jung-2000-journal of molecular recognition

ReviewInsight into protein structure and protein–ligandrecognition by Fourier transform infraredspectroscopy

Christiane Jung*Max-Delbruck-Center for Molecular Medicine, D-13125 Berlin, Germany

An overview of the application of Fourier transform infrared spectroscopy for the analysis of the structureof proteins and protein–ligand recognition is given. The principle of the technique and of the spectraanalysis is demonstrated. Spectral signal assignments to vibrational modes of the peptide chromophore,amino acid side chains, cofactors and metal ligands are summarized. Several examples for protein–ligandrecognition are discussed. A particular focus is heme proteins and, as an example, studies of cytochromeP450 are reviewed. Fourier transform infrared spectroscopy in combination with the various techniquessuch as time-resolved and low-temperature methods, site-directed mutagenesis and isotope labeling is ahelpful approach to studying protein–ligand recognition. Copyright # 2000 John Wiley & Sons, Ltd.

Keywords:Fourier transform infrared spectroscopy; proteins; protein–ligand recognition; heme proteins; cytochromeP450

Received 9 June 2000; accepted 9 June 2000

INTRODUCTION

Molecular recognition is a very complex phenomenon.Proteins, substrates and ligands are part of an ensemble ofvarious components of the solution or the cell which alltogether contribute to the recognition process. How can onefigure out and separate all the different interactions and findthe interaction that is most relevant for the function? Thereare many experimental and theoretical methods to approachthe problem. One of them is Fourier transform infraredspectroscopy (FTIR). The application of FTIR spectroscopyto proteins is a broad field. This review will focus onselected topics which are connected with protein–ligandrecognition and will take cytochrome P450 (P450) as theprimary example. P450s represent a big superfamily ofheme-type monooxygenases which catalyze the conversionof diverse substrates (Lewis, 1996). In contrast to mostheme proteins, like myoglobin and hemoglobin, P450s face

the problem of different recognition phenomena. How theprotein recognizes the dioxygen (and other related smallheme iron ligands) and how it depends on the substratebound in the heme pocket are important questions foruncovering the catalyzing function of this class of enzymes.Interpretation of the infrared spectra of P450 benefitssignificantly from the knowledge of infrared spectroscopyon many other proteins which has been accumulated in theliterature over more than three decades. This will be brieflyreviewed and recognition phenomena, in particular in hemeproteins, will be discussed. Because there are so manypapers about FTIR studies, only the more recent ones will berefered to the particular subject.

METHODOLOGY OF FOURIERTRANSFORM INFRAREDSPECTROSCOPY

Instrumental principle

In contrast to the dispersive spectrometer, where theradiation from an infrared source is made monochromatic,the Fourier transform spectrometer uses polychromaticradiation. The collimated radiation from the infrared lightsource is partially reflected and partially transmitted by thebeamsplitter (BS) [Fig. 1(A)]. The beam originallytransmitting the beamsplitter is then reflected from a fixedmirror (FM) while the beam originally reflected from thebeamsplitter is reflected from a movable mirror (MM). Bothmirror-reflected beams are recombined and split again at thebeamsplitter, where one of the newly split beams goes back

JOURNAL OF MOLECULAR RECOGNITIONJ. Mol. Recognit.2000;13:325–351

Copyright# 2000 John Wiley & Sons, Ltd.

* Correspondence to: C. Jung, Max-Delbru¨ck-Center for Molecular Medicine,Research Group Protein Dynamics, Robert-Ro¨ssle-Strasse 10, D-13125Berlin, Germany.E-mail: [email protected]/grant sponsor:Deutsche Forschungsgemeinschaft;contract/grantnumber: Ju229/1-1; contract/grant number: Ju229/3-1; contract/grantnumber:Sk35/3-1,2.Contract/grant sponsor: European Commission;contract/grant number:BIO2-CT942060.

Abbreviations used: Adx, adrenodoxin; ATP, adenosine 5'-triphosphate; CD,circular dichroism; DHAP, dihydroxyacetone phosphate; FTIR, Fouriertransform infrared spectroscopy; GAP,D-glyceraldehyde 3-phosphate; Pdx,putidaredoxin; P450, cytochrome P450; P450cam, P450 from camphor-hydroxylatingPseudomonas putida.

to the source while the other one passesthe sampleandreachesthe detector. The recombination of the mirror-reflectedbeamsleadsto acomplex interferencepatternwiththe highest intensity in the center burst and decreasingintensity to the sides of the center with increasing ordecreasingdistance (x) betweenthemovable mirror andthebeamsplitter. The detector measuresthe intensity [I(x)] ofthe resulting radiation as a function of the distance (x)(interferogram) [Fig. 1(B)].

A calibrating beamof a He–Nelaser goesthesamepathas the infrared radiation. Because of the monochromaticHe–Ne laser light, its interferogramis a sinefunction. Thedistancebetween two zerocrossing pointsis simplyhalf thewavelengthl of the laserlight which is usedasan internalfrequency calibration. The relation betweenthe intensityI(x) at the mirror position x and the intensity of amonochromatic spectralline S(n) at the wavenumber n (=1/l) is given by eq. (1).

I �x� � S�� cos�2��x� �1�Theresultof thedataacquisition is adigitized interferogramI(nDx), where n is the counting number of the distancedifferenceDx betweentwo zerocrossingpointsof the He–

Ne laser light. This interferogramis Fourier transformedinto thespectrum S(kDn) [eq. (2)] with k usedasa countingnumberof thefrequency incrementDn. N is thetotalnumberof digitized points.

S�k�� XNÿ1

n�0

I �n�x� exp�i2�nk=N� �2�

�� 1=�N�x�S(kDn) represents the so-called single channel spectrum[Fig. 2(A)]. The ratio between two single channel spectra(Ssample/Sreference) givesthe transmission spectrum which istransformedto theabsorptionspectrum [Fig. 2(B)] by takingthe negativedecadic logarithm.

FT infrared spectroscopyhas various advantages com-pared to thedispersivemethod:(i) absorbancedifferencesofonly 10ÿ4 at anabsolute absorbance of 0.5–1canclearly beresolved with few scans;(ii) the measurementtime is veryshort— a time resolution of severalmilli seconds with amirror velocity of 320 kHz anda resolution of 8 cmÿ1 canbe realizedin the rapid-scan mode; and (iii) the step-scantechnique of moving the mirror step-wise opens new

Figure 1. Principle of the experimental set-up of Fourier transform infraredspectroscopy coupled with various applications of time-resolved, low-temperature and high-pressure methods. (A) Right upper corner: interferom-eter with FM = ®xed mirror; MM = movable mirror; BS = beam splitter;x = distance MM moves (scanner drive); MCT = HgCdTe detector; A/D = ana-log±digital converter; PC = personal computer; Nd±YAG = laser for photo-initiated processes, 1064 nm or 532 nm; M = mirror; ir-source = infrared lightsource (globar). (B) Interferogram and (C) time trace for the rebinding of thephotodissociated CO ligand in (1R)-camphor-bound cytochrome P450cammonitored at a speci®c MM mirror position (170 Dx increments apart from thecenter burst); 50 mM potassium phosphate D2O buffer, pD 7; 50% (v/v)glycerol-d3, c(P450) = 0.79 mM, 23.7 mM (1R)-camphor (Contzen and Jung,1998).

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Copyright# 2000JohnWiley & Sons,Ltd. J. Mol. Recognit.2000;13:325–351

applications for time-resolved measurements. At eachmirror position, a chemical reaction or conformationalchangeis induced, for example by a laserflash.The timeevolution of the intensity at the detector after the flash isrecorded [Fig. 1(C)]. Under the condition that the flash-induced perturbation is completely relaxedto equilibriumbefore the next flash is initiated, this experimentcan berepeatedat anothermirror position.Af ter manysteps,a setof time-digitized transients for each mirror position isobtainedwhich canbere-sortedinto a setof interferogramsfor eachtime point. Af ter Fourier transformation,completeinfrared spectra for each time point can be obtained(Uhmann et al., 1991; Palmeret al., 1993; Rammelsberget al., 1997). Other time-resolved(not FT) techniquesexist,whichhavebeenrecentlyreviewedby SlaytonandAnfinrud(1997).

Time-resolvedexperiments are triggered or initiated byvarious methods. Most of the studies are done withflashphotolysis where a pulsedlaser(for examplethe Nd–YAG laser)is used.Thelaserflashleadsto thedissociationof theligand,for examplefrom thehemeiron or afunctionalimportantagent(for example ATP) is releasedfrom a cage(Cepuset al., 1998a,b). Light-induced structural changesconnected with protonation equilibria of the differentintermediatesin the photocycle of bacteriorhodopsinhasbeenextensively studiedby time-resolvedFTIR (Rammels-berg et al., 1997; Rodig and Siebert,1999). Temperaturejump experiments, in which a fast heatingof the sampleisiniti atedby a laser(Phillips et al., 1995)or by a fastmixingwith a hot solution (Backmann et al., 1995) have beenreported to studyprotein dynamics and protein unfolding/folding phenomena. The pressure jump (or release) tech-niquehasalso beencombinedwith FTIR (Frauenfelderetal., 1990).

FTIR studies at low temperatures down to 10K areusually performedwith closedcycleheliumcryosystemsorbathcryostates.High-temperaturestudies(up to 100°C) arecommonly applied to study protein unfolding. Proteinunfolding is also studied extensively using high-pressureanvil cells (Wong, 1987; Heremans, 1997). Vibrationalcircular dichroism is one of the newestdevelopmentsandgives detailedstructural information aboutmolecules withchiral properties. For details see the recent review byKeiderling (1996). Extensively used is also the ATR(attenuated total reflection) technique which allows thestudy of thestructureof proteinsadsorbedon solid surfacesor in lipids (Menestrinaet al., 1999).

Spectra analysis

Most of the infraredbandsof proteinsarerelatively broad.Resolutionenhancementcanbeachievedby deconvolutionof thespectra(SurewiczandMantsch, 1996).Thebasic ideais that the spectrum S(n) is regardedasa convolution S'(n)* L(n) of a delta function S'(n) = �(nÿ no) and a Lorentzfunction L(n) = a/(p[a2� n2]) with a being theLorentzhalf-width. A deltafunction correspondsto a cosinefunction inthe interferogram, S'(n) → I'(n) = cos(2pnox), while a Lor-entz function is described by an exponential in theinterferogram,L(n) → `(x) = exp[ÿ2pajxj]. In the interfer-ogramdomain, theconvolution product representsa simplemultiplication operation I(x) = I'(n)`(x) = cos(2pnox)exp[ÿ2pajxj]. Resolution enhancementis reachedby dividingthe interferogramI(x), obtained by inverse Fourier trans-formation of the spectrum, by the interferogram of theLorentz function. If the resulting interferogram is againFourier transformed,a spectrum with a higher resolution isobtained.Usingthisprocedure, thebroadbandswith aweakfine structurecanberesolved moreclearly [Fig. 3(A)]. Thefrequencyof the bandmaximain the deconvolutedspectrashould correspond to the frequencyof the minima in thesecond derivative of the undeconvoluted spectrum [Fig.3(A)].

Nonlinear least-squarecurve fitting with given bandshapes is an alternative and/or additional method todecomposebroad structuredbands.Different line shapesareuseddepending on thekind of spectraandexperiments.

Figure 2. Infrared spectrum of substrate-free cytochromeP450cam-CO. (A) Single-channel intensity spectra of the proteinsample (Ssample) in 100 mM potassium phosphate D2O buffer, pD7, 20°C (Mouro et al., 1997), and reference buffer (Sreference). (B)Top: absorption spectrum obtained from the single channelspectra in (A), ÿlog(Ssample/Sreference); middle: water spectra with0% H2O, 25% H2O and 100% H2O taken with CaF2 windows and a5 �m te¯on spacer; and (C) water vapor spectrum with a smallimpurity of CO2.

