Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG geförderten) Allianz- bzw. Nationallizenz frei zugänglich.This publication is with permission of the rights owner freely accessible due to an Alliance licence and a national licence (funded by the DFG, German Research Foundation) respectively.We report herein the vibrational spectroscopic characterisation of a catalytic intermediate formed by the reaction of H2O2 with DyP-type peroxidase immobilised on a biocompatible coated metal support. The SERR spectroscopic approach is of general applicability to other peroxidases which form relatively stable catalytic intermediates.DFG, EXC 314, Unifying Concepts in Catalysi

Hildebrandt, Peter

Martins, Ligia O.

Todorovic, Smilja

Physical Chemistry Chemical Physics

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11954 | Phys. Chem. Chem. Phys., 2015, 17, 11954--11957 This journal is© the Owner Societies 2015Cite this:Phys.Chem.Chem.Phys.,2015, 17, 11954Surface enhanced resonance Raman detection ofa catalytic intermediate of DyP-type peroxidase†Smilja Todorovic,*a Peter Hildebrandtb and Lı´gia O. MartinsaWe report herein the vibrational spectroscopic characterisation of acatalytic intermediate formed by the reaction of H2O2 with DyP-typeperoxidase immobilised on a biocompatible coated metal support.The SERR spectroscopic approach is of general applicability to otherperoxidases which form relatively stable catalytic intermediates.Dye-decolourising peroxidases (DyPs) are novel heme peroxidases,the primary sequence, structural and mechanistic properties ofwhich are unrelated to those of other known peroxidases.1–10They lack the distal histidine that is highly conserved in classicalperoxidases and which acts as an acid/base catalyst in thereduction of hydrogen peroxide to water. DyPs are classified intofour phylogenetically distinct classes (A–D); their physiologicalrole is not fully established yet and it appears to be subfamily-dependent.1–10 DyP from Pseudomonas putida MET94 (PpDyP) isan extremely versatile B-type DyP, capable of efficient oxidation of awide range of anthraquinonic and azo dyes, phenolic substrates, thenon-phenolic veratryl alcohol and evenmanganese and ferrous ions.8In the reaction with H2O2, PpDyP forms a stable Compound I (CpdI) at a rate of 1.4  0.3  106 M1 s1, comparable to those ofclassical peroxidases and other DyPs.8 The intermediate is, like inother DyPs, surprisingly long-living, with a half-life ofB60 min, asdemonstrated by electronic absorption spectroscopy.9 Uponimmobilisation on biocompatiblemetal supports, PpDyP is capableof efficient electrocatalytic activity in the presence of hydrogenperoxide. The enzyme is therefore a promising candidate for thedesign of bio-electronic devices with unique DyP-type peroxidasesubstrate specificity.9,10 Resonance Raman (RR) spectroscopy hasbeen extensively used in the detection and characterisation ofperoxidase and catalase-peroxidase catalytic intermediates, andRR spectroscopic fingerprints of Cpd I (two equivalent oxidisedresting ferric state) and Cpd II (one equivalent oxidised restingferric state) are well established.11–16 However, a strong fluores-cence impeded the RR studies of PpDyP intermediates. Here, wehave probed catalytic intermediate species of immobilisedPpDyP by surface enhanced RR (SERR) spectroscopy. The SERRspectra of heme proteins immobilised on nanostructured Agsurfaces exclusively display the cofactor signals of the adsorbedmolecules. In addition, close proximity of the molecule to themetal surface can efficiently quench fluorescence.17 Like in RRspectroscopy, SERR signals include, inter alia, the core-sizemarker bands (e.g. n4, n3, n2, n10) which are specifically enhancedupon excitation in resonance with the Soret absorption band ofthe porphyrin. The frequencies of these modes are indicative ofthe redox and spin states and the coordination pattern of theheme iron.We have previously shown that the high frequency region ofRR spectra of the resting PpDyP in solution displays broadmarker bands, indicative of the co-existence of several spinpopulations.9,10 Component analysis of the spectra reveals wellresolved bands of the n4, n3, n2, n10 and nCQC modes at 1376,1502, 1572, 1626 and 1637 cm1 (Fig. 1a, green trace) and at1373, 1494, 1564, 1621 and 1632 cm1 (Fig. 1a, blue trace),which are attributed to quantum mechanically