HemeandpH-dependentstabilityofananionic horseradishperoxidase AnaSofiaL.Carvalho, a EduardoPinhoeMelo, a,b, * BrunoSommerFerreira, a MariaTeresaNeves-Petersen, c SteffenB.Petersen, c andMariaRaquelAires-Barros a a Centro de Engenharia Biol ogica e Qu  ımica, Instituto Superior T ecnico, Av. Rovisco Pais, Lisboa 1049-001, Portugal b Centro de Biomedicina Molecular e Estrutural, Universidade do Algarve, Campus de Gambelas, Faro 8000-117, Portugal c Biostructure and Protein Engineering Group, Department of Life Sciences, University of Aalborg, Sohngaardsholmsvej 49, Aalborg DK-9000, Denmark Received18March2003,andinrevisedform19May2003 Abstract HorseradishperoxidaseA1thermalstabilitywasstudiedbysteady-statefluorescence,circulardichroismanddifferentialscanning calorimetryatpHvaluesof4,7and10.Changesintheintrinsicproteinprobes,tryptophanfluorescence,secondarystructure,and hemegroupenvironmentarenotcoincident.The T m valuesmeasuredfromthevisibleCDdataarehigherthanthosemeasuredfrom Trpfluorescenceandfar-UVCDdataatallpHvaluesshowingthatthehemecavityisthelaststructuralregiontosuffersignificant conformationalchangesduringthermaldenaturation.Howeverejectionofthehemegroupleadstoanirreversibleunfoldingbe- havioratpH4,whileatpH7and10refoldingisstillobserved.Thisisputativelycorrelatedwiththetitrationstateoftheheme pocket.ThermaltransitionsofHRPA1showedscanratedependenceatthethreepHvalues,showingthatthedenaturationprocess waskineticallycontrolled.Thedenaturationprocesswasinterpretedintermsoftheclassicscheme,N $ U ! Dandfittedtofar- UVCDellipticity.Agoodagreementwasobtainedbetweentheexperimentalandtheoretical T m valuesandpercentagesofirre- versibility.HowevertheequilibriumbetweenNandUisprobablymorecomplexthanjustatwo-stateprocessasrevealedbythe multiple T m values. Ó 2003ElsevierScience(USA).Allrightsreserved. Keywords: HorseradishperoxidaseA1;Thermaldenaturation;pH-dependentstability;Activationenergy Peroxidases (EC 1.11.1.7) are heme-containing en- zymesthatcatalyzetheoxidationofalargenumberof substrates by reduction of hydrogen peroxide. The ap- plications of peroxidases range from analytical and biosensor systems [1], protein-based time-temperature- integrators [2], through industrial effluent treatment [3] to commercial synthesis of peroxidatively-produced phenolic resins [4,5]. The most widely used peroxidase comes from horseradish roots, HRP, which belongs to the plant peroxidase superfamiliy, more specifically to class III, secretory plant peroxidases [6,7]. HRP com- prises a set of isoenzymes, a total of 15 [8]. Several isoenzymeshavealreadybeensequenced,suchasHRPC [9], HRPE5 [10] and HRPA2 [6]. HRPA2 amino acid sequence shows 54% of identity to HRPC and around 80% to the partially sequenced HRPA1 [6]. The se- quenceidentitiesamongplantperoxidasesarereported torangefrom33%to96%[11]. Three-dimensional structures of several plant perox- idasesbelongingtothethreeidentifiedclasseshavebeen reported[12–17].Inallplantperoxidasesthehemegroup islocatedbetweenhelicesB(positionedabovetheheme) onthedistalside,andF(positionedbelowtheheme),on theproximalside.ClassIIIperoxidaseshaveextraheli- cesthatplayaroleinaccesstothehemeedge[12–14], two calcium ions, an N-terminal signal peptide for ex- cretion and four conserved disulphide bridges [11,18]. TheonlytryptophanresiduepresentinclassIIIperox- idasesisfoundintheextendedstructureconnectinghe- licesDandD 0 andislikelyaconservedresidue[12,13,16]. Thetryptophan(Trp)fluorescenceisquencheddueto intramolecular tryptophan–heme energy transfer in the nativeHRPC[19,20].ThedistancebetweentheTrpand ArchivesofBiochemistryandBiophysics415(2003)257–267 www.elsevier.com/locate/yabbi ABB * Correspondingauthor.Fax:+11-351-21-841-9062. E-mail address: emelo@ualg.pt (E.P.Melo). 0003-9861/03/$-seefrontmatter Ó 2003ElsevierScience(USA).Allrightsreserved. doi:10.1016/S0003-9861(03)00275-3