FTIR AND PROTEIN–LIGANDRECOGNITION 327


GaussandLorentzline shapesareusedfor fitting theproteinamidebands[Fig. 3(B)]. For analysis of the stretch modespectraof theCO iron–ligandtheVoigt function [convolu-tion of a Lorentz and Gauss function; Fig. 3(C)] and thelogarithmic normal distribution function [Fig. 3(D)] havealso been used. The Lorentz line shape represents thehomogenousbroadening of a spectral line and the half-width is a measure of the lifetime of the excitedstate.Thehomogenousline width of thestretchmodeof theCOligandin hemeproteinslies in the rangeof 0.2–2cmÿ1 (Rectoretal., 1997). The Gaussian distribution results from theinteraction of the vibrating group with the environmentresulting in many microstructures for the vibrating group(inhomogenousbroadening effect). Because the width ofCO stretchmodesin hemeproteins is significantly largerthan 2 cmÿ1 (7–20cmÿ1) one can concludethat inhomo-

genousbroadeningis alwayspresent. Thebandshapemustbe describedby the Voigt function. A shapefactor [Fig.3(C)] allows a smoothchangefrom a Lorentz to a Gaussfunction whenit is variedduringthefitting. It is thereforeameasureof theextentof inhomogenousbroadening.Seriouscarehasto betakenwhenfitting complex spectrawith manybands. The minimum number of bands resulting in areasonablefit with well-distributed positive and negativewingsin theresidualsover thewholespectralregionshouldbe considered[Fig. 3(B)]. Sometimesthe absorption bandsare asymmetric in shapewithout a clear shoulder and theresult of fitting with two or more Gaussians is notunequivocal and depends on the starting parameters. Inthese cases, the bandscan be fitted with the logarithmicnormal distribution function(Jungetal., 1996a). Thereis nophysicalmeaningbehindthis line shapebutonecanuseit as

Figure 3. Analysis of infrared spectra. (A) Bottom: original amide I' band ofcytochrome P450cam from Fig. 2 and side-chain absorption spectrum calculatedaccording to the amino acid composition of cytochrome P450cam using the standardspectra provided by Chirgadze et al. (1975); middle: side-chain corrected spectrumcorresponding to the difference of the original spectrum minus the side-chainspectrum, deconvoluted spectrum of the side-chain corrected spectrum; top: secondderivative of the side-chain corrected spectrum. (B) Result of a nonlinear least-square®t of the deconvoluted amide I' band using Gaussians and assignment to secondarystructure elements. (C) Comparison of the line shapes of Gauss [E(n) =Aexp[ÿ4ln2((nÿ no)/Dn1/2)2]], Lorentz [E(n) = A/[1� 4((nÿ no)/Dn1/2)2]] and Voigt[E(ni) = (1/p)

�exp[ÿ(nÿ ni)

2/X22][X1/(X1

2� (nÿ no)2)]dn, with X1 = 12Dn1/2(Lorentz); X2 =

12(ln2)ÿ 1/2Dn1/2(Gauss) and X1/X2 = shape factor; X1/X2 = 0 for Gauss; X1/X2 → ? forLorentz]. (D) Line shape of the logarithmic normal distribution function [E = (A/X)exp[lnX(1ÿ ln2lnX/ln2a)] with X = [(nÿ no)(a2ÿ 1)� Dn1/2a]/Dn1/2a and a = asymmetryparameter; a< 1 � asymmetry at the lower-energy side; a> 1 � asymmetry at thehigher-energy side] for an asymmetry parameter of 1.4. [n = wavenumber; Dn1/2 = halfwidth; no = wavenumber of band maximum; E(n) = absorbance; A = amplitude].

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mathematical description for an ensemble of bandswhichmight beassignedto anensemble of conformational states.Variousothermethodsareusedto decompose thecomplexinfraredspectra(for example factoranalysis,(Gilletteetal.,1983;Lee et al., 1990)which will not be further discussedhere.

A very powerful methodfor analyzing completesetsofFTIR spectrafor variousperturbationsli ke H/D exchange,varying concentrations of a chemical agent, temperaturechangeor even time-resolved processes is the recentlydeveloped two-dimensionalFTIR spectroscopy(2D-FTIR)which will certainly improve the analysis of complexprocessessignificantly (NabetandPezolet, 1997).

Before all thesefitting anddeconvolution procedurescanbeappliedto theexperimental spectra,two correctionshaveto be made. The first correction is necessary if the infraredspectrometer is not purged carefully with dry air and thewatervaporabsorption overlayswith the samplespectrum[Fig. 2(B)]. The watervaporspectrum canbe interactivelysubtractedusingsoftware.The second correction concernsthebaseline.For example, for studiesof thedependenceofthe iron ligand CO stretch modes on temperature in therange20–297K,seriousbaselineshiftsmaybepresentif thereferencespectrum is not takenat thesametemperature.Abaselinecorrection canbeperformedby fitting thespectrumontheleft sideandtheright sideof theCObandsby acubicpolynomial. Baseline problems are not so serious inflashphotolysis or electrochemical experiments becausethe spectrato be substracted are produced in the samesample. Therefore, signals with a very weak absorption(10ÿ5) arestill well detected.

INFRARED SIGNAL ASSIGNMENT

Proteins

Infrared spectroscopic studies on proteins have beenreviewedextensively (Surewitz andMantsch, 1996;Arron-do andGoni, 1999).Besidesthe ‘fingerprint’ vibrationsoffunctional groups of chemicals in general, which aretabulatedin varioustextbooks, therearespecificvibrationsresulting from the peptidegroup(Table1).

Amide I band. Thebeststudied infraredbandof proteinsis

the amide I bandappearing between 1600 and 1700cmÿ1

with a maximum for most proteins at around 1654–1674 cmÿ1 [Figs. 2(B) and3(A,B)], which arisesprimarilyfrom the stretch vibration of the peptide C=O group.Normal mode analysis reveals that the C=O stretchingcouplesslightly with CN stretching,CCN deformationandNH bending.Unfortunately, the HOH bendingmotion ofwater almostcoincideswith the amideI band[Fig. 2(B)]and makesstudiesin protonic aqueous solution difficult(Rahmelow andHubner,1997).This problemis overcomeby using D2O as solvent. The D2O substitution leadsto ashift of theamideI band(amideI') between 2 and9 cmÿ1 tolower frequencies depending on the particular protein(Tu,1986).

Because the C=O group is involved in differentsecondary structureelements via hydrogen bonding to thepeptide NH group, the experimentally observedamide Ibandenvelopesa multitude of single bandswith differentfrequencieswhich canberesolved asdescribedabove[Fig.3(B)]. Thelargenumberof infraredspectroscopic studiesincombination with crystal structure and NMR analysesonvariousproteins revealed some variability of the frequencyof theassignedcomponentamideI bands.However, onecangenerally classify the components between 1649 and1658 cmÿ1 to a-helices,1620–1635 cmÿ1 to intramolecularb-sheets,and 1665–1690 cmÿ1 to turns. b-Sheetsshow aweak high-frequency component at around 1672cmÿ1

which will, however, not significantly contribute to theabsorption in this region(Byler andSusi,1986). However,aggregated proteins show intermolecular antiparallel b-sheets with infrared absorption around 1614–1624 and1684 cmÿ1 (Ismail et al., 1992;Panicket al., 1999). Someexceptions from this general assignment have beendiscussed in the recent review by Arrondo and Goni(1999)andby Heimburget al. (1999).Table2 summarizesthe assignmentof the amide I components to secondarystructure elementsfor cytochrome P450cam— the onlyP450 for which the amide I band spectrum has beenpublishedso far.

Secondarystructure composition can be estimatedfromtherelativeareaof thesingle bandsassignedto thedifferentstructuresassuming that the extinction coefficient for thepeptide CO stretchvibration is the samein all hydrogenbonding structures. This is of coursean approximation.Nevertheless, the agreement with the secondary structure

Table 1. Amide vibrations of the peptide group in proteins (Susi, 1972; Krimm and Badekar, 1986; Baello et al.,1997)

Wavenumber(cmÿ1)

Band In H2O In D2O Assignment

Amide A 3250–3300 N—H stretch,in resonancewith amideII overtoneAmide I 1600–1700 1600–1700 Mainly C=O stretching,slightly coupledwith CN stretching,CCN

deformation,NH bendingAmide II 1550 1450 N—H bendingcoupledwith CN stretching.Amide III 1230–1330 NH bendingandCN stretchingAmide IV 625–767 OCN bending,coupledwith othermodesAmide VI 640–800 Out-of-planeN—H bendingAmide VII �200 Skeletaltorsion



composition obtained from CD measurements of theelectronic transitionsof the peptidechromophor and fromcrystal structureanalysesis reasonably good (Byler andSusi, 1986). For camphor-boundP450cam, for example,54%a-helix� 310-helix, 21%b-sheetsand21%turnshavebeenestimatedby FTIR (Mouro et al., 1997),which is ingood agreementwith 53%, 19% and 16%, respectively,determined from the crystal structure. Quantitation ofsecondarystructurecompositionsfor variousproteinshavealsobeenperformedfrom second derivative spectra(Donget al., 1990)andfrom factor analysis (Lee et al., 1990).

Amide II band. TheamideII bandarisesfrom NH bendingcoupled with CN stretching. The bandis observed around1550cmÿ1 in H2O andshiftsto about1450cmÿ1 [amideII ',Fig. 2(B)] in D2O (Susi, 1972). Because of the strongsensitivity of theamideII bandfrequencyupondeuterationthis band is usedto monitor H/D exchangebetween theprotein coreandthesolventduring unfoldingof theproteinstructure induced by various perturbationsas for exampletemperature or pressure(Surewicz and Mantsch, 1996;Heremans,1997).

Amide III band. The amideIII bandis found in the rangebetween 1220and1330cmÿ1 andresults from NH bendingandCN stretching.Thebandposition is verysensitive to thesecondarystructure. a-Helix is reflected in theregion1293–1328cmÿ1, b-sheet in 1225–1250 cmÿ1 and unorderedstructures in 1257–1288cmÿ1 (Griebenow and Klibanov,1995).However, becauseof its low intensity the amideIIIband observed with infrared spectroscopy is of lowerdiagnostic value. Some progressin improving the signifi-cance of the structural information from this band hasrecently beenreported usingvibrational circular dichroismmeasurements(Baello et al., 1997).

Modes of amino acid side chains. Infrared absorption ofaminoacidsidechainsalsoappearsin thespectralregionofthe amide I and II bands and makes the extraction ofstructural properties from the amide bandsmore compli-cated.Depending on the amino acid composition of theprotein and on the pH, the contribution of the side chainabsorptioncanvarydramatically andmaycauseup to about30%of theamideI bandintegralintensity.Standardspectrafor the side chain absorption have been published by

Chirgadze et al. (1975) for D2O solutions and byVenyaminov and Kalnin (1990) for H2O solutions. If theaminoacidcompositionis known thesespectramaybeusedto correcttheproteinamideI bandbeforedeconvolutionanddecomposition in the single bandsto obtain the contentofthe different secondarystructureelements [Fig. 3(A)].

For estimationof thesecondarystructurecompositionofproteins the side chain absorption may be regarded asdisturbing contribution. However, for the analysis ofrecognition phenomena, side chain infrared bandsare ofhighdiagnosticvalue,e.g.to detectsaltlinks by theinfraredabsorption of theCOOHgroupin aspartic or glutamic sidechains.In theprotonatedstatetheCO stretchvibrationof aCOOH group appears in the region around 1760cmÿ1 inH2O and is shifted to 1750 cmÿ1 in D2O (Dollinger et al.,1986). This position is significantly loweredby hydrogenbonding andsignals at different frequencies between1695and 1740 cmÿ1 have been observed in various proteins(Lubben et al., 1999; Iliadis et al., 1994; Hellwig et al.,1999). The deprotonated COOÿ group shows the asym-metric vibration between 1540 and 1620cmÿ1 while thesymmetric vibration is observed between 1300 and1420 cmÿ1 (Chirgadze et al., 1975; Venyaminov andKalnin, 1990). The simultaneous appearance of bandsbetween 1750–1695 cmÿ1 and 1540–1620 cmÿ1 or 1300–1420 cmÿ1 with oppositesignin differencespectraobtainedfrom perturbation-induced conformational changes is astrongindicationof achangein theprotonation state(Iliadiset al., 1994). In contrast, a shift of the signal or theappearance of a derivative-shape differencesignal in thenarrower spectral rangebetween 1700 and 1740cmÿ1 iscaused by a changein the strength of the hydrogen bond(Lubbenet al., 1999; Contzen andJung,1999).

Histidinemay alsobe involved in salt bridge formationwith aspartates.Three bandsof histidine are sensitivetoprotonation of the imidazole nitrogen in hemoglobins(Table 3, Gregoriou et al., 1995). C—H and C—Nstretching modes of histidine imidazole have also beenassigned(Table 3).

Tyrosine C—C ring vibration is observedat around1515 cmÿ1 in D2O andis a very goodmarkerbandbecauseof sharpnessandalmostisolatedspectral location.Thisbandcan be used to normalize infrared spectra of proteins(Arrondoand Goni, 1999). In addition, hydrogen bondingfrom thetyrosineto othergroupsleadsto ashift of thisband.

Table 2. Component band frequencies of the amide I' band of cytochrome P450cam (Mouro et al., 1997)

(1R)-Camphor-bound Substrate-free

Position(cmÿ1) Population(%) Position(cmÿ1) Population(%) Assignment

1599.6 2 1555.5 3 Arg sidechain1611.1 2 1610.8 4 Arg, tyr sidechain1622.1 9 1621.8 8 b-Sheet1631.8 12 1634.6 23 b-Sheet1640.7 14 — — a-Helix, 310-helix1648.8 16 1648.2 24 a-Helix1658.2 24 1658.6 20 a-Helix1666.1 2 — — Turns1673.2 17 1672.5 17 Turns1689.3 2 1688.7 1 Turns

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The S—H sulfhydryl group of cystein absorbs between2400and2700cmÿ1 in H2O andaround 1857cmÿ1 in D2O(Tu, 1986; Gregoriou et al., 1995; Guarrera et al., 1999).S—HgroupswhicharehydrogenbondedshowsignificantlylowerS—Hstretchmodesdownto 2290cmÿ1 (Sellmannetal., 1991; Boormanet al., 1992). Table 3 summarizes theinfraredsignalsobservedfor aminoacid sidechains.

Cofactors

Proteins may havevariouscofactors suchas flavins, ironsulfur cluster, retinal or heme.On onehand,their infraredabsorptionmaycomplicatetheinterpretation of theinfraredspectraof proteins but on the other hand their specificsignals may be very helpful for the analysis of recognitionphenomena.

Retinal. Retinal is the cofactor in bacteriorhodopsin. Thesignal of high diagnostic value is the Schiff baseC=Nstretchingmodelocated atabout1640cmÿ1 whoseconcrete

position depends on the protonation state of the nitrogen.Other importantsignalsaretheC—C stretchmodeat about1188 cmÿ1 and the ethylenic C=C stretching at about1527 cmÿ1 of thecarbons13,14,15nextto theC=N group,whichareinfluencedby theisomerizationstateof theretinal(Bouscheet al., 1992).

Heme.Metal porphyrinsshowseveral vibrational modes ina broadspectral region (Boucher and Katz, 1967; Alben,1978; Spiro, 1983). Only the in-plane vibrations of thesymmetry Eu and the out-of-plane vibrations of thesymmetry A2u of the porphyrin ring are infraredactive. Inmost hemeproteins thechromophoreis protoporphyrin IX,which has two vinyl groups.Infrared bandsfor the vinylgroup have been reported by Spiro (1983), the mostimportant one being the C=C stretch mode at about1640 cmÿ1. Thesevibrationalmodes aredifficult to seeintheinfraredspectraof hemeproteinsbecauseof theoverlapwith theamidebandsof theprotein.However, in differencespectra obtained from photochemical or electrochemicalreduction or ligand photodissociation,small signals might

Table 3. Amino acid side chain infrared absorption signals

Wavenumber(cmÿ1)

Amino acid H2O D2O Assignment Reference

Asparticacid 1716 1713 CO stretchin COOD,pH(D) <4 VenyaminovandKalin (1990);Chirgadzeet al. (1975)1574 1584 COOÿ asymmetric

Glutamicacid 1712 1706 CO stretchin COOD,pH(D)< 4 VenyaminovandKalin (1990);Chirgadzeet al. (1975)1596 1567 COOÿ, asymmetric

Asparaginyl 1678 1648 CO stretch VenyaminovandKalin (1990);Chirgadzeet al. (1975)1622 ÿNH2 deformation

Glutaminyl 1670 1635 CO stretch VenyaminovandKalin (1990);Chirgadzeet al. (1975)1610 ÿNH2 deformation

Arginyl 1673 1608 ÿCN3H5� asymmetricstretch VenyaminovandKalin (1990);

Chirgadzeet al. (1975)1633 1586 ÿCN3H5

� symmetricstretch VenyaminovandKalin (1990)Tyrosyl 1518 1515 Ring motion,pD< 9 VenyaminovandKalin (1990);

Chirgadzeet al. (1975)1615 pD< 91498 1500 Ring motion,pD> 101602 1603 pD> 10

1240–1280 C—Oÿ stretching Roepeet al. (1987)Histidine 1596 Ring motion VenyaminovandKalin (1990)

1611 Protonated Gregoriouet al. (1995)1575 Protonated Gregoriouet al. (1995)1410 Protonated Gregoriouet al. (1995)1630 Neutral Gregoriouet al. (1995)1530 Neutral Gregoriouet al. (1995)1412 Neutral Gregoriouet al. (1995)

3135-3145 C—H stretchhistidineimidazole Puustinenet al. (1997)1087 1197 C—N stretchin Nt(4-MeIm) Noguchiet al. (1999)1104 1103 C—N stretchin Np(5-MeIm)1089 1108 C—N stretchin Nt, Np1101 1101 (MeImH(D)�)1090 1096 C—N stretchin (MeImÿ)1106 1107 C—Nt (DL-His)

C—Np (DL-His)Tryptophan 3486–3491 IndoleN-H stretch Maedaet al. (1992)Cysteine 2400–2700 1857 SH stretch Tu (1986)



originate from porphyrin or its substituents (Schlereth andMantele,(1992); Mill er andChance,1994).

Protoporphyrin IX hastwo propionic acid groupswhichhave characteristic infrared bands depending on theprotonation stateof the COOHgroup,asalreadydiscussedabovefor the COOH group in aspartateor glutamate.Thestretching modeof the CO groupin the methyl propionateesterhasbeenobserved at 1738cmÿ1 (in chloroform) andbetween 1725and1740cmÿ1 for variousother substitutedporphyrins (Alben, 1978). Two signals at 1735 and1700cmÿ1 existwhen theesteris split (Boucher andKatz,1967).The CO stretchis coupled with the O—H in-planebending in the COOH. Hydrogen bonding from thepropionic acid OH groupor from a hydrogen donor to theCO propionic group by basicamino groupsin the proteinshifts the value to lower frequencies, 1697–1740 cmÿ1

(Behr et al., 1998).

Ligands

Metal ligands. Infrared absorption of small heme ironligands like CO, O2, NO, CNÿ, SCNÿ, N3

ÿ have beenextensively studied for varioushemeproteins over severaldecades,but other metalcentersin proteinsarealso abletobind such ligands. Before drawing conclusions aboutprotein–ligand recognition in heme proteins one has toremember theprefered modeof bindingof theseligandsinprotein-free metal complexes which follow the rules fororbital hybridization. Binding of the ligand to the metalchanges the ligand stretch modefrequency [Fig. 4(A)].

Carbon monoxide. CO binds to ferroushemeand hasalinear geometry. The sp hybridization of the CO valence

orbitals would bestdescribe the structure.

Dioxygen.O2 alsobindsonly to hemewith ferrousiron andis in a bent configuration with a typical angle ofapproximately 110–130° because of the sp2 hydridization(Godboutetal., 1999).Then(O—O)stretchvibration of thefree ligand is not infrared-active becauseof a zero-dipolemoment. After binding to the hemeiron the O—O bondbecomes asymmetric and a nonzero dipole moment isinducedgiving rise to a weak infraredabsorptionintensity.O2 complexes have not been so extensively studied byinfraredspectroscopybecauseseveral other components oftheproteinandsolution absorbin thespectralregionof theO—O stretch modeand the O—O band intensity is low.ResonanceRaman spectroscopy is an alternative method(Bajdor et al., 1984:Macdonaldet al., 1999).

Nitri c oxide. Iron–NO complexes usually show a bentgeometry if the iron is in the ferrousstate[rule given byFeltham and Enemark (1981): {M NO}n, M = metal,n = numberof the electronsin the metald-orbitals andthep* orbital of theNO ligand;for n� 7 M—N—O is bent]. InthiscaseNO canacceptanelectronfrom iron(II) by formingthe resonance structure (Fe3�NOÿ) with NOÿ beingisoelectronic with O2. The n(N—O) stretch vibration ofthe bound NO is therefore expected to be in a lowerfrequencyrangedown to 1550cmÿ1 of the free NOÿ. Incontrast, ferric NO complexes prefer a linear iron–ligandorientation(case n� 6) by donating an electronto the irondp-orbitals and forming NO� which is isoelectronic withCO. In this casen(N—O) should be in the rangeof 2150–2400 cmÿ1 of free NO�. In real complexeshowever thestretchfrequency liesbetween thefrequency of freeNO andeitherNO� or NOÿ.

Figure 4. Sketch of the binding mode of heme iron ligands and structural parameters: (A) in protein-free heme ironcomplexes and assignment of the range of the ligand stretch mode frequencies (Collman, 1977; Alben, 1978; Yu, 1986;Bof® et al., 1997; Obayashi et al., 1997), and (B) in heme proteins indicating the parameters which in¯uence the stretchmode frequency of heme-bound ligands (p and s indicate the iron and ligand orbitals assigned to the p- and s-orbitalsystem. The arrows point in the direction of the electron density donation in the respective orbital system. X = Y is thedistal iron ligand while `5. ligand' means the proximal iron ligand. � and ÿ indicate the sign of the electrostaticpotential).

332 C. JUNG


Cyanide. CNÿ can bind to either ferric or ferrous heme(Yoshikawaet al., 1985;Yu, 1986;Boffi et al., 1997).TheCNÿ stretch vibration frequencyof the freeCNÿ andHCNare observedat about 2078 and 2092cmÿ1, respectively.Whenboundto ferric complexesit shiftsupbecauseof s- aswell asp-donationto theiron, but shiftsdownat bindingtoferrous heme because of increased iron-p-backdonation.Undisturbed cyanidecomplexeshave a linear Fe—C—Ngeometry.

Azide. Free azide is linear with symmetric N—N bonds.Whenboundthe metal complex N3

ÿ remainsin the linearfashion but theN—N bondsbecomeasymmetric.ThelinearN3ÿ ligandbindsin abentmodeto themetal.For iron heme

complexesit bindsto the ferric form.

Environmental effects. The concrete value of the ligandstretchmodefrequencyis determinedby variousparameters[Fig. 4(B)]. Themostimportantoneis thebalancebetweenelectron densitydistributionwithin thes- andp-systemsofthe metal–ligandunit. This hasbeenextensively discussedin theliteraturefor theCOandO2 complexesasthesynergiceffect of ligand-s-donation and iron-p-backdonation (Yu,1986).This balanceis sensitiveto variousotherparameterssuchas the natureof the proximal (5th) ligand, the anglebetween the iron–ligand and the intra-ligand bonds,hydrogen bonding, electrostatic interaction between theligand andaminoacids, substratesandwateron the distalsideof theporphyrin plane.Also theporphyrin substituentsinfluence the stretch frequency (cis-effect) due to theirelectronic effect on the porphyrin-p-system (Alben andCaughey,1968;Alben, 1978).

Studies on pyridine–heme–CO complexesshowed thatthen(CO) decreaseswhenthebasicity of the trans-pyridineligand increases(trans effect) (Alben andCaughey,1968).The sametrend has been observedin model complexeswhen the proximal imidazole or mercaptane are deproto-nated (Mincey and Traylor, 1979; Chang and Dolphin,1976).The reasonis that the strongercharge donationintotheantibonding orbitalsof theCO or O2 ligandsleadsto anincrease of the ligand net chargeanda decreaseof the p-bondorder. Yoshimura(1983)hasshownfor NO complexesof ferrous protoporphyrin IX with nitrogenousbasesasproximal ligand that, with increasingthe polarity of thesolvent, the stretch mode frequencies decrease. Theseeffects of the proximal ligand and of the polarity of theenvironment observedin porphyrin complexes representalreadythe rangeof effectswhich turn out to be the mostimportant ones for the stretch mode frequencies of theligandsboundin hemeproteins,asdiscussed below.

Other ligands

Bound water. Infrared studies of proteins in protonicaqueous solution are problematic because of the HOHbendingabsorptionatabout1650cmÿ1 whichoverlapswiththe amide I band as discussedabove.However, specificwatermolecules play an importantrole for the structureofproteins and have functional significance for protontranslocation(Yamazakiet al., 1995).Onemayunderstandthese water molecules as ‘ligand’. Binding of water

moleculesto the protein is mediatedby hydrogen bondingwhichsignificantly influencestheO—H stretchvibrationofH2O. The antisymmetric O—H stretch mode of waterappears at about3550cmÿ1 [Fig. 2(B)] but mayshift up ordown depending on the microenvironment in the protein.Specific waterO—H stretchchanges canonly be observedin thin films andin differencespectrainduced,for example,by illumination asstudied in bacteriorhodopsin(Maedaetal., 1993).

Phosphate. ATPases, kinases,and ion pumpsbind phos-phate andnucleotides.Phosphate buffer is commonly usedfor proteinsolution. Knowledgeof the infrared absorptionsignalsof phosphate is thereforeimportant.Theasymmetricand symmetric stretch vibrations of PO2

ÿ in H2PO4ÿ are

located at 1158 and 1068cmÿ1, respectively. The sym-metric stretch and the degenerate stretch of PO2

2ÿ inH2PO4

2ÿ appearat 992and1068cmÿ1, respectively.Thesesignals are influencedwhen phosphatebinds to proteins(Cepuset al., 1998a,b).

Recognitionphenomena

Recognition phenomena in hemeproteins withhistidine asproximal iron ligand

Hemoglobins and myoglobins. CO binds to protein-freeheme with an affinity which is approximately 20000timeshigher than that for O2. However, in proteins such ashemoglobin and myoglobin this ratio is dramaticallylowered to about 25–200. How can hemoglobins andmyoglobins as important oxygen carrier and storageproteins discriminate betweendioxygen and other smallligandswhich act aspoison suchasCO, CNÿ andNO?

CO stretch vibration: Fe—C—Obending or electrostaticpolarization?Fora long time it wasthought thattheproteindestabilizes the CO bond to the heme iron by sterichindrance.This had beenconcludedfrom the Fe—C—Ogeometry, seenin the crystal structureof heme proteins,which deviates from the expected linear orientation.Infraredspectroscopic studies seemed to support this ideaat first view. In myoglobin, essentiallythree CO stretchmodebands[Ao (�1965 cmÿ1), A1,2 (�1945–1954 cmÿ1),andA3 (�1932cmÿ1)] areobserved with different relativeintegral intensities of the bands(Ansari et al., 1987). Inhemoglobin from varioussources,a majorCO stretchbandbetween 1948and1951cmÿ1 andminorbandsin theregionaround 1930,1943and1970cmÿ1 areobserved(Potteretal., 1990).Frauenfelderandcoworkershaveexplainedthedifferent infrared bandswith conformational substates(Astates) of the protein (Frauenfelder, 1997). Ormos et al.(1988)andMoore et al. (1987)assignedthemto differentFe—C—O angles by measuring the linear dichroismfollowing photoselective flashphotolysis of the CO ligandusing infrared spectroscopyat low temperaturesor time-resolved. However, recent infrared dichroism and time-resolved infraredpolarization spectroscopicstudieson CO-myoglobin revealedanangleof �7° from thenormal hemewithout differencesfor thesubconformerA states(Ivanovetal., 1994;SageandJee,1997;Lim etal., 1995a). Therecentcrystal structureat near-atomic resolution (Vojtechovsky et



al., 1999)is in agreementwith thisobservation.Thiscrystalstructureandinfraredstudieswhere aminoacidshavebeenmutated on the heme proximal side (L89I and H97F)demonstratethatthedifferentA substatesarepredominantlycaused by thedistalside(Abadanet al., 1995)andonly thepopulationof A3 is affectedby theinfluenceof theproximalside,asshown for themutantH93G(Decaturet al., 1996).Variousmutationsonthedistalsideof differentmyoglobinsdemonstratedthatthefrequencyrangefor thethreebandsismaintained and only the populationof the subconformerstatesis affected(Braunsteinet al., 1993;Cameronet al.,1993;Li et al., 1994;Andertonet al., 1997;Uchidaet al.,1997a,b; Hildebrandetal., 1998).Forsomeof thesemutantsthecrystalstructurehasbeenresolved, revealingalmostthesameFe—C—O geometry.

At present most of the data indicate that the distalhistidine (His64) in its threeorientationsof the imidazolemodulatesthe dipole momentof the bound CO ligand byhydrogen bonding and/or electrostaticinteractions withouthaving a significant effect on the Fe—C—O angle(Ray etal., 1994;Springeret al., 1994; Rectoret al., 1997).Recenttheoretical studiessupport this conclusion (Kushkuley andStavrov, 1996, 1997a,b). Ao has been assignedto theprotonated histidine in its swung-outconformation [Vojte-chovsky et al., 1999; Fig. 5(A)]. A1 is identified as ahistidine conformation with the lone pair of the imidazolenitrogen atom pointing to the CO ligand with a nitrogen–oxygen distance of 3.2A causing predominantly polarinteraction.A3 shouldcorrespond to thehistidineconforma-tion with theNH pointing in thedirectionof theCOoxygen

atomat a distanceof 2.7A andforming a hydrogen bond.Alternative assignmentsof the Ao, A1 and A3 statesto thehistidine conformationshavebeenperformedby differentauthors, as discussed by Vojtechovsky et al. (1999). Thedeviation of the Fe—C—O geometry from the linearity(til ting andsmall bending),asgenerallyobservedin hemeproteins,hasbeenexplainedusingab initio calculationsbythe effect of the proximal ligand (Jewsbury et al., 1994).

CO —a goodmodelfor O2? Studiesof CO complexesofmyoglobins and hemoglobins are motivated by regardingCO as a model for O2 becauseboth ligands bind to theferroushemeat thesamecoordinationposition.Howeverincontrastto CO,O2 generally bindsin abentorientation[Fig.4(A)]. Potter et al. (1987) have found two O—O stretchmodesignalsat 1150 and 1120cmÿ1 for bovine oxymyo-globin with a minor componentat about1103–1107 cmÿ1.Mill er and Chance (1994) have recently shown byphotolysis differenceinfrared spectroscopythat the O—Ostretch band at around 1103cmÿ1 overlaps with C—Hbending modeof the imidazolein histidine.The two majorsubstatesat 1150and1125–1135 cmÿ1 havebeenrecentlyassignedby high-resolution crystallography to subconfor-mationsof thedistal histidine in myoglobin (Vojtechovskyet al., 1999). The 1135cmÿ1 signal shouldbelong to thedistal histidine conformation which may form a hydrogenbond to theterminaloxygenatomof theO2 ligand(A3 in COcomplex) while the other O—O mode is assignedto thehistidine conformation which inducespolar contact[A1 inCO complex; Fig. 5(A)]. An O—O stretch mode, corre-sponding to theA0 conformationwithout H-bond andpolar

Figure 5. Sketch of the heme pocket in heme proteins indicating the interaction of theCO ligand with distal side groups and the assignment of the CO stretch mode. (A)Myoglobin (according to VojteÏ chovskyÂ et al., 1999); (B) horseradish peroxidase (thepropionic group HOOCprop. is involved in a hydrogen bond network with Gln176,Ser73, Ser35 and Arg31, according to Gajhede et al., 1997); and (C) cytochrome coxidase; the lower frequency belongs to the iron-bound CO and the higher frequencyto the copper-bound CO (according to Mitchel et al., 1996).

334 C. JUNG


contact,doesnot seemto exist.Thereis consensusbetween the various researchersthat

steric constraintson theboundCO ligandby theprotein arenot relevant for discrimination from O2 binding. Thepreferred explanation is that dioxygen can be betterstabilized by hydrogen bonding or enhancedelectrostaticinteractionsbecauseof the largernegativenetchargeat theterminal oxygenatom(Olson andPhillips, 1997;Borman,1999).

H bondingand/orelectrostatic interactions for NO, N3ÿ

andCNÿ ?NO canbindto theferric aswell asferrousstate.Crystal structureanalysesof NO-ferrousform of hemoglo-bins andmyoglobins revealeda bentFe—N—O geometrywith an anglebetween112° and147° (Harutyunyanet al.,1996;Brucker etal., 1998)whichmatchestheangleof 131–149° reportedfor NO ferrous tetraphenylporphyrin com-pounds (Scheidt et al., 1977). Corresponding to thisgeometry and in agreement with the rule by FelthamandEnemark (1981) the NO stretch vibration observedliesbetween that of NO and NOÿ (Fig. 4; 1607–1615 cmÿ1;Mill er et al., 1997).However for ferric myoglobin, theNOstretch vibration has beenobserved at 1927cmÿ1 in thedifferenceinfraredspectrum producedby photodissociationof NO (Miller et al., 1997). This value is in between thefrequency of NO and NO�. This frequencydecreasesto1904cmÿ1 when His64 is replaced by leucine. In contrast,thestretchmodeof theCOligandin theferrousmyoglobin,which is isoelectronic to the ferric NO complex, shifts thepopulationfrom thelower-frequencymodeA1 to thehigher-frequency modeA0 uponmutation. Thisexampleshowsthathydrogen bonding and electrostatic interactions are veryimportant. Ferric NO complexes may be described by aformal charge distribution of FedÿNOd� in contrast toFed�COdÿ. An inverseresponseof the ferric NO and theferrous CO complexes on His64 mutation is thereforeexpected.

Azidemyoglobin andhemoglobinexistin anequilibriumof iron(III) low-spin andhigh-spin states,which is reflectedin the N3

ÿ-vibration mode. Studies by Alben and Fager(1972)andMcCoy andCaughey (1970)assignedthebandsat about2023and2046 cmÿ1 to the low-spinandthehigh-spinstates, respectively.TheN3

ÿ stretch frequency showsasmall shift of the low-spin state bandto lower frequencieswhen a positivechargeis introducedon theprotein surfacenearthe hemepocket(Val67Arg) or to higher frequenciesfor a mutation to a negativecharge(Lys45Glu); Bogumil etal., 1994).This indicatesthatelectrostatic interactionsmaystabilize or destabilize the azide ligand binding, respec-tively. Mutations of the distal His64 induce strongerchanges of the low-spin statecorresponding stretch mode(down-shift by 5–8cmÿ1 for theThr andtheIle mutantsandasignificant increaseof thebandwidth). Thissuggeststhatahydrogen bond between the distal histidine and the azideligand in the low-spin stateshouldexist, which is affectedby mutations. In contrast to N3

ÿ complexes,however, theCO stretch modeis moreaffectedby mutations,asrecentlyobserved for a quadruple mutant (Thr39Ile/Lys45Asp/Phe46Leu/Ile107Phe) of horseheartmyoglobin (Hildebrandet al., 1998), indicating that CO is a bettersensorfor thepolarity on the distal side.

Boffi et al. (1997) have studied the Fe(II) and Fe(III)cyanidecomplexesof homodimeric Scapharca inaequival-

vis hemoglobin. The CN stretch mode for the Fe(III)complex is observedat 2132cmÿ1 and for the Fe(II)complex at 2058cmÿ1. Uchida et al. (1997a) found forvariousmutants at position29 of Fe(III)-myoglobin theCNstretch modebetween 2125and2135cmÿ1. Thereis litt levariation in the CN stretch frequency in the Fe(III)complexesof hemeproteins (Reddyet al., 1996) becausethe CN binding to Fe(III) is dominatedby the s-donationandlittle contributionof p-backdonation exists(KushkuleyandStavrov,1997b). In contrast, the additionalelectron intheFe(II) complexesis distributedoverthes andp* orbitalsof CN resulting in a larger negative net charge on thenitrogen atom of CN, which favors electrostatic interac-tions, andin the decreaseof n(CN).

Summarizing, the ligand stretchmode for the differentdistal iron ligands is predominantly determined by theirgeneral bindingmodeto themetalporphyrinsbutfine-tunedby hydrogenbonding and electrostatic interaction to thedistalsidehistidinein thedifferentorientations.Amongthedifferent ligands CO seemsto be most sensitive to thepolarity on the distal sidewhile hydrogenbondingmay bemore important for O2. Theotherligandsarein betweenorlessaffected.However, it is difficult to estimatetheconcreterelative contribution of theseboth interactionsfor stabiliz-ing the boundligand.

Peroxidases.Peroxidases form a large family of hemeproteinswhich catalyze the oxidationof varioussubstratesusing hydrogen peroxideas oxidizing agentwhose O—Obond is heterolytically split (Dunford andStillman, 1976).Two recognition phenomenaoccur—the binding of hydro-gen peroxide to the heme iron, and the binding of thesubstrate.Thereis no high substrate specificity. Chemicaland crystallographic data indicate that the accessof thehemepocketfor smallsubstratesis restricted.H2O2 bindingto the heme iron is the more important recognitionphenomenom. The split of the O—O bond in hydrogenperoxide is an acid–base catalyzedprocesswherehistidineon the distal hemesideservesasthe proton acceptor. TheH2O2/protein recognition cannotbe studiedwith conven-tional methodsbecauseof the short life time of the Fe—H2O2 complex (Miller et al., 1994).Therefore, iron ligandslike CO,O2, NO andCNÿ havebeenusedasspectroscopicprobes to study thecapability of thedistalsideto donateoraccept a proton.

Amongtheperoxidases,horseradish peroxidaseis oneofthe best studied proteins. Infrared studies on differentisozymes indicated two CO stretch modes, around1905 cmÿ1 at low pH and around1932cmÿ1 at high pH(Barlow et al., 1976). H2O/D2O exchange (Smith et al.,1983) andpH dependenceinfraredstudiesclearly indicatedthat the low-frequency mode corresponds to a hydrogenbond betweenCO andan aminoacid sidechainwhich hasbeenshown to be a histidine [Gajhedeet al., 1997; Fig.5(B)]. Both conformersareinterconvertible eitherby pH orby binding of the substrate (benzhydroxamate; Uno et al.,1987). Distal andproximal sidesarestronglycoupled by ahydrogenbondnetwork involving variousaminoacid sidechains,awatermolecule andthehemepropionate [Gajhedeet al., 1997;Fig. 5(B)]. This maycontributeto thestronglyloweredCO stretch modefrequencyat low pH. Thestretchmodeof the CNÿ ligand hasbeentaken asa betterprobe



than the CO ligand becauseit approchesthe iron asHCNand donates the proton to the distal histidine whencoordinating to iron. This mimics the situation whenhydrogen peroxide binds. Bound CNÿ exists in twoconformations (almost linear and tilted, respectively)suggested from resonance Raman studies. In both con-formers CNÿ is suggested to be hydrogen bondedto thedistal histidineand/orto other distal groups(Tanaka et al.,1997). The CN stretchmode frequency is therefore verysensitive whenstructural changes areinducedby mutationsaround the histidine.

FTIR studieson yeastcytochromec peroxidase,anothermember of the peroxidase family, show that similar tohorseradishperoxidasetwo conformersin the CO complexexistwhich interconvertby changing thepH. However,theCO stretch frequency of the conformer at low pH,corresponding to the 1905cmÿ1, conformer in horseradishperoxidase,is up-shifted to 1922cmÿ1, indicating a muchweaker hydrogen bond to the distal histidine (His52;Smulevich et al., 1986).

Oxidases. Oxidasescatalyze the reductionof dioxygentowater (Gennis,1998). The active site consists of a hemewith a coppercentervery close (�4.5–5.2A) to the hemeiron (binuclearreaction center;Michel et al., 1998). Theinteresting recognition phenomena are the binding ofdioxygen in the binuclear reaction centerand the transferof electronsandprotonsto O2 (Yamazakiet al., 1999).Thebinuclearreaction centerhasbeenextensivelycharacterizedalsoby FTIR usingCO,NO, CNÿ andN3

ÿ asspectroscopicprobes (Tsubaki et al., 1996, 1997; Zhao et al., 1994).Dependingon the redoxstate of thedifferentredoxcenterswithin the bovine heart cytochrome c oxidase,the stretchfrequency of the CO ligand is observedbetween 1959and1965cmÿ1 (Yoshikawa et al., 1977; Yoshikawa andCaughey,1982).Themost striking finding is thevery smallwidth of the CO stretch band <4 cmÿ1 (compared to

hemoglobin >8 cmÿ1), which has beenexplained with avery stableand restricted CO binding arrangement in anapolar environment (Fiamingo et al., 1982). Two COconformers are observedin the bacterial aa3-type cyto-chrome c oxidase from Rhodobacter sphaeroides withresonanceRamanaswell as infrared studies (Wanget al.,1995). The higher frequency n(CO) modeat �1966cmÿ1

correspondsto then(Fe—CO)stretchingmodeat519cmÿ1,named the a-form, while the lower-frequency n(CO) modeat �1955cmÿ1 is assigned to the n(Fe—CO) stretchingmode at 493cmÿ1 (b-form). Surprisingly, the a-form ofcytochromec oxidasefrom differentsourcesshowsunusualpropertiesregarding therelation between n(CO) andn(Fe—CO). For heme carbonyl complexes with histidine asproximal ligand thereis an inverselinear relation betweenn(Fe—CO) andn(CO) (Fig. 6; Li andSpiro, 1988;Ray etal., 1994;Andertonetal., 1997).Cytochromec oxidaseliesoff from this line and is shifted to higher CO stretchfrequencies, normally observedonly for weak proximalligands or ligand-free carbonyl porphyrin complexes,althoughhistidine as proximal ligand has beenidentifiedin this protein.

Theproximity of thecoppercentercanbeprobedwhentheCOligandisphotodissociatedfromthehemeiron[Fig.5(C)].ThedissociatedCOis trappedon thecopperandshowstwoCO stretch modes for the copper-bound CO at 2037–2039 cmÿ1 (corresponding to the b-form) and at 2061–2064 cmÿ1 (corresponding to thea-form) in caseof bovineheart cytochromecoxidaseandof theaa3-typecytochromecoxidase from Rhodobacter sphaeroides (Fiamingo et al.,1982; Wanget al., 1995).The a- andb-form differ in thepolarity around thebinuclear center (Mitchell et al., 1996).Iwaseet al. (1999) have recently reported time-resolvedFTIR studies of the CO photodissociation of bovinecytochrome c oxidase. The light minus dark differencespectrum indicated the CO stretch of CuB—CO at2063 cmÿ1. A negative bandat 1737cmÿ1, resultingfromthe CO stretchin protonatedCOOH, and a positive bandbetween 1550and1600cmÿ1, corresponding to the asym-metricstretchmodeof COOÿ, suggeststhatdeprotonationofa carboxylate(presumably Glu242) is accompaniedby thephotoinducedCOtransfer from thehemeiron to thecopper.

Puustinenet al. (1997) have recently shown in a veryelegant way how FTIR can help to uncover recognitionphenomena. The light minus dark difference infraredspectrum at 80K has been measured for the carbonmonoxide complex of cytochrome bo3 oxidase fromEscherichia coli and its Glu286Asp and Glu286Cysmutants.Signalsin differentspectral regions,correspondingto differentstructuralsitesin theprotein,aresimultaneouslymonitored:(i) CO stretchmodeat 1960andat 2065cmÿ1,for CO boundat the iron andthecopper, respectively [Fig.7(A)]; (ii) a derivative-shapespectral changearound1724–1731 cmÿ1 assigned to a changed hydrogen bondedcarboxylic acid C=O [inset in Fig. 7(A)]; (iii) a spectralshift at 3145–3135 cmÿ1 assignedto weakening of ahistidine imidazole C—H stretching;and (iv) a derivativespectral changeat 3020and3080cmÿ1 resultingfrom theC—H stretch of the heme vinyl group or methine bridge[Fig. 7(B)]. Signals (ii) are sensitive against mutations atposition 286demonstratingahydrogen-bondedconnectivitybetween Glu286and a histidine copperligandwhich may

Figure 6. Correlation of the iron-bound CÐO and the FeÐCOstretch mode frequencies for various heme proteins andporphyrin±iron±CO complexes. Data are taken from Table 1 inRay et al. (1994) for the weak proximal ligand (benzene,tetrahydrofuran) and for various hemoglobins and myoglobinsfor the imidazole ligand and from Legrand et al. (1995) for thethiolate ligand (cytochrome P450cam±CO in the presence ofvarious substrates).

336 C. JUNG


play a role in theprotontranslocation andtherefore for thedioxygenreductionto water.Analogousstudies havebeenperformed on bd-type ubiquinol oxidase from Escherichiacoli using CNÿ asprobemolecule. Thesestudiesshowthat

CNÿ is releasedfrom thecomplex uponphotoreduction andthat a changeof thehydrogenbondingof a cysteineS—H,monitored at the shift of the S—H stretchmode, occurs(Yamazakiet al., 1999).

Figure 7. FTIR `light minus dark' difference spectrum for the CO complex ofwild type cytochrome bo3 at 80 K (reprinted with permission from Puustinen etal., 1997, copyright (1997) American Chemical Society). (A) spectral region ofthe CO metal ligand stretch mode. [The CO stretch mode in the iron-boundform and copper-bound form are seen as a negative band at 1960 cmÿ1 and apositive band at 2065 cmÿ1, respectively. Simultaneously, a derivative-shapespectral change is observed at 1724 cmÿ1 (negative signal) and at 1731 cmÿ1

(positive signal; inset) assigned to a weaker hydrogen bonded carboxylic acidC=O in the complex when CO is bound to the copper center. This spectralfeature shifts to 1756 and 1761 cmÿ1, respectively, when Glu286 is substitutedby Asp and is completely lost for the Glu286Cys mutant.] (B) A spectral shift isobserved at 3145±3135 cmÿ1 upon CO binding to copper CuB, which can beassigned to weakening of a histidine imidazole CÐH stretching. In addition, aderivative spectral change is seen at 3020 and 3080 cmÿ1 resulting from theCÐH stretch of the heme vinyl group or methine bridge.



Table 4. CO stretch mode frequencies for the CO complex of cytochromes P450 at room temperature (Gauss curve®t data in case of overlapping bands)

n(CO) (Dn1/2, population)(cmÿ1)

Protein H2O D2O Reference

P-450lm2(CYP2B4) 1949(15–16) 1949(14) Bohm et al. (1976)P-450lm4 1954(15), 1961(s) 1954(13–14),1961 Bohm et al. (1979)P-450rlm2(CYP2B1) 1948(25) 1948(20) Bohm et al. (1979)P-450rlm2(CYP2B1) 1948 O’Keefeet al. (1978)P-450rlm4 1952 1952(21) Bohm et al. (1979)P-450rlm4 1954(30) O’Keefeet al. (1978)P-450scc(CYP11A1)

Substrate-free,ÿAdx 1952.5(12.6) Tsubakiet al. (1992)Substrate-free,�Adx 1951.5(12.4) Tsubakiet al. (1992)Cholesterol,ÿAdx 1953.7(12.9) Tsubakiet al. (1992)Cholesterol,�Adx 1951.9(12.5) Tsubakiet al. (1992)25-OH-Cholesterol,ÿAdx 1954.7(9.9) Tsubakiet al. (1992)25-OH-Cholesterol,�Adx 1954.6(10.4) Tsubakiet al. (1992)22(R)-OH-Cholesterol,ÿAdx 1951.8(12.6) Tsubakiet al. (1992)

1934.5(11.6)22(R)-OH-Cholesterol,�Adx 1951.7(12.6) Tsubakiet al. (1992)

1933.4(12.6)22(S)-OH-Cholesterol,ÿAdx 1946.8(10.4) Tsubakiet al. (1992)22(S)-OH-Cholesterol,�Adx 1946.2(10.7) Tsubakiet al. (1992)20(S)-OH-Cholesterol,ÿAdx 1949.5(17.0) Tsubakiet al. (1992)20(S)-OH-Cholesterol,�Adx 1946.2(17.7) Tsubakiet al. (1992)20,22-(OH)2-Cholesterol,�Adx 1937.2(13.6) Tsubakiet al. (1992)22-Ketocholesterol,ÿAdx 1950.6(13.0) Tsubakiet al. (1992)22-Ketocholesterol,�Adx 1949.0(13.2) Tsubakiet al. (1992)

P-45011b (CYP11B1)Deoxycorticosterone,ÿAdx 1937.3(8.8) Tsubakiet al. (1992)Deoxycorticosterone,�Adx 1937.2(9.1) Tsubakiet al. (1992)

P-45015b (CYP106A2)Substrate-free 1937.5(13.4,0.156) Simgenet al. (2000)

1946.3(12.5,0.581)1957.4(14,6,0.231)1968.6(6.9,0.023)1984.0(7.4,0.009)

deoxycorticosterone 1936.0(17.7,0.568) Simgenet al. (2000)1947.2(9.4,0.068)1960.1(18.0,0.289)1976.6(11.1,0.075)

P-450lin(CYP111)Substrate-free 1944.3(9.5) JungandMarlow (1987)

1955.8(6.5)1965.9(22.9)

Linalool-bound 1953.0(10.0) JungandMarlow (1987)P-450cam(CYP101)

Substrate-free 1942(19–21) O’Keefeet al. (1978)1963(11–12)

(1R)-Camphor 1940(13)Substrate-free,type 1 1939.8(12.0,0.52) 1938.4(18.6,0.44) Junget al. (1996b);Legrandet al.

(1995)1949.4(10.4,0.07) 1953.1(11.1,0.05)1955.3(19.2,0.41) 1962.6(14.3,0.51)

(1R)-Camphor 1939.8(12.7) 1940.2(12.2,1.00) Junget al. (1996b);Legrandet al.(1995);Schulzeet al. (1996)

(1S)-Camphor 1940.7(9.5) 1941.4(10.2,1.00) Junget al. (1996b);Legrandet al.(1995);Schulzeet al. (1996)

(1R)-Camphorquinone 1940.5(10.6) Junget al. (1996b)(1S)-Camphorquinone 1939.6(9.1) Junget al. (1996b)

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Recognition phenomenain hemeproteins withcysteinateas proximal iron ligand

Three hemeproteins have a deprotonated cysteine as theproximal iron ligand:cytochromesP450,NO synthasesandchloroperoxidase.Althoughthehemecomplexcoordination

is very similar, the secondaryand tertiary structures arecompletely different (Hasemann et al., 1995;Craneet al.,1999; Fischmann et al., 1999; Li et al., 1999; Sundar-amoorthy et al., 1993). In contrast to myoglobins andhemoglobins, for which the recognition of the iron ligandsthemselvesis thepointof interest, in thehemethiolateheme

Table 4. continued.

n(CO) (Dn1/2, population)(cmÿ1)

Protein H2O D2O Reference

Adamantanone 1941.5(9.0) Junget al. (1996b)1942.3(9.1) Junget al. (1992b)

Adamantane 1955.0(11.7,1.00) Junget al. (1996b)1928.6(41.4,0.162) Junget al. (1992b)1939.9(16.3,0.241)1955.0(14.1,0.597)

1-Azidoadamantane 1945.5(13.2) Junget al. (1996b)1-Chloroadamantane 1943.8(8.3,0.88) Junget al. (1996b)

1952.6(10.0,0.12)1-Bromoadamantane 1943.1(8.4,0.60) Junget al. (1996b)

1950.8(13.1,0.40)1-Iodoadamantane 1943.2(9.9,0.16) Junget al. (1996b)

1954.6(11.6,0.84)1-Bromodimethyl-adamantan 1941.6(11.1,0.20) Junget al. (1996b)

1955.2(10.9,0.80)1-Chlorodimethyl-adamantane 1943.4(12.2,0.14) Junget al. (1996b)

1955.3(10.9,0.86)Fenchone 1944.5(13.8) 1944.1(10.8) Junget al. (1996b);Legrandet al.

(1995)1944.7(11.1) Junget al. (1992b)

Endo-borneolallyl ether 1944.0(10.5,0.74) Junget al. (1996b)1952.2(12.4,0.26)

Camphane 1941.5(15.7,0.60) 1942.9(19.4,0.29); Junget al. (1996b);Legrandet al.(1995)1955.3(10.8,0.27) 1951.9(10.8,0.71)

1961.9(21.0,0.13)1940.9(10.5,0.119) Junget al. (1992b)1953.7(14.0,0.881)

Norcamphor 1946.1(10.3) 1946.4(9.8) Junget al. (1996b);Legrandet al.(1995)

1947.0(10.1) Junget al. (1992b)Norbornane 1953.0(10.0) Junget al. (1996b)3-Endo-norborneol 1951.5(9.0) Junget al. (1996b)Endo-borneolpropyl ether 1944.7(8.0,0.20) Junget al. (1996b)

1954.7(12.0,0.70)1965.0(12.6,0.10)

Tetramethylcyclohexanone 1933.3(10.9) Junget al. (1996b)1934.2(9.7) Junget al. (1992b)

3-Bromocamphor 1933.7(10.6)P-450cammutantD251N,

1R-camphorbound1938.2(10.4) ContzenandJung(1998)

P-450cam� Pdx 1932 Unnoet al. (1997)P-420

P-420lm2 1966(20) Bohm et al. (1976)P-420lm4 1972 Bohm et al. (1979)P-420rlm2 1966 Bohm et al. (1979)P-420rlm4 1970 Bohm et al. (1979)P-420cam 1964 Mouro et al. (1997)

Adx = adrenodoxin;Pdx= putidaredoxin.



proteins the substrate binding and recognition is anadditional importantphenomenon.

Cytochromes P450. First infrared spectroscopic studieswere already carried out on a microsomal P450 in 1976(Bohm et al., 1976) andon the bacterialP450camin 1978(O’Keefeet al., 1978).Later,manystudieshavebeendoneon P450camas function of substrate binding, solvent,temperature and pressure. A summary of the CO stretchmodes, observedso far, is given in Table4.

P450camCO ligand stretchmodeandsubstrate binding.In the absence of any substrate a broad, structuredbandisobserved which results from the overlap of varioussubbands (Jung and Marlow, 1987; Jung et al., 1992b,1996a). P450camhasno aminoacidsidechainon thedistalside which could specifically interactwith the CO ligandand therefore cause the sub-bands as is the case inmyoglobin with the distal histidine. However, substratebinding has a remarkable effect on the CO stretchmode(Junget al., 1992b,1996b; Fig.8). The numberof stretchmode bands is restricted compared to the substrate-freeprotein andthebandfrequenciesstrongly differ. Inspectionof the three-dimensionalcrystal structure of P450cam-COin the (1R)-camphor complex (Raag and Poulos, 1989)revealsthatthesubstrateis in vanderWaalscontactwith theCO ligand.Similar to the hemeproteinswith imidazole asproximal ligand, the CO ligand is tilted (�9°) andslightlybent166° (Fig. 9).

In the first study on various substrate-P450cam com-plexes,Junget al. (1992b) suggested that watermoleculesin the hemepocketmay interfere with the CO ligand andmight, therefore, be responsible for the different Fe-COconformers. The different subconformers were originally

assignedto differentFe—C—O anglesin analogyto dataofOrmoset al. (1988)for myoglobin. Froma studyof a largenumber of substratecomplexes,however, it turnedout thatthere existsanalmostlinearrelationbetweenthecontentoftheiron high-spinstateof thecomplex in theferric form andthe effective CO stretch frequency n'(CO) at roomtemperatureas shownin Fig. 10(A) (Junget al., 1996b).Complexeswith asmallhigh-spinstatecontenttendto havea CO stretch mode at higher frequencies. The high-spincomplex of P450shas been regardedas representing anapolarandthelow-spincomplexapolar hemeenvironment.The increase of the CO stretchfrequency when the hemeenvironmentbecomesmore polar is unexpected. Usually,for protein-free heme carbonyl complexes the inversebehavior is observed. Polar solvents induce a decreaseoftheCOstretchmodewavenumberdueto adirectinteractionbetween the solventmolecules and the CO ligand.There-fore, an indirect effect of the polarity of the hemeenvironmentin P450camshouldexist. It wasproposedthatthe electrostatic field of the watermolecules present in thelow-spin state might partially compensate the supposedpositive electrostatic potential in the I-helix groovearoundThr252 nearthe CO ligand (Junget al., 1996b). Hydrogenbonding between theCO ligandandaminoacid residuesinthe vicinity of the heme has been excluded from pH-dependencestudiesof the CO stretchmode in camphor-bound P450cam(Schulzeet al., 1994b).Hydrogenbondingwould be indicated by an population exchange betweensubconformers when the pH is changed, as seen formyoglobin (Muller et al., 1999)or horseradish peroxidase(Barlow et al., 1976). Such effectshavenot beenobservedfor P450camin the presenceof (1R)-camphor, althoughasmall pH-dependentshift of the n(CO) by about1.5cmÿ1

with a pKa of about6.2 is visible. This pKa is in agreement

Figure 8. CO stretch mode infrared spectra of various substratecomplexes of cytochrome P450cam. (1R)-camphor (1); (1S)-camphor (2); (1S)-camphor quinone (3); (1R)-camphor quinone(4); adamantanone (5); 1-azidoadamantane (6); 1-chloroadaman-tane (7); fenchone (8); 1-bromoadamantane (9); endo-borneolallyl ether (10); camphane (11); 1-iodoadamantane (12); norcam-phor (13); norbornane (14); adamantane (15); 1-bromodimethyl-adamantane (16); 3-endo-norborneol (17); 1-chlorodimethyl-adamantane (18); endo-borneol propyl ether (19); tetramethyl-cyclohexanone (20) (Jung et al., 1996b).

Figure 9. Active site of (1R)-camphor-bound cytochromeP450cam-CO. The coordinates are taken from the PDB entrycode 3cpp (Raag and Poulos, 1989). Sketch of camphor analogs.

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with the pKa of 6 observed for the high-spin/low-spinequilibrium.

P450camCO ligand stretchmodeand chargedonationfrom the proximal cysteinate ligand. Since the discoverythat a negatively charged sulfur of a cysteine is the fifthligand, the uniquefunction of P450to activate molecularoxygenhasbeenseenin thechargedonationfrom thesulfurto the dioxygenligand. Quantumchemical calculations ofthe O2 aswell CO complexesof porphyrin iron complexeswith either imidazole or sulfur clearly indicated that thecharge donationto O2 or CO is more pronouncedfor thenegativesulfur ligandthanfor imidazole.Therefore,alowerCO or O2 stretch modefrequency shouldbe expected forP450s compared to myoglobins or hemoglobins. Theexperimental studies were,however, disappointingat first,revealing CO stretchmodes in the samewide frequencyrangeasalso observedfor hemeproteins with histidine asthefifth ligand.Forhemeproteinsandtheir mutantsandforiron porphyrin complexeswith imidazoleasfifth ligand,aninverse linear correlation between the CO stretch modefrequency andtheFe—CO stretchmodefrequency hasbeenobserved from resonanceRamanand infrared studies,asalready discussed above (Fig. 6). For weak proximalligands, as observed in some model complexes andcytochromec oxidase, or in the absence of the proximalligand, the line is shifted to higher frequencies. Such aninverserelationbetweenn(CO) andn(Fe-CO) hasalsobeenshown for various substrate complexes of P450cam(Legrandet al., 1995). However the line is shiftedto lowern(CO) values comparedwith the line for myoglobins and

hemoglobins. So, the stronger charge donation from thecysteineproximal ligandis not directly seenin theabsolutevaluesof n(CO) but in the shiftedline.

P450cam CO ligand stretch mode and relation tofunction. CO rebinding after flashphotolysis has recentlybeencarried out by Contzen and Jung(1998) for severalsubstrate complexes of P450camusing step-scan time-resolved FT infrared spectroscopy. The CO rebinding isveryfastfor substratecomplexeswhicharecharacterizedbyasmallhigh-spincontent, by afastentryof watermoleculesinto the hemepocket(Schulze et al., 1997)andby a highfrequencyof theCO stretchmodein theCO complex (Junget al., 1996b). Studieson theP450camsubstrate complexesusing FTIR andseveralothermethodsrevealed a consistentpicture that substratesmissingor having disturbedhydro-phobic contactsto Val295,Asp297andVal247(Fig. 9) aremore mobile in the active center (Contzen et al., 1996;Schulzeet al., 1996;Schlichting et al., 1997),allow higheraccess of the protein for water molecules(Schulzeet al.,1997), induce a high compressibility or volume fluctuationof theprotein (Junget al., 1995),causea high apparentCOstretch modein the CO complex (Junget al., 1996b)andshowa prefered formation of hydrogenperoxide instead ofthe substrate hydroxylation. Figure 10(B) showsa correla-tion betweenthe apparent CO stretchmode frequency atroom temperature for various P450cam–CO substratecomplexes with the percentage of consumed dioxygenwhich is converted to hydrogenperoxide in activity studies.The H2O2 formation represents a sidereactionin the P450reaction cycle after O2 has bound to the heme iron. It

Figure 10. Relation of the CO ligand stretch mode frequency in cytochromeP450cam with two further functional important parameters. (A) Correlation of theapparent CO stretch mode frequency in the CO complex of various substratecomplexes of P450cam (Fig. 8) with the high-spin state content induced by thesubstrates in the Fe3� form (Jung et al., 1996b). The inset indicates the heme ironcoordination sphere in the Fe3� form for the substrate-free protein with 100% low-spin-state population and the (1R)-camphor-bound protein with 100% high-spinstate population (Poulos et al., 1987). The intermediate high-spin state contentsfor the various substrate complexes can be explained by the mobility of thesubstrate inside the heme pocket, which allows a certain accessibility of the hemepocket for water molecules (Raag and Poulos, 1991; Jung et al., 1996b; Schulze etal., 1997). (B) Correlation of the apparent CO stretch mode frequency in thecytochrome P450cam±CO complex in the presence of various substrates (Jung etal., 1996b) with the percentage amount of dioxygen which is converted in thereaction cycle of P450cam to hydrogen peroxide (%H2O2; uncoupling sidereaction) in activity studies using the complete monooxygenase system (Kozinand Hui Bon Hoa, 2000, in preparation). (1R)-camphor (1); (1S)-camphor (2);camphor-N-methylimine (3); camphor-oxime (4); fenchone (5); norcamphor (6);camphane (7); endo-borneol propyl ether (8).



competes with the expected formation of the iron-oxointermediate which hydroxylatessubstrates (Lewis, 1996).Whetherthen(O—O) stretchvibration modecorrelateswiththeH2O2 formationsimilarily to theCOstretchmodeis notknown. FT infraredstudiesof theO2 complex of P450havenot been carried out becauseof the instability of thecomplex and its difficult handling with FTIR. FewresonanceRaman studiesexist which revealed the O—Ostretch mode for (1R)-camphor-bound P450cam at1140cmÿ1 (Bangcharoenpaurpong et al., 1986; Hu et al.,1991; Macdonald et al., 1999) which is in the regionexpectedfor a bent-on Fe—O—O geometry (Schlichting etal., 2000).

CO ligand stretch modes of steroid converting P450s.Results of FTIR studies on other P450s are not sostraightforward. Correlationsas seenin Figs 6 and 10(A)fail to hold within the various substrate complexes ofP450scc–CO(Tsubaki et al., 1992;Table4). Thesubstrate-free protein (almost low-spin) and the cholesterol-boundprotein (almost high-spin), show a similar CO-stretchfrequency at around 1952cmÿ1. Equally surprising arerecent infrared studies on P45015b, which is completelylow-spin in the substrate-freeas well as substrate-boundstate (deoxycorticosterone; Simgen et al., 2000). In theabsence of the substrate, severalCO stretchmodesexistwith a major band at about 1946cmÿ1 (Table 4). Thepopulation of the lower-frequency mode at about1936cmÿ1 is strongly increasedwhendeoxycorticosteroneis bound.In contrast to substratesof P450cam,the steroidsubstrates for P450scc and P45015b are rather bulky.Therefore steric constraints may be more relevant in theseP450sthan the compensating electrostatic effect of watermoleculesdiscussed for P450cam.

NO synthase.Thebiogenesisof nitric oxideis catalyzedbyNO synthases(NOSs) which form L-citrulline and NOthrougha stepwiseNADPH andO2 dependentoxidation ofL-arginine (Stuehr, 1999).The first FTIR study of the COstretchvibrationmodeof theinducible nitric oxidesynthaseoxygenasedomain (iNOSox)hasrecently beenreportedbyJung et al. (2000) for the temperature rangefrom 20 to298K. iNOSox in the absenceof arginine reveals atemperature-dependentequilibrium of two majorconforma-tional substateswith CO stretch bandscenteredat about1945 and 1954cmÿ1. This behavior is not qualitativelychanged whentetrahydrobiopterin (H4B) is bound.Argininebindingchanges thespectrum significantly by formationofa sharp CO stretch mode band at about 1904cmÿ1,indicating the formation of a hydrogen bond to the COligand.It is suggested thatthearginineitself maydonatethehydrogen. It is an open question whether the proposedhydrogen bond is also present for the dioxygen complexbecauserecent resonanceRaman measurementsof theO—O stretch vibration, observed at 1135cmÿ1, did not revealan influenceof the arginine binding (Coutureet al., 2000).

Chloroperoxidase. Chloroperoxidase isolated from Cal-dariomycesfumagohasbeenshownby variousmethodstohavea cysteinate proximal hemeiron ligand (DawsonandSono, 1987; Sundaramoorthy et al., 1993). This enzymecatalyzesthehydrogenperoxide-dependentoxidationof Iÿ,Brÿ and Clÿ with the resulting formation of a carbon–

halogen bond with various compounds. The vibrationalproperties of the heme complex havemainly beenstudiedby resonanceRaman spectroscopy(Hu andKincaid, 1993).Infrareddataarereported by O’Keefeetal., (1978),giving aCO stretch modeat 1942cmÿ1 for pH 3 andat 1958cmÿ1

for pH 6.Scholl (1991)hasshown thattheinfraredspectrumof theCOligandstretchmodeis verycomplex,with severalbandsbetween1930 and 1970cmÿ1 which are stronglydependentonthepH, temperatureandpressure. BecausethepH dependenceis completely different from P450camonemay conclude that the interactions with CO on the distalside are very different. Indeed, the crystal structure(Sundaramoorthy et al., 1993) revealsa glutamic acid inthe active sidewhich can inducehydrogen bondingto theCO ligand. In this regard, chloroperoxidaseis similar toother peroxidaseswhich have a distal histidine hydrogenbonding to the CO ligand.

Recognition phenomena in non-hememetal centers inproteins

Ca2� binding. Various proteins havemetal centerswhichstabilize theprotein structure(Jacksonet al., 1991)or haveother functions (Ermler et al., 1998). The effect of Ca2�

bindingonthesecondarystructuremonitoredontheamideIbandhasrecentlybeenreviewedby Surewicz andMantsch(1996)andArrondoandGoni (1999).Metal ionsareheldinplace by coordinating histidines or carboxylatesidechainsfrom glutamateor aspartate. Naraet al. (1994)haveshownfor pike parvalbumin, which belongs to the large class ofCa2� bindingproteins,that theasymmetricstretchmodeofCOOÿ can be usedas markerbandfor metal binding. Inneutral D2O, the b–COOÿ of aspartate showsa band at1584 cmÿ1 and the –COOÿ group of glutamate at1567 cmÿ1. These signals are down-shifted to 1547 and1551 cmÿ1, respectively, when coordinating Ca2� in adidentateform. Ca2� binding in horseradish peroxidasehasalso been monitored using the antisymmetric stretchvibration observedat 1554cmÿ1 (Kaposi et al., 1999).Dzwolak et al. (1999)usedthis spectralmarkerto study theeffectof Ca2� on thebarostability of bovinea-lactalbumin.The symmetric stretch vibration observed at about1388 cmÿ1 has also beenshown to be affectedby Ca2�

binding to a-lactalbumin and lysozyme (Mizuguchi et al.,1997).

Hydr ogenases. Hydrogenasesform a family of enzymesfound in various microorganisms which split molecularhydrogen (Ermler et al., 1998). Two types of classesofhydrogenasesareknown: theNi/Fe hydrogenaseswhereNiand Fe form a binuclearcenterand the Fe hydrogenaseswhichcontainonly iron in theactive center. In 1994Bagleyet al. reported infrared spectra of Ni/Fe hydrogenases,revealingunusualsignalsat1944cmÿ1 (strong) andat2081and2093cmÿ1 (weaker) in theoxidized form whichshift to1929, 2060 and 2069cmÿ1 when reducedwith ascorbateunder CO atmosphere. Van der Spek et al. (1996) haveshown thatsuchsignalsareexclusively observedfor Ni/Fe-hydrogenasesand Fe-hydrogenasesand not seenin othernon-metal-hydrogenases or various iron–sulfur proteins.Basedon 13C and15N labelingof Ni/Fe-hydrogenase from

342 C. JUNG


Desulfovibrio gigas, Happeet al. (1997) have found thattwo internal cyanidegroupsandoneinternalCO moleculeareboundto theFeion in theNi–Febinuclearcenter,whichcauses the infrared bandsat 2093, 2083 and 1945cmÿ1,respectively.

Substrate binding. Substratebinding and recognition bytheenzymecanbefollowedby FTIR in differentways.Onecan monitor a spectroscopic probe molecule near thesubstrate binding site as demonstratedabovefor P450orthe substrates itself have infrared active modes of highdiagnostic value which are affected by the binding. Forexample triosephosphate isomerasecatalyzes the conver-sion of dihydroxyacetonephosphate (DHAP) to D-glycer-aldehyde 3-phosphate (GAP). The carbonyl group ofunboundDHAP appears at 1730cmÿ1 and is shifted to1715cmÿ1 when boundto the enzyme.This shift indicatesthatthecarbonyl groupof thesubstrate is morepolarizedinthe bound state by forming electrostatic or hydrogenbonding interactionswith the active site histidine His95(Zhanetal., 1999).Increasedpolarization is required for thefunctionally importantprotonshuttle.

The effect of substrateand ligand binding on thesecondary structurecan also be followed on the amide Iband. Usually, data about global structural changes areobtainedwhich areless informativefor specific recognitionprocesses. Further examples of monitoring substrate-specificinfraredprobes andeffects of substrate binding on

protein unfolding are discussed by Surewicz and Mantsch(1996).

Study of protein–ligand dynamicsusing low-temperature, high-pressure and time-resolvedFTIR

FTIR spectroscopyhasbeenusedto studythedynamics ofproteins and protein–ligand interactions. Two approachesareapplied: (i) analysis of the changeof the population ofconformational substates with changing the temperature(and/or pressure);and (ii) following the ligand rebindingprocess or conformational relaxation using time-resolvedFTIR. Most of the studieshavebeenperformedon hemeproteins.

Static low-temperature and high-pressure studies ofheme protein CO complexes. With lowering of thetemperature (and/or increasing the pressure) a populationexchangebetweenconformational substates,reflected in thedifferent CO stretch modes,is observed.This exchangegenerally stopsrathersharplywhen passingthetemperaturerangebetween200and170K, that is the liquid/glassphasetransition of the solvent/co-solventmixture (Jung et al.,1996a) where the population equilibria ‘freeze-in’ (Mayer,1994a) or the substate transitionsare strongly ‘damped’because of the high viscosity. Figure 11 shows thetemperature and pressure dependence of the CO stretchmodeinfraredspectraof P450camin theabsenceandin the

Figure 11. CO stretch mode infrared spectra of cytochrome P450cam±CO. In the absence(top) and in the presence of (1R)-camphor (buttom) as a function of the temperature(left), of the hydrostatic pressure (middle) and for selected times after a pressure jump(fast release from 200 to 40 MPa) (right). Experimental conditions are described inSchulze et al. (1994b), Scholl (1991) and Jung et al. (1992a, 1996b).



presenceof thesubstrate(1R)-camphor. In theabsenceof asubstrate, decreasingtemperatureshifts the substate equili-brium to the lower-frequency mode while increasinghydrostatic pressure favors the substate with the higher-frequency mode (Junget al., 1996a).The kinetics of thesubstate transitionshasbeenstudied with thepressurejump(fastreleasefrom 200to 40MPa)technique(Frauenfelder etal., 1990;Scholl, 1991; Junget al., 1992a).WhenP450camrecognizes the substrate (1R)-camphor, the number ofsubstatesis significantly restricted.However, low-tempera-ture and high-pressure studies reveal that at least twosubstatesarepresentwhoseinterconversioncanbefollowedby the pressurejump technique (Schulze et al., 1994b;Scholl, 1991,Junget al., 1992a).

The CO stretch mode frequency and width of the COstretch bands are also affected by the temperature(pressure). The frequencyis shifted by several wavenum-bers(�5 cmÿ1 from 298to 200K)with almostnochangeora changewith a much smaller slope when further cooleddown to 20K (JungandMarlow, 1987; Schulze, 1997).Inmyoglobin, for example, a frequency shift to highervaluesis observedfor modeAo andmodeA1 (Ansariet al., 1987).ModeA3 tends to shift downwith cooling (Mayer,1994b).However, in hydrated films a sharp breakdown of thechangesin populationandfrequencyataround 180–200K isnot observed(Mayer, 1994b) so water as an importantcomponent in the protein strongly enhancesthe internalmobility of theprotein (FischerandVerma,1999)resultingin asharp changeof theinfraredparametersataround180K.In P450cam, in the presenceof various substrates, adecreaseof the CO stretch modefrequencyis observed oncooling from 298Kto about180Kwith amuchsmallerslopebelow this temperature (Schulzeet al., 1994b; Schulze,1997). However for substrate-free P450cam, which isknown to have more water in the protein, the higher-frequency CO stretchmodeat about1954cmÿ1 shifts upwith cooling. For this and also for the mode at around1963cmÿ1, an inverseeffect of hydrostatic pressureand

glycerol (interpretedasosmotic pressure)on the frequencyhas been observed (Jung et al., 1996a). The inverseinfluence of hydrostatic and osmotic pressure on the COstretch modes indicatesthat these higher-frequencymodesreflect subconformations which are more accessible forwater.

Thus, the effect of temperature andpressureon the COstretchmodesis determinedby abalanceof severalphysicaleffectssuchasvolumecontraction which forceshydrogenbonding (Demmel et al., 1997), dielectric constant (Jungetal., 1996a)andpH changes of the solvent (Schulzeet al.,1994a,1997), whichmayinfluencethelocal environmentoftheCO ligand.This should beconsideredwhen recognitionphenomenaarestudiedusing the CO stretch mode.

CO ligand flashphotolysis. Most of the studiesuse theheme Soret electronic absorption band to follow therebindingprocess.TheSoretbandis, however, notsensitiveenoughto distinguishbetweentherelaxationbehaviorof theconformational substates and its possible influence ofsubstrates.FT infrared spectroscopycan,however, do thisand has advantages for following structural changes indifferentpartsof the protein.

A simplified model describes the photodissociation andrebindingin four mainsteps.After breakingtheiron–ligandbond (step1) by light absorptionof the heme,the ligandmoves to a docking site within the heme pocket (step2).Thenit migratesthroughtheprotein(step3) (probably fromonesiteto another)beforeit escapesto thesolvent(step4).Rebindingfrom thesolventmight follow thesamepathwayor others.At low temperature(<160K) thephotodissociatedCO ligand,dockedto sitesneartheheme,cannotleavetheheme pocket.Only geminate rebindingoccurs.At very lowtemperatures(<60K) thegeminaterebindingis soslowthatone can easily observe the CO stretch mode of thedissociatedCO ligand(theso-calledB-state) in theinfrareddifferencespectrum to the bound state (the so-calledA-state). Figure 12 shows the light minus dark infrared

Figure 12. Difference infrared spectra (`light minus dark') for photo-dissociated cytochrome P450cam±CO in the presence of two differentsubstrates. (1R)-camphor and 1-iodoadamantane at 20K; 50 mM potas-sium phosphate buffer, pH 7, 60% (v/v) glycerol, CaF2 windows, 100 �mspacer, c(P450)�1 mM, photodissociation induced by the Nd±YAG laserwith 532 nm (Jung, unpublished).

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differencespectra of P450camfor two different substratecomplexes as a typical example. Analogous spectraformyoglobin andvariousmutants aregiven by Braunstein etal. (1993).

TheCO stretchmodes of theB-statesareobservedin therange between2100 and 2150cmÿ1 (Fig. 12) near thestretch modeof thefreeCO (2143cmÿ1). Usually,multipleA-states correspond to multiple B-states (Mourant et al.,1993;Junget al., 1992a).The integral intensity of the totalB-statebandspectrumis approximately17–30timesweakerthan that of the A-statesignals. The concrete value of theCO stretch modefrequency of theB-stateis determinedbythe microenvironmentof the docking site which polarizesthe CO dipole.The B-stateshavebeenstudied in detail onwild-type myoglobin (Mourant et al., 1993; Lim et al.,1995a,b, 1997; Nienhauset al., 1994),various myoglobinmutants (Braunsteinet al., 1993),on hemoglobin (Lim etal., 1995a), on leghemoglobin (Stetzkowskietal., 1985),oncytochromeP450(Junget al., 1992a)andon NO synthase(Jungetal., 2000).Usinganultrafasttime-resolvedinfraredspectrometer Lim et al. (1995a,b) have found that thephotodissociated CO molecule is hostedin a rotationallyconstrained environment. From measurements of thepolarization anisotropy decay with femtosecond timeresolution, Lim et al. (1997) concluded that the two B-statesin myoglobinobservedwith FTIR maycorrespondtothe samedocking site but with a rotated CO moleculeshowing rotational time constantsin therangebetween 200and 500 fs (Anfinrud et al., 1999). Molecular dynamicssimulationsfor spermwhale myoglobin by Meller andElber(1998)haveassignedthedocking siteto beabovethehemeand between Leu29, Val68 and Ile107 in agreementwithsites identified in the crystal structureof the photodisso-ciated myoglobin (Hartmannet al., 1996). Other dockingsites in horseheart myoglobin and mutants haverecentlybeendetected(Chuet al., 2000;Brunori et al., 2000).

The CO stretchsignals monitor local events.To under-standhow the apparentlocal changes areaccompaniedbystructural changes in the whole protein, further spectro-scopicprobesareneeded.Infraredspectroscopyis an idealmethodto follow simultanously the broad spectral rangewhere various signals of the different absorbing groupsappear. An examplehasbeendiscussed abovefor oxidases(Puustinen et al., 1997). Static differenceinfrared spectrabetween the CO bound and deoxy hemoglobin in D2O-dialyzed buffer indicatedthat distinct changes canbe seenalsoin the amideI andamideII bandregion(Gregoriou etal., 1995).Thesechanges arevery smallcomparedwith theabsolute intensity of the amide bands.A very accuratesubtractionof the referencesampleis required, which is avery tricky task.This problemdoesnot existwhen thestep-scantime-resolvedFTIR spectroscopy is used.This tech-nique hasbeenapplied first to myoglobin (Plunkett et al.,1995)andhemoglobin (Hu et al., 1996).Contzen andJung(1998) usedthis technique to study the effect of substratebindingon therebindingkineticsof theflashphotolyzedCOligand.

Figure 13 showsa typical step-scaninfrared differencespectrum for selectedtimeswith P450camasan example.Structural changes in the amideI regionappear, indicatingsmallchanges in thesecondarystructure, butchangesof theheme or other side groups of the protein may also be

considered.Theappearance of thesignalaround 1719cmÿ1

is outsidethecrowdedamideI regionandis thereforemorespecific andis assignedto achangein asaltli nk between theheme 7-propionateand Asp297 and Arg299. A changeinthe region between 1724 and 1713cmÿ1 has also beenobserved in infrared differencespectrainducedby photo-reduction and assignedto the salt bridge betweenthe 6-propionateof the heme and Arg112 and His355 (ContzenandJung,1999).Thischangein thesalt link is notobservedin the substrate-free protein, indicating that the substratebinding affectsnot only the distal side structurenear theheme ligand but may also have a more global effect. Inaddition, simultaneous monitoring the infrared signals,reflecting different partsof the protein, may not showthesame kineticswhich could pinpoint to a different dynamicbehavior of the protein substructures (Contzen and Jung,1998).

Light-induced proton translocation and O2 evolution.Uncovering the mechanismof proton translocation intransmembranepumps has benefited significantly frominfraredstudies.Bacteriorhodopsinis a light-driven protonpump. Proton translocation is the consequence of thephotoisomerizationof theretinal chromophore(Oesterheld,1998). Thephotocyclehasvariousintermediates.Themostinteresting point is to understandthe structural differencesbetween these intermediates.Using light-inducedinfrareddifferencespectroscopycoupledwith low-temperatureandtime-resolvedtechniques,isotopelabeling andsite-directedmutagenesis,manygroups(Tyr, Trp, Asp,Pro) involved inproton movement havebeenidentified (Rothschild et al.,

Figure 13. Time-resolved absorption difference infrared spectra(`light minus dark') for the rebinding of the ¯ash-photolyzed COligand of (1R)-camphor-bound cytochrome P450cam taken withthe step-scan time-resolved FTIR spectroscopy. 50 mM potassiumphosphate D2O buffer, pD 7; 50% (v/v) glycerol-d3,c(P450) = 0.79 mM, 23.7 mM (1R)-camphor (Contzen and Jung,1998). The time trace at a speci®c mirror position is shown in Fig.1. The peak assignment follows Table 2 and the explainationgiven in the text for salt links.



1992;Maedaet al., 1997;Hesslinget al., 1997).Photosynthetic oxygen evolution takes place on the

electron-donorsideof photosystemII (PSII). The catalyticcenterconsistsof a tetranuclearMn cluster wheretwo watermolecules are oxidized in a light-driven cycle of fiveintermediatesto form onedioxygenandfour protons.Usinglight-induced difference infrared spectroscopy variousamino acid side chain groupshave beenidentified to beinvolved in the reaction.For example, using 15N-labeledspinachandPSII corecomplexesfrom Synechocystis cellsNoguchi et al., (1999)haveassignedC—H stretchandN—H modes of histidine(Table3).

Although proton translocationand O2 evolution are notwithin the scopeof this review, it is importantto mentionthesestudies becausemany infraredsignals of aminoacidside chainshave beenassigned, which is very helpful inunderstanding corresponding signals observed in protein–ligand recognition.

CONCLUDING REMARKS

FTIR spectroscopy is one of the few methodswhich canprobe various local and global structural properties ofproteins and is therefore appropriate to study recognitionphenomena.The secondary structureof the proteincanbestudiedusing theamideI band.Localstructural changescanbe analyzedby the specificsignalsof the amino acid side

chains whereby the carboxylate groups have a highdiagnostic value for salt bridges.Cofactors are accessibleto study by their specific vibrational modes.The stretchmode of heme ligandsmay be used to probe the activecenter in heme proteins.Thestretch modeof theCO ligandis asensitive markerfor thepolarity of thehemepocketandmay alsobe usedasa probemolecule for the study of theprotein dynamics. It is possibleto study water moleculesbound to the protein. Because FTIR spectroscopycan becombined with time-resolved,low-temperature and high-pressure techniques, a broad field for studies of thedynamics of the folding and unfolding of the proteinstructure, and of the influence by ligand and substratebinding is opened.FTIR spectroscopyis apowerful methodwhichcancontributesignificantly to analysisof recognitionphenomena when combined with isotope labeling, site-directedmutagenesisandcomplementedwith crystallogra-phy andfurther spectroscopicmethods.

Acknowledgements

The former and presentPh.D. studentsJorg Contzen,Heike Schulze,NathalieLegrand,CorinneMouro andEric Deprezfrom the author’sandcooperatinglaboratoriesare gratefully acknowledged for their work oncytochromeP450.The authorthanksRebeccaWadefor carefully readingthemanuscript andfor manyhelpful comments.

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