mixed-spin (QS)and 5-coordinated high spin (5cHS) species, respectively.9,10 Inaddition, small contributions of a 6-coordinated HS (6cHS)species with n4 at 1366 cm1 and n3 at 1483 cm1 are also presentin the spectra. The SERR spectra of PpDyP immobilised on Agsupports coated with mixed amino- and hydroxyl-terminatedalkanethiol self-assembled monolayers (SAM), employing amino-octanethiol (AOT) and mercaptohexanol (MOH) in a 1 : 3 (M/M)ratio, reveal essentially the same band frequencies as RR spectra(Fig. 1a and b). This indicates that the protein structure ispreserved, on the level of the heme group, upon adsorption.We observe a slight alteration of relative contributions of QS and5cHS. The former species, which is dominant in solution,appears to be less abundant in the immobilised state. Thisfinding is related to a highly flexible heme cavity observed ina Instituto de Tecnologia Quı´mica e Biolo´gica (ITQB), Universidade Nova de Lisboa,Av. da Repu´blica, 2780-157 Oeiras, Portugal. E-mail: smilja@itqb.unl.ptb Institut fu¨r Chemie, Sektr. PC14, Technische Universita¨t Berlin,Strasse des 17. Juni 135, D-10623 Berlin, Germany† Electronic supplementary information (ESI) available: Experimental details ofprotein purification and spectroscopic experiments, together with SERR spectraof the immobilised PpDyP upon addition of H2O2 in the 600–1700 cm1 range.See DOI: 10.1039/c5cp01283jReceived 4th March 2015,Accepted 6th April 2015DOI: 10.1039/c5cp01283jwww.rsc.org/pccpPCCPCOMMUNICATIONPublished on 09 April 2015. Downloaded by TU Berlin - Universitaetsbibl on 12/06/2017 14:09:19. View Article OnlineView Journal  | View IssueThis journal is© the Owner Societies 2015 Phys. Chem. Chem. Phys., 2015, 17, 11954--11957 | 11955DyPs (and other bacterial and plant peroxidases) in which thetemperature, pH and physical state of the enzyme (i.e. solutionvs. crystalline form) induce a typically reversible inter-conversionof the spin populations.9,10,18,19Upon addition of 1 equivalent of hydrogen peroxide tothe SERR cell holding the enzyme-loaded metal support, wellreproducible spectral changes occur instantaneously: a pronounceddecrease of signal intensity, accompanied by an upshift of n4 and n3modes and a broadening of n2, n10 and nCQC bands (Fig. S1, ESI†).Component analysis of the spectra suggests an equilibrium of twopredominant species, one of which is characterised by n4 and n3 at1373 and 1494 cm1 and the other newly formed one with n4 and n3at 1378 and 1508 cm1 (Fig. 1c, blue and red trace, respectively). Weattribute the former population to the 5cHS (vide supra) and thelatter to the Cpd I – like species. This assignment is consistent withthe frequencies reported in the literature for catalytic intermediatesof classical peroxidases.11–16 In the absence of the reference RRspectra of Cpd I and Cpd II in DyPs, the assignment of the1378 cm1 and 1508 cm1 modes to the oxyferryl Fe(IV)QOporphyrin p cation is supported by the evidence from ourprevious work: (i) electronic absorption spectra, which reveala fingerprint of stable Cpd I in the reaction of PpDyP with1–3 equivalents of H2O2 in solution;8–10 and (ii) electrochemicaldata which demonstrate that PpDyP immobilised on AOT/MOHSAMs is electrocatalytically active and capable of H2O2reduction.9 We can therefore rule out the possibility thatinactive Cpd III (Fe(III)–O2 or Fe(II)–O2) is formed upon additionof H2O2 to the immobilised PpDyP. However, distinguishingbetween Cpd I and Cpd II is not straightforward at this point(note that some DyPs are, like lignin and versatile peroxidases,prone to spontaneous reduction of Cpd I to a Cpd II species).8The n4/n3 values that we observe here are slightly higher thanthose reported for Cpd I in the literature,11 which may be anintrinsic property of DyPs, or an indication of Cpd II. Forinstance, n4 and n3 modes of Cpd I in HRP were found at1373–1376 and 1505 cm1, respectively, but at 1379 and1510 cm1 in Cpd II.14 Nevertheless, RR measurements of theseintermediates in solution are in most of the cases accompaniedby experimental difficulties that frequently lead to the for-mation of photoreduced species.14 Bands of these side productsmay partially mask those of the catalytic intermediates. We couldactually identify traces of a population with n4 at 1361 cm1 inthe SERR spectra of the immobilised PpDyP that most likelyoriginates from a photoreduced Cpd I (Fig. 1c, turquoise trace).Additional support for the assignment of the ‘new’ species toCpd I comes from the attenuated SERR signal intensity uponaddition of H2O2 (Fig. S1, ESI†). It is most likely related to adecrease and moderate blue shift of the Soret absorption band(lSoret = 405 nm for the resting state PpDyP and 401 nm for Cpd I),which are characteristic of Cpd I,8–10 and may cause a distinct lossof resonance enhancement in the spectra upon excitation at413 nm (note that partial desorption of the enzyme cannot beruled out). Formation of Cpd II, on the other hand, causes asignificant red-shift of the Soret band to 417 nm,8–10 withoutpronounced intensity changes, which should actually have anopposite effect on (SE)RR spectra, i.e. an increase of the bandintensities. The intermediate of the immobilised PpDyP is quitestable, with a half-life of B15 min (Fig. 2). With time, the SERRsignals decay, without apparent protein denaturation or conversioninto Cpd III, most likely due to protein desorption (Fig. 2c).A comparison of deconvoluted SERR spectra of PpDyP recordedin the absence and presence of H2O2 reveals a conversion of QS andFig. 1 High frequency region of the RR and SERR spectra of ferric PpDyPin the absence/presence of hydrogen peroxide. Experimental and com-ponent RR spectra of PpDyP (a), SERR spectra of PpDyP immobilised onAOT/MOH SAM in the absence (b) and presence of 1 equivalent of H2O2(c). Inset: SERR spectra of the immobilised resting state PpDyP (black trace)and Cpd I (red trace) in the n4/n3 mode region. After polynomial baselinesubtraction, the experimental spectra (black trace) were subjected tocomponent analysis.9 The fits (dotted black trace) are based on thecomponent spectra of 6cHS (blue dashed trace), 5cHS (blue solid trace)and QS (green trace) species (a and b), compound I (red trace) andphotoreduced (turquoise trace) species (c), together with non-assignedand/or spin state insensitive bands (grey trace). RR and SERR spectra wererecorded with 413 nm excitation, 5 and 1.1 mW laser power and 60 and20 s accumulation time, respectively; SERR spectra were recorded at theopen circuit. RR experiments were carried out with 100 mM (SERR with0.4 mM) protein in buﬀered solution (50 mM Tris-HCl + 200 mM NaCl,pH 7.6). Two RR spectra (a) and four (b) or eight (c) SERR spectra wereco-added to increase S/N. Note that no assignments are given for thebands in the n2/n10 envelope (c) due to the lack of reference RR data oncatalytic intermediates of DyPs.Communication PCCPPublished on 09 April 2015. Downloaded by TU Berlin - Universitaetsbibl on 12/06/2017 14:09:19. View Article Online11956 | Phys. Chem. Chem. Phys., 2015, 17, 11954--11957 This journal is© the Owner Societies 2015HS species into putative Cpd I and HS populations, shedding morelight onto disputed physiological relevance of QS species.11,18–20Namely, the origin of this unusual heme configuration, alsoobserved in class III plant peroxidases, cytochromes c0 and catalaseperoxidases (e.g. KatG), is not well understood.11,18–20 We havepreviously demonstrated that it could be observed in B-, but not inA-type DyPs, and that its abundance sensitively depends on the pH,temperature, and physical state of the enzyme.10 Here we providedirect experimental evidence that the QS species is capable ofreacting with H2O2 and that it is therefore, most likely catalyticallycompetent in PpDyP.In conclusion, we presented a vibrational-spectroscopicfingerprint of a catalytic intermediate of the reaction betweenimmobilised PpDyP and H2O2. To our best knowledge, this isthe first report of a catalytic intermediate of a peroxidaseobtained by SERR spectroscopy. The technique can thereforebe regarded as an important alternative for RR spectroscopicstudies of the catalytic cycle of peroxidases11–16,22 when fluores-cence impedes solution measurements. As a further advantage,SERR requires only small (sub-micromolar) amounts of protein,due to high sensitivity which results from both the plasmonicand resonance enhancements of the signal. In addition, SERRdetection of catalytic intermediates is possible at ambienttemperature in contrast to EPR spectroscopy that is restricted tocryogenic conditions. Thus, SERR spectroscopy avoids temperature-dependent changes of spin state distributions,9,10,18,19 and isapplicable to the EPR-silent Cpd II.21 As a drawback, SERRspectroscopic identification of intermediates is restricted tospecies with lifetimes on the minute time scale (e.g. cytochromec peroxidases and DyPs)1–9,23 and can only be used if theenzyme is immobilized under preservation of its native stateand function. These conditions are fully satisfied for someDyPs.9 Thus, SERR in particular represents a promising approachfor a more detailed mechanistic analysis of DyPs from differentsubfamilies, stabilizing either Cpd I or Cpd II.1–8 We have recentlyobserved Cpd I with t1/24 60 h in distal PpDyP variants D132N andN136L, in which aspartate 132 was replaced by asparagine andasparagine 136 by leucine, respectively.24 Future studies of thesesystems will contribute to a better understanding of both, mole-cular determinants of catalytic activity in DyP-type peroxidases, andof their mechanistic properties in the immobilised state, which is aprerequisite for application of these catalytically versatile andefficient enzymes in the construction of bioelectronic devices.AcknowledgementsFinancial support from DFG (UniCat PH and ST) is acknowl-edged. We thank S. Mendes for help with protein purification.Notes and references1 X. Liu, Q. Du, Z. Wang, D. Zhu, Y. Huang, N. Li, T. Wei, S. Xuand L. Gu, J. Biol. Chem., 2011, 286, 14922.2 Y. Sugano, R. Muramatsu, A. Ichiyanagi, T. Sato andM. Shoda, J. Biol. Chem., 2007, 282, 36652.3 Y. Sugano, Cell. Mol. Life Sci., 2009, 66, 1387.4 J. N. Roberts, R. Singh, J. C. Grigg, M. E. P. Murphy,T. D. H. Bugg and L. D. Eltis, Biochemistry, 2011, 50, 5108.5 R. Singh, J. C. Grigg, Z. Armstrong, M. E. P. Murphy andL. D. Eltis, J. Biol. Chem., 2012, 287, 10623.6 E. van Bloois, D. E. Torres Pazmino, R. T. Winter andM. W. Fraaije, Appl. Microbiol. Biotechnol., 2010, 86, 1419.7 S. J. Kim and M. Shoda, Appl. Environ. Microbiol., 1999,65, 1029.8 A. Santos, S. Mendes, V. Brissos and L. O. Martins, Appl.Microbiol. Biotechnol., 2014, 98, 2053.9 M. Sezer, T. Genebra, S. Mendes, L. O. Martins andS. Todorovic, Soft Mater., 2012, 8, 10314.10 M. Sezer, A. Santos, P. Kielb, T. Pinto, L. O. Martins andS. Todorovic, Biochemistry, 2013, 52, 3074.11 S. M. Kapetanaki, S. Chouchane, S. Yu, R. S. Magliozzo andJ. P. M. Schelvis, J. Inorg. Biochem., 2005, 99, 1401.12 K.-J. Paeng and J. R. Kincaid, J. Am. Chem. Soc., 1988,119, 7913.13 V. Palaniappan and J. Terner, J. Biol. Chem., 1989,264, 16046.14 W. J. Chaung and H. E. Van Wart, J. Biol. Chem., 1992,267, 13293.15 C. M. Reczek, A. J. Sitter and J. Terner, J. Mol. Struct., 1989,214, 27.16 S. Hashimoto, J. Teraoka, T. Inubushi, T. Yonetani andT. Kitagawa, J. Biol. Chem., 1986, 261, 11118.17 F. Siebert and P. Hildebrandt, Vibrational Spectroscopyin Life Sciences, Wiley-VCH Verlag GmbH&Co. KGaA,Weinheim, 2008.18 G. Smulevich, A. Feis and B. D. Howes, Acc. Chem. Res.,2005, 38, 433.Fig. 2 Time evolution of SERR spectra of Cpd I of immobilised PpDyP.The spectra showing the n4 and n3 modes were recorded: 2 (a), 15 (b) and20 (c) minutes after the addition of H2O2 to the SERR cell including thePpDyP-loaded Ag support, coated by AOT/MOH. Experimental conditionsas in Fig. 1.PCCP CommunicationPublished on 09 April 2015. Downloaded by TU Berlin - Universitaetsbibl on 12/06/2017 14:09:19. View Article OnlineThis journal is© the Owner Societies 2015 Phys. Chem. Chem. Phys., 2015, 17, 11954--11957 | 1195719 B. D. Howes, C. B. Schiodt, K. G. Welinder, M. P. Marzocchi,J.-G. Ma, J. Zhang, J. A. Shelnutt and G. Smulevich, Biophys.J., 1999, 77, 478.20 M. M. Maltempo, T. H. Moss and M. A. Cusanovich, Biochim.Biophys. Acta, 1974, 342, 290.21 A. Ivancich, H. M. Jouve, B. Sartor and J. Gaillard, Biochemistry,1997, 36, 9356.22 S. Hu and J. R. Kincaid, J. Am. Chem. Soc., 1991, 113,7189.23 M. A. Miller, G. W. Han and J. Kraut, Proc. Natl. Acad. Sci.U. S. A., 1994, 91, 11118.24 S. Mendes, V. Brissos, A. Gabriel, T. Catarino, D. L. Turner,S. Todorovic and L. O. Martins, Arch. Biochem. Biophys.,2015, DOI: 10.1016/j.abb.2015.03.009.Communication PCCPPublished on 09 April 2015. Downloaded by TU Berlin - Universitaetsbibl on 12/06/2017 14:09:19. View Article Online

Surface enhanced resonance Raman detection of a catalytic intermediate of DyP-type peroxidase

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Surface enhanced resonance Raman detection of a catalytic intermediate of DyP-type peroxidase

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