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.Surface enhanced vibrational spectro-electrochemistry of endonuclease III provides direct evidence that the [4Fe-4S] cluster is responsible for the enzyme redox activity, and that this process is not exclusively DNA-mediated, as currently proposed. We report the first surface enhanced resonance Raman spectrum of a [4Fe-4S](2+) cluster containing enzyme.DFG, EXC 314, Unifying Concepts in Catalysi

Hildebrandt, Peter

Moe, Elin

Sezer, Murat

Todorovic, Smilja

Chemical Communications

Redox activation of the [4Fe–4S] cluster in DNA repair enzyme endonuclease III does not exclusively rely on interaction with DNA.</p

Elin Moe

Murat Sezer

Peter Hildebrandt

Smilja Todorovic

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Surface enhanced vibrational spectroscopic evidence for an alternative DNA-independent redox activation of endonuclease III

Surface enhanced vibrational spectro-electrochemistry of endonuclease III provides direct evidence that the [4Fe-4S] cluster is responsible for the enzyme redox activity, and that this process is not exclusively DNA-mediated, as currently proposed. We report the first surface enhanced resonance Raman spectrum of a [4Fe-4S](2+) cluster containing enzyme

SEZER, Murat

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This journal is©The Royal Society of Chemistry 2015 Chem. Commun., 2015, 51, 3255--3257 | 3255Cite this:Chem. Commun., 2015,51, 3255Surface enhanced vibrational spectroscopicevidence for an alternative DNA-independentredox activation of endonuclease III†Elin Moe,a Murat Sezer,b Peter Hildebrandtb and Smilja Todorovic*aSurface enhanced vibrational spectro-electrochemistry of endo-nuclease III provides direct evidence that the [4Fe–4S] cluster isresponsible for the enzyme redox activity, and that this process isnot exclusively DNA-mediated, as currently proposed. We report thefirst surface enhanced resonance Raman spectrum of a [4Fe–4S]2+cluster containing enzyme.Deinococcus radiodurans is an extremely radiation and desiccationresistant bacterium, which can withstand 200-fold higher irradia-tion doses than other bacteria.1,2 The resistance mechanism is notknown, but an eﬃcient DNA repair machinery is considered toplay a key role in it. Endonuclease III (EndoIII) is a [4Fe–4S] clustercontaining DNA repair enzyme from the helix–hairpin–helixfamily of DNA glycosylases, crucial for removal of oxidationdamaged bases in DNA.3,4 Early evidence indicated that Fe–Sclusters in these enzymes are not amenable to oxidation orreduction in solution and suggested structural or regulatory rolesof the cofactor.5,6 More recent electrochemical studies of glyco-sylases immobilised on DNA-modified electrodes indicated thatthe [4Fe–4S] cluster is activated towards oxidation upon bindingto DNA. It was proposed that the cluster exerts a function in DNA-mediated signalling for detection of DNA lesions,7–12 but based ondata obtained by electrochemical approaches, which cannotprovide any information about the molecular origin of the electro-chemical signal. Here we employed surface enhanced reso-nance Raman (SERR) spectroscopy and surfaced enhanced IRabsorption (SEIRA) spectro-electrochemistry to demonstratethat the cluster of the immobilised EndoIII from D. radiodurans(DrEndoIII) is prone to reduction, which is not necessarilyDNA-mediated.DrEndoIII was electronically coupled to metal electrodes mod-ified with bifunctional alkanethiol-based self assembledmonolayers(SAMs).13 The cluster integrity of DrEndoIII immobilised on the11-mercapto-undecanoic acid (MUA)-coated nanostructured Agworking electrode was probed by SERR spectroscopy. ForDrEndoIII, which has a broad electronic absorption bandcentered at 410 nm, both the plasmonic and the resonance enhance-ment conditions are well matched using 413 nm excitation.14 Thenegatively charged MUA surface readily interacts with the highlypositively charged DNA binding site of DrEndoIII (vide infra) thatincludes the active site pocket and the [4Fe–4S] cluster (Fig. S1, ESI†),which is in glycosylases optimised for a fast and precise accommo-dation of the negatively charged DNA substrate.4,15–17Upon protein immobilisation and removal of the looselybound molecules by rinsing with buﬀer, the functionalised flatdisk Ag electrode was inserted into a pre-cooled cryostat thatwas rapidly cooled to 77 K. The SERR spectrum of the Ag–MUA–DrEndoIII construct was measured at 77 K since the intrinsi-cally weak RR bands of Fe–S clusters can only be detected at lowtemperatures.18,19 The spectrum shows well defined modes at337, 363 and 384 cm1 (Fig. 1a), indicative of a [4Fe–4S] clusterin the +2 oxidation state, which in proteins can change either to+1 or +3 state as in, e.g. ferredoxins and HiPIPs, respectively.It closely resembles the RR spectral pattern observed in frozenDrEndoIII solution (Fig. 1b), which reveals bands at 337 cm1,384 cm1 and 390 cm1 (Fe–S stretching involving the bridging S)and at 359 cm1 and 366 cm1 (Fe–S stretching involvingthe terminal S), demonstrating the structural integrity of thecofactor upon enzyme immobilisation. The electrode constructwas afterwards brought to ambient temperature, reducedchemically with 1 mM sodium dithionite, rinsed with buffer,frozen again and measured by SERR spectroscopy. No featuresof the [4Fe–4S]2+ (or [3Fe–4S]+) cluster were observed in thespectra (Fig. 1c), indicating either its reduction to a RR-silentstate which lacks resonance enhancement at 413 nm excitationor degradation/desorption.a Instituto de Tecnologia Quimica e Biologica (ITQB), Universidade Nova de Lisboa,Av. da Republica (EAN), 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 cloning, expression and purification, spectroscopic and CV experimentsand generation of the homology model, together with surface charge distributionof DrEndoIII (Fig. S1), control CVs (Fig. S2–S4) and SEIRA spectra of the Au/NTA/DrEndoIII/DNA construct (Fig. S5). See DOI: 10.1039/c4cc09498kReceived 27th November 2014,Accepted 12th January 2015DOI: 10.1039/c4cc09498kwww.rsc.org/chemcommChemCommCOMMUNICATIONPublished on 13 January 2015. Downloaded by TU Berlin - Universitaetsbibl on 15/06/2017 11:16:47. View Article OnlineView Journal  | View Issue3256 | Chem. Commun., 2015, 51, 3255--3257 This journal is©The Royal Society of Chemistry 2015Subsequent cyclic voltammetry (CV) measurements of thesame RR-silent Ag–MUA–DrEndoIII construct at ambient tem-perature revealed that the protein was still bound to theelectrode surface and redox active, with transition at E00 B 10 mV(Fig. S2, ESI†). The redox transition occurs at the same potential asin the case of DrEndoIII immobilised on MUA coated Au electrodes(Fig. 2), which reveals an apparent redox potential, E00, of 20 10 mV and an electron transfer (ET) rate constant, kET, of 3.1 0.4 s1, demonstrating that the [4Fe–4S] cluster of the proteinwhich was exposed to the laser beam in SERR experiment, wasin the reduced, native, RR-silent state. Our data thereforesuggest that the Fe–S cluster in immobilised DrEndoIII is proneto reduction, and that voltammetric signals can be ascribedto the [4Fe–4S]1+/2+, rather than to the [4Fe–4S]2+/3+ couple ascurrently proposed.7–12 In a control experiment DrEndoIII wasimmobilised on Au electrodes modified with 15 base pairdouble-stranded DNA (15 bp dsDNA) terminated SAMs, pre-viously employed in electrochemical studies of other DNAglycosylases.7–11 The redox properties of DrEndoIII on DNA-terminated SAMs (E00 B 10 mV, kET B 1.4 s1) (Fig. S3, ESI†)are comparable with those on MUA. Moreover, they are con-sistent with data reported for EcEndoIII, MutY and AfUDGglycosylases immobilised on DNA-modified Au electrodes(E00 in 50–100 mV range, kET in 1–10 s1 range)7–11 andEcEndoIII attached to DNA-functionalised graphite electrodes(E00 = 20  10 mV).12 No CV signals were observed whenDrEndoIII was immobilised on Ni2+-NTA SAMs via a His tagintroduced at the N-terminus (Fig. S4, ESI†).20,21The orientation of DrEndoIII with respect to the electrodecoated with the employed SAM was probed by ATR-SEIRAspectroelectrochemistry.14,20–22 DrEndoIII was adsorbed onfunctionalised gold film deposited on silicon crystals, whichat the same time serves as a working electrode and an amplifierof the spectroscopic signal of the immobilised protein mole-cules.14,22 The spectra indicate that DrEndoIII adsorbs on MUA,15 bp dsDNA and Ni2+-NTA terminated SAMs, adopting distinctorientations relative to the electrode surface (Fig. 3).14,18,22 Thisis concluded from the variations of the intensity ratios ofthe amide I (B1650 cm1) and amide II (B1550 cm1) bands.A relatively low Iamide I/Iamide II ratio (1.06) observed on carboxyl-and DNA-terminated SAMs corresponds to a predominantlyparallel alignment of DrEndoIII helices with respect to theFig. 1 SERR and RR spectra of DrEndoIII. (a) SERR spectrum of DrEndoIIIimmobilised on MUA, upon (c) reduction with sodium dithionite; (b) RRspectrum of 0.5 mM resting state DrEndoIII. Spectra are measured with413 nm excitation at 77 K using 1.5 mW and 8 mW in the SERR and RRexperiment, respectively.Fig. 2 CV of DrEndoIII immobilized on MUA (vscan 50 mV s1, Ag/AgClreference and Pt counter electrode) (a) and a control MUA coated Auelectrode (b).Fig. 3 SEIRA spectra of DrEndoIII immobilized under diﬀerent orienta-tions. The transmission FTIR spectrum of 0.7 mM protein in solution isshown for comparison.Communication ChemCommPublished on 13 January 2015. Downloaded by TU Berlin - Universitaetsbibl on 15/06/2017 11:16:47. View Article OnlineThis journal is©The Royal Society of Chemistry 2015 Chem. Commun., 2015, 51, 3255--3257 | 3257electrode surface, suggesting that the SAMs interact with thehighly positively charged DNA binding site of the enzyme. Thehigh ratio (3.6) determined for Ni2+-NTA SAM indicates that thea-helices, and thus the long axis of the protein, are largelyoriented perpendicular to the electrode surface, correspondingto a close proximity of the cluster to the electrode and the DNAbinding cleft exposed to the solvent. The FTIR spectrum of theenzyme in solution, measured in transmission mode (Fig. 3)reflects a random orientation distribution of the protein mole-cules (Iamide I/Iamide II = 1.57). Taken together, CV and SEIRAresults demonstrate that favourable orientation of the proteinwith respect to the electrode, which is achieved only for DNAand MUA SAMs, is crucial for heterogeneous ET in DrEndoIII.Moreover, orientation, E00 and ET rate constants are all similarfor DrEndoIII immobilised on DNA and COOH modified elec-trodes, indicating that heterogeneous ET in EndoIIIs is notnecessarily DNA-mediated. Other negatively charged moleculescan induce redox activity of the [4Fe–4S] cluster in the immo-bilised enzyme.We further tested if the presence of DNA can induce the redoxactivity of DrEndoIII immobilised on NTA, which leaves the DNAbinding cleft exposed to the solvent (Fig. 3). An excess of normalor damaged 15 bp dsDNA was added to the SEIRA spectro-electrochemical cell accommodating immobilised DrEndoIII.SEIRA bands of the protein immobilised on Ni2+-NTA undergosignificant changes upon addition of DNA (Fig. S5, ESI†): theamide I downshifts from 1666 cm1 to 1660 cm1, accompaniedby an intensity decrease and the appearance of an additionalcomponent at 1641 cm1. Further bands at high frequencies canbe assigned to C–H and N–H vibrations originating from theDNA duplex, indicating its binding to the protein. No CV signalswere observed upon addition of undamaged or damaged DNA(data not shown), indicating that the presence of DNA alone isnot sufficient for redox activation of immobilised DrEndoIII, ascurrently believed.7–12 The orientation of the enzyme relative tothe electrode surface (or the physiological redox partner) has tobe correct, which ensures the directionality of ET and thusexplains the lack of redox activation in solution even in thepresence of DNA.8,9,18 Addition of damaged or undamaged DNAhad no effect on redox activity of DrEndoIII immobilised onMUA SAMs, since the electrode surface docking engages the DNAbinding site and makes it unavailable for DNA.In conclusion, we show that redox activation of DrEndoIIIdoes not rely exclusively on DNA binding. We propose thatin vivo the FeS cluster may be redox-activated by other chargedspecies (e.g. proteins or ROS) and take part in cellular processesother than DNA damage search and identification. Clearly,further studies are required to test this hypothesis. The obtainedinsights will have a general impact on the understanding of therole of [4Fe–4S] clusters in DNA glycosylases, including thecentral enzyme for repair of ROS damage in humans, hNTH1,for which DrEndoIII represents a bacterial homologue. The firstSERR spectroscopic characterisation of a Fe–S containingenzyme immobilized on a biocompatible surface in its nativestate that we report here will open new possibilities for studies ofsimilar systems.Financial support from Intra-European Fellowship (IEF) withinthe FP7 Marie Curie actions (EM), DAAD-CRUP (A43/12) bilateralaction (ST and PH), and the DFG (UniCat – PH) is acknowledged.We also thank M. Rosa, C. Roma˜o and M. Teixeira for the helpwith protein preparation, and I. Zebger and S. Katz for usefuldiscussions.Notes and references1 M. M. Cox and J. J. Battista, Nat. Rev. Microbiol., 2005, 3, 882.2 J. R. Battista, Annu. Rev. Microbiol., 1997, 51, 203.3 M. Dizdaroglu, Mutat. Res., 2005, 591, 45.4 M. M. Thayer, H. Ahern, D. Xing, R. P. Cunningham and J. A. Tainer,EMBO J., 1995, 14, 4108.5 R. P. Cunningham, H. Asahara, J. F. Bank, C. P. Scholes, J. C.Salerno, K. Surerus, E. Munk, J. McCracken, J. Peisach and M. H.Emptage, Biochemistry, 1989, 28, 4450.6 O. A. Lukianova and S. S. David, Curr. Opin. Struct. Biol., 2005, 9, 145.7 A. K. Boal, E. Yavin, O. A. Lukianova, V. L. O’Shea, S. S. David andJ. K. Barton, Biochemistry, 2005, 44, 8397.8 A. K. Boal, J. C. Genereux, P. A. Sontz, J. A. Gralnick, D. K. Newmanand J. K. Barton, Proc. Natl. Acad. Sci. U. S. A., 2009, 106, 15237.9 E. M. Boon, A. L. Livingston, N. H. Chmiel, S. S. David andJ. K. Barton, Proc. Natl. Acad. Sci. U. S. A., 2003, 100, 12543.10 M. A. Grodick, H. M. Segal, T. J. Zwang and J. K. Barton, J. Am. Chem.Soc., 2014, 136, 6470.11 E. Yavin, A. K. Boal, E. D. A. Stemp, E. M. Boon, A. L. Livingston,V. L. O’Shea, S. S. David and J. K. Barton, Proc. Natl. Acad. Sci. U. S. A.,2005, 102, 3546.12 A. A. Gorodetsky, A. K. Boal and J. K. Barton, J. Am. Chem. Soc., 2006,128, 12082.13 J. C. Love, L. A. Estroﬀ, J. K. Kriebel, R. G. Nuzzo and G. M. Whitesides,Chem. Rev., 2005, 105, 1103.14 F. Siebert and P. Hildebrandt, Vibrational Spectroscopy in Life Sciences,Wiley-VCH, Weinheim, 2009.15 I. Leiros, E. Moe, A. O. Smalas and S. McSweeney, Acta Crystallogr.,Sect. D: Biol. Crystallogr., 2005, 61, 1049.16 E. Moe, I. Leiros, A. O. Smalas and S. McSweeney, J. Biol. Chem.,2006, 281, 569.17 C. D. Mol, A. S. Arvai, R. J. Sanderson, G. Slupphaug, B. Kavli,H. E. Krokan, D. W. Mosbaugh and J. A. Tainer, Cell, 1995, 82, 701.18 S. Todorovic, S. S. Leal, C. A. Salgueiro, I. Zebger, P. Hildebrandt,D. H. Murgida and C. M. Gomes, Biochemistry, 2007, 46, 10733.19 W. Fu, S. O’Handley, R. P. Cunningham and M. K. Johnson, J. Biol.Chem., 1992, 267, 16135.20 K. Ataka and J. Heberle, J. Am. Chem. Soc., 2003, 125, 4986.21 S. Todorovic, A. Verissimo, M. Pereira, M. Teixeira, P. Hildebrandt,I. Zebger, N. Wisitruangsakul and D. H. Murgida, J. Phys. Chem. B,2008, 112, 16952.22 J. Kozuch, C. Steinem, P. Hildebrandt and D. Millo, Angew. Chem.,Int. Ed., 2012, 51, 8114.ChemComm CommunicationPublished on 13 January 2015. Downloaded by TU Berlin - Universitaetsbibl on 15/06/2017 11:16:47. View Article Online

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Surface enhanced vibrational spectroscopic evidence for an alternative DNA-independent redox activation of endonuclease III

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