Iron L-edge X-ray absorption spectra of the active centre of myoglobin in the
met-form, in the reduced form and upon ligation to O2, CO, NO and CN are
presented. The strength of ligation with the iron centre is finger-printed
through the variation of the L3 : L2 intensity ratio. Charge Transfer
Multiplet calculations are performed and give qualitative information about
oxidation states as well as charge transfer

Aziz, Emad F.

Bonhommeau, Sébastien

Golnak, Ronny

Lange, Kathrin M.

Institutional Repository of the Freie Universität Berlin

This journal is c The Royal Society of Chemistry 2013 Chem. Commun., 2013, 49, 4163--4165 4163Cite this: Chem. Commun.,2013,49, 4163Ligand discrimination of myoglobin in solution:an iron L-edge X-ray absorption study of theactive centre†‡Kathrin M. Lange,a Ronny Golnak,b Se´bastien Bonhommeaud and Emad F. Aziz*bcIron L-edge X-ray absorption spectra of the active centre of myo-globin in the met-form, in the reduced form and upon ligation toO2, CO, NO and CN are presented. The strength of ligation with theiron centre is finger-printed through the variation of the L3 : L2intensity ratio. Charge Transfer Multiplet calculations are per-formed and give qualitative information about oxidation statesas well as charge transfer.Myoglobin is a metalloprotein found in muscle cells, where it isresponsible for the storage and the transport of oxygen.1,2 Asthe simplest globin it serves as a model system for investigatingthe relationship between structure, dynamics and function ofthe ligand binding reaction.1,3,4 The binding of the myoglobin(Mb) to ligands takes place at its active centre, a heme pros-thetic group, consisting of an iron atom which is fourfoldcoordinated to a porphyrin ring. On the proximal side of theheme an imidazole group serves as an axial ligand (proximalhistidine). Via the sixth coordination site of the heme iron,ligands like O2, CO, NO or CN can bind reversibly to the activecentre. Their binding aﬃnity is influenced by a distal histidine(His64) which can form e.g. in the case of O2-binding stabilizinghydrogen bonds to the FeO2 complex.5 A wide range of studieshave been carried out in order to understand the discrimina-tion mechanism for diﬀerent ligands by the Mb active centre.6Here we investigate the electronic structure of the iron activecentre of Mb in solution upon ligation with CO, CN, O2, and NOas well as in comparison to the deoxidized form and in the met-form (schematic presented in Fig. 1). The electronic structure isrevealed at the L-edge of iron via total fluorescence yield (TFY)X-ray absorption spectroscopy (XAS), which allows the mole-cular orbital structure of the d-orbitals to be probed. Suchvalence d-orbitals of the iron are involved in bond-buildingand breaking as well as charge transfer with ligand.7–9 Note thatTFY spectra of a concentrated sample suﬀer normally fromsaturation eﬀects,10 even for concentrations down to 500 mM.11,12Nevertheless, this eﬀect is negligible in this study for tworeasons; first, the measured concentration is 2 mM and second,the concentration was preserved for all measurements. TheL-edge XAS for 3d-transition metals (e.g. iron) lies in the softX-ray regime. This is the reason why most previous soft X-raystudies on proteins were carried out on solid, hydrated orfrozen samples.9,13 Just recent spectroscopic developments alsoallowed the investigation of proteins in solution.7–9,14 For this,samples are separated from the vacuum by Si3N4 membranes of150 nm thickness, which allow the soft X-rays to pass. In orderto prevent X-ray induced sample damage the sample solutionwas constantly flowed through the sample flow-cell (schematicpresented in Fig. 1). The samples have been prepared as shownpreviously.13,15–17 Their oxidation states and ligation weremonitored via UV-Vis spectroscopy. As shown before by Haradaet al.18 and Chergui and co-workers,17 such control is suﬃcientenough to guarantee the proper preparation of the sample,namely a +II oxidation state for the central iron atom indeoxyMb, MbO2, MbNO and MbCO but a +III oxidation statein metMb and MbCN. Note that we cannot exclude the presenceof a small Fe(II) fraction (i.e. deoxyMb fraction) in metMb andMbCN. However, it should be negligible based on our UV-visspectra (see Fig. S1, ESI‡). Especially the Q-band of the UV-Visspectra is sensitive to the oxidation state and even smallchanges would alter it markedly.19,20 Details of sample prepara-tion and ligand control using UV-Vis are described in ESI.‡In Fig. 2 iron L-edge TFY-XA spectra of Mb with diﬀerentligands are presented together with the reduced and the met-form. The spectra are normalized to the L2 intensity. Thisnormalization was chosen, since it was previously shown thatthe L3 intensity is more sensitive to changes in the local electronicstructure than in the L2 intensity.21,22 Among Mb–Fe2+ proteins,aMax-Born-Institut, Max Born Straße 2A, 12489 Berlin, Germany.E-mail: kathrin.lange@mbi-berlin.de; Tel: +49 30 63921248b Joint Ultrafast Dynamics Lab in Solutions and at Interfaces (JULiq),Helmholtz-Zentrum Berlin fu¨r Materialien und Energie, Albert-Einstein-Str 15,12489 Berlin, Germanyc Freie Universita¨t Berlin, FB Physik, Arnimallee 14, D-14195 Berlin, Germany.E-mail: emad.aziz@helmholtz-berlin.de; Tel: +49 30806215003d Universite´ de Bordeaux, Institut des Sciences Mole´culaires, CNRS UMR 5255,F-33400 Talence, France† This article is part of the ChemComm ‘Emerging Investigators 2013’ themed issue.‡ Electronic supplementary information (ESI) available. See DOI: 10.1039/c3cc37973fReceived 3rd November 2012,Accepted 4th February 2013DOI: 10.1039/c3cc37973fwww.rsc.org/chemcommChemCommCOMMUNICATIONPublished on 05 February 2013. Downloaded by Freie Universitaet Berlin on 27/01/2015 10:16:55. View Article OnlineView Journal  | View Issue4164 Chem. Commun., 2013, 49, 4163--4165 This journal is c The Royal Society of Chemistry 2013deoxyMb leads to the highest L3 intensity. Upon ligation withO2, NO and CO, this intensity decreases. It looks comparablefor MbNO and MbO2 and reaches its lower value for MbCO.Among Mb–Fe3+ proteins, metMb shows the highest L3 inten-sity, which is dramatically reduced upon ligation to CN. Suchreduction of the L3 intensity in TFY-XA spectra at the L-edgeof 3-d transition metals in aqueous solution has recentlybeen correlated with ultrafast charge-transfer processes to thesolvent.23–25 Orbital mixing leads to electron delocalizationbetween the valence d-orbitals of the transition metal and thevalence bands of the solvent molecules, quenching some of themetal X-ray fluorescence bands.26 It was argued that electrondelocalization which contributes to the non-radiative process isfaster than L3 core-hole life time (2 fs)14 and slower than the L2one (0.7 fs),14 aﬀecting accordingly mainly the L3-edge.23 In thecurrent study, the strength of orbital hybridization between themetal active centre and the ligand reflects directly on the L3 : L2intensity. Indeed, this observation is similar to the recentobservation by Carniato et al.,27 where they show that theL3 : L2 ratio is directly correlated with the strength of thechemical bond. According to the earlier observation,21,23,25the reduction of L3 intensity relative to L2 is due to strongerinteraction between the ligand and the iron centre and theincrease in the strength of orbital hybridization causes anincrease in the non-radiative channel under L3 reducing itsradiative intensity relative to L2.23 Note that the analysis ofTFY-XA spectra from transition-metal aqueous solution is com-plicated due to competing signal contributions from solute andsolvent, causing sub-background intensity.28 In the currentstudy, therefore, the concentration of the solute (myoglobin)versus solvent (water) was kept constant for all samples in orderto ensure that the relative changes are not caused by back-ground eﬀects.21,22In Fig. 3 iron L-edge XA spectra of metMb and deoxyMbcalculated using Charge Transfer Multiplet Theory (CTMT),which includes ligand-to-metal charge transfer (LMCT) andmetal-to-ligand charge transfer (MLCT) but does not accountfor the aforementioned fluorescence quenching, are plotted(see ESI‡ for more details). The good agreement betweenexperimental and theoretical XA spectra suggests that the L3of the TFY-XAS in metMb and deoxyMb is little aﬀected by non-radiative channels. Therefore, these proteins can be consideredas benchmark molecules for further interpretation of Mb–Fe3+and Mb–Fe2+ samples, respectively. For other ligated proteins,we could not reproduce the intensity decrease at the L3-edge(Fig. 2) and leave unaltered intensity and lineshape at the L2-edge,Fig. 1 X-ray absorption measurements were carried out on myoglobin, the oxygen transport and storage protein of the muscle cells, for six diﬀerent ligation states ofthe active centre (deoxyMb, metMb, MbO2, MbNO, MbCO, MbCN). The ligation stability of the sample solutions was monitored before and after X-ray irradiation viaUV-Vis spectroscopy.Fig. 2 Fe L-edge X-ray absorption spectra of myoglobin in various ligandconfigurations (deoxyMb, metMb, MbO2, MbNO, MbCO, MbCN). The spectrawere normalized to the L2-edge intensity.Fig. 3 Fe L-edge X-ray absorption spectra of metMb and deoxyMb from Fig. 2compared with spectra calculated using CTM theory. Calculated spectra werenormalized to each other with respect to their L2-edge intensity.Communication ChemCommPublished on 05 February 2013. Downloaded by Freie Universitaet Berlin on 27/01/2015 10:16:55. View Article OnlineThis journal is c The Royal Society of Chemistry 2013 Chem. Commun., 2013, 49, 4163--4165 4165which can be ascribed to the occurrence of fluorescencequenching in the presence of O2, NO, CO and CN ligands.In the frame of our CTMT calculations, the ground stateconfigurations are 6.7% d4L + 65.0% d5 + 28.3% d6L+ formetMb and 94.3% d5L + 5.6% d6 + 0.1% d7L+ for deoxyMb,where d and L/L+ are related to metal orbitals and an electron/a hole localized on the ligand, respectively. These configura-tions are in line with the expected +III oxidation state formetMb and +II oxidation state for deoxyMb. The low MLCTcontribution (o10% d4L) for metMb is congruent with formerobservations in the met-form of haemoglobin7 and some modelFe heme complexes.29 In contrast, the significant MLCT (d5L)contribution for deoxyMb is quite unusual when comparedwith literature data about solid samples.29 This hints that, inthis protein, an electron is almost fully localized to the waterligand and that the observed XA spectrum is mainly due to theprotein solvation. Likewise, for MbO2 and MbNO, the limitedintensity decrease at the L3-edge compared with the caseof deoxyMb supports a proposal by Weiss30 (originally forhaemoglobin) that the ligation process produces Mb–Fe3+-likespecies bound to negatively charged O2 and NO ligands,respectively. In fact, TFY measurements are seemingly essen-tially sensitive to the large MLCT to these ligands, which mightexplain the similarity of the corresponding XA spectra althoughNO is described in the literature as a much stronger ligandthan O2.27For MbCO and MbCN, a strong fluorescence quenchinghappens which reflects additional charge transfers. For MbCNin particular, an increase in MLCT might contribute to lowerthe intensity of the most prominent spectral feature at the L3-edge (Fig. S2 in ESI‡). From the decreasing L3 : L2 intensity ratioin Fig. 2, we can sort ligands with respect to the increasingstrength of their interaction (involving fluorescence quenching)with the iron active centre of Mb, namely CNE CO > O2E NO.In brief we have presented here the first study of liganddiscrimination by myