection,

the peaks from the carboxylates also do not shiftwith temperature. (Note that it may lead to a falseconclusion of resolved ¢ne structure that a small`hole' appears in the spectrum at 1653 cm31. Thisis due to overcorrection for water vapor in the in-strument, and this feature arises if during the meas-urement the purge of the instrument changes. Thehole does serve to indicate the resolution of the spec-trum.)

3.3. Temperature dependence of the H2O/glycerolsolvent

The OH stretch occurs in the spectral region of3100^3600 cm31. The OH stretch results from resid-ual H2O in the D2O and perdeuterated glycerol, andbecause the anti-symmetric and symmetric stretchesfound in H2O or D2O are decoupled in solvents thatare isotopically diluted, this transition represents asimple OH stretch [27]. It is well known that thepeak from the OH stretch shifts to lower frequency

Fig. 2. IR spectra of EDTA and HRP. Bottom spectrum: theEDTA concentration was 100 mM in D2O. Next spectrum: 100mM EDTA and 200 mM CaCl2, pD 7.0. Incubation and con-centration conditions for the protein are given in Fig. 1. Ali-quots of a stock Na-EDTA solution in D2O were added togive the EDTA concentrations indicated. The measurementswere made in the ATR cell at 20³C. Inset: carboxylate regionfor the protein, the sequence of the spectra is the same as forthe main ¢gure.

Fig. 3. E¡ect of pD on the IR absorption of HRP. Incubationand concentration conditions are given in Fig. 1. The pD waslowered by adding DCl to the sample. The sample pD's are in-dicated on the ¢gure. The measurements were made in theATR cell at 20³C.

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as the temperature decreases, consistent with in-creased H bonding [10,11]. The spectra in this regionare shown in Fig. 5. Around V230 K, at the glasstransition of the solvent, the peak sharpens, but thepeak continues to shift as temperature is lowered.From 80 K to 10 K there was no further change inpeak maximum.

The small peaks in Fig. 5 in the range of 2850^3000 cm31 arise from CH, CH2 and CH3 stretchingmodes [22]. The perdeuterated glycerol has no ab-sorption in this range, and therefore the peaks seenat these frequencies come from the protein. Thesepeaks showed no detectable shift in temperature.

Fig. 5. Temperature dependence of the OH stretch region. Thesample conditions are given in Fig. 4C. Spectra were measuredat 10 degree intervals from high to low. Temperatures are indi-cated on the ¢gure. Not shown are the spectra between 20 and70 K, since there was no signi¢cant change in the spectra inthis region.

Fig. 4. Temperature dependence of HRP in D2O/glycerol glass.The protein was incubated as described in Fig. 1. Prior tomeasurement perdeuterated glycerol was added to give a 50:50v/v solution. Temperatures are indicated on the ¢gure. (A) Noadditions. Protein concentration was 4.8 mM. (B) 4.8 mMBHA added to 4.8 mM HRP. (C) 18 mM EDTA added to 3.8mM HRP. The Te£on spacer was 25 Wm wide for A and B,and 15 Wm for C.6

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3.4. Comparison of spectral shifts and linewidths as afunction of temperature

The temperature pro¢les obtained from data rep-resented by Figs. 4 and 5 are summarized in Fig. 6.In the present context, we neglected the fact that theamide IP peak is a composite band [2] and we ap-proximated the peak by one single Gaussian. Thespectral position of this band is given by the centerof the ¢tted Gaussian in the ¢gure. For all temper-atures, amide IP shifted to lower frequency and theamide IIP peak to higher frequency as temperaturedecreases. The presence of the substrate BHA pro-duced no detectable change in the peak positions.

4. Discussion

HRP has 308 residues, of which 27 amino acidsare from dicarboxylic amino acids. Of these, fourAsp and one Glu residues can be directly in£uencedin Ca binding. For reference, the two Ca bindingsites of HRP with respect to the presence of carbox-ylate groups that may be a¡ected by coordination

Fig. 7. Ca binding site in HRP. The ¢gure was prepared by theInsight II program (Molecular Simulations) using the 1ATJ ¢lein the Brookhaven National Laboratory PDB data base.

Fig. 6. Gaussian ¢tted peak center positions of the amide bondsand absorbance of the OH region as a function of temperature.Conditions for HRP (triangle), HRP+BHA (circle) andHRP+EDTA (diamond) are given in the legend of Fig. 4 forA, B and C, respectively.6

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are shown in Fig. 7 [6]. The heme is also shown inthe ¢gure. The ¢rst Ca is 13 Aî from the heme Fe,and the second is 16 Aî . The long distance from theheme would indicate that the metal has no direct rolein the catalytic function. The crystal structure of theenzyme^substrate complex [7] shows that the sub-strate BHA binds near the heme in a hydrophobicregion, not directly related to carboxyl groups andnot necessarily interfering with the heme structure(not shown in the ¢gure).

4.1. E¡ect of Ca binding

The IR spectra of the resting enzyme (substratefree and with bound Ca) shows amide regions char-acteristic of mainly K-helical proteins. The interior ofthe protein is monitored by other markers ^ the Tyrring stretch arising from Tyr-7, -185, -201, -233 and-234 and in the region of 2900^3000 cm31 the reso-nances from C^H, C^H2 and CH3 can also be seen(Fig. 6). The frequencies, however, of these lattervibrations cannot be identi¢ed with a particular res-idue within the protein and thus they cannot be usedin structural studies. A point can be made that thesebands do not change with temperature. This is ofcourse expected from the simplest view of chemistry:vibrational frequency is una¡ected by temperature, ifthe e¡ect of thermal expansion is negligible [28].However, IR frequencies will be temperature-de-pendent, when bonding and/or structure changesare temperature-dependent, or a particular vibration-al mode is coupled to low frequencies phononmodes. The same comment can be made for theTyr ring stretch. Its frequency did not change withtemperature. We also notice that in spite of that factthat there are ¢ve Tyr in the protein, this transition isrelatively narrow. This can be taken to indicate theinsensitivity of this transition to environment.

The removal of the Ca can be directly monitoredby the IR spectra of the carboxylate region (Fig. 2),where well distinguishable peaks can be related tospeci¢c vibrational modes of the carboxylate group.The peak around 1400 cm31 is known as the sym-metric stretching mode, while the two modes at 1554and 1580 cm31 can be attributed to anti-symmetricmodes of the group in Ca-coordinated (B) and in Ca-free (F) condition, respectively [23]. The position ofpeak B can be interpreted as that of the carboxylate

group involved in bidentate coordination to Ca2�

[23]. This type of binding was shown to shift thefrequency that is around 1600 cm31 of the carboxy-late group signi¢cantly downward. In our system, webelieve the position at 1580 cm31 to correspond withthis ionic state of the carboxylates as its contributiondoes not change with the removal of the Ca. Thespectrum of free EDTA does not contribute to thespectrum of the protein in the range of its two men-tioned anti-symmetric carboxylate vibrations. The in-creased sharp absorption peak at 1413 cm31 is at theposition of the symmetric stretch of the carboxylategroup also seen in the spectrum of EDTA. It gainsintensity, at higher EDTA concentrations in Fig. 2and also the distortion of the amide IP band on thelow frequency side. Both can be interpreted as in-creased contributions from vibrations of EDTA.The spectral changes thus clearly show the removalof Ca from the structure.

The carboxylate vibrations were shown to studythe structural change of Ca removal in other Cabinding proteins like parvalbumins [23,24] and otherproteins [29,30], and this spectral feature may proveto be generally useful to monitor cation binding tocarboxylates. In general, Ca2� is required to main-tain the heme environment and it is also required forthe proper in vitro folding of denatured apoprotein[31,32]. However HRP is a relatively large protein,and the Ca binding areas encompass a relativelysmall amount of the total structure. To tell whetherthere are changes in the protein structure indicatedby the amide bands, further analysis of the bands,including deconvolution, would be needed as wasdone for other Ca binding proteins [29,33].

The IR spectrum of HRP was not a¡ected by thepresence or removal of the substrate BHA. Crystallo-graphic data would indicate that BHA has little ef-fect on the structure of the protein [7]. BHA bindsnear the heme and optical spectroscopy on the hemeshowed characteristic changes upon binding [34]. Op-tical studies upon binding of the analogous substratenaphthohydroxamic acid (NHA) to free base por-phyrin and metal-porphyrin substituted HRP alsoindicated signi¢cant changes [18,35]. These electronictransitions, however, are extremely sensitive to envi-ronment, and changes in electric ¢eld or positions ofatoms near the heme may induce relatively largechanges in the heme optical absorption, without pro-

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ducing overall changes in the entire protein confor-mation that would be detectable by IR. Consistentwith the lack of change within the total protein, in astudy of the optical transition by high resolution holeburning, it was found that addition of NHA, did nota¡ect the compressibility of Mg-substituted HRP[14].

4.2. Temperature e¡ect

The amide bands in proteins have been extensivelyexamined for sensitivity to environment. The bandsshow in£uence from Coulombic and dipolar interac-tions and from H-bonding. For H-bonding, the bandpositions will be temperature dependent when themode is coupled to low frequency modes that canbe thermally activated. The vibrations of water canbe considered also from this aspect, as water has lowfrequency vibrational modes with energies in therange of kT. This would interpret the results shownin Fig. 5.

A question arises whether the temperature-depend-ent changes within the protein are slaved to changesin the solvent. While there is a shift to lower fre-quency with lowering the temperature, the OHstretching region is quite complex as seen in Fig. 5.The situation is complicated because OH can be as-sociated with water and with two distinct positionson the glycerol, and in addition, the protein is gly-cosylated and these residues have OH contributions.Furthermore, each hydroxyl group may be H-bonded, and there may be a distribution of OH-bond angles and distances. So instead of analyzingthe spectrum, we monitored the absorption intensityon the low and high energy side of the band (Fig. 6,bottom). Above 160³C the solvent and the proteinbands show marked temperature dependence. Below150 K, the high energy side (3500 cm31) of the bandremains almost constant, but the solvent still changesbecause the low energy side (3150 cm31) continues toshow temperature dependence.

In all cases, a shift of the amide IP peak to lower,and the amide IIP to higher frequency occurs withdecreasing temperature (Fig. 6). This behavior isalso seen in myoglobin [36]. The suggestion is thatas temperature decreases, there is increased H-bond-

ing by the water to surface exposed amide groups.Increased H-bonding is expected because as temper-ature decreases the bond becomes stronger as thegeometry for optimal bonding increases [10,11]. Theamide IP peak mainly comprises a CNO stretch, andas this group becomes bonded, its frequency de-creases. Amide IIP is mainly a NH stretch, with tor-sional contributions from C^N. Its frequency goeshigher with hydrogen bonding. The overall bandwidth determined from Gaussian ¢tting increaseswith temperature re£ecting the inhomogeneous na-ture of the band. Both amide bands continue toshow spectral shifts, below the glass transition rangeand we conclude that there may be changes in thebonds even below 100 K. Our data convincinglyshow that the structure of HRP is maintained from290 to 10 K in the three conditions tested. The con-ditions used to measure the IR spectra are the sameas often used for optical studies, and therefore IRmeasurements show that the optical results obtainedat cryogenic temperature has validity for the proteinat physiological temperatures.

4.3. Conclusion

It was shown by IR spectroscopy that the proteinHRP retained its structure from room temperature to10 K. Shifts in the amide bands were attributed toincrease in H-bonding for amide groups exposed towater as temperature decreases. The carboxylatestretch region could be used to indicate the presenceor removal of bound Ca in the resting enzyme. Nosigni¢cant overall conformational changes could beseen either by the addition of the substrate BHA orby the removal of the bound Ca in the IR spectra.

Acknowledgements

This work was supported by Hungarian GrantsFKFP 1191/1997 and OTKA T25545 (J.F.) and Na-tional Institutes of Health Grant GM 55004(J.M.V.). Travel was supported by cooperativegrants from the National Science Foundation INT98123221 and Hungarian Academy of Science 122/1998.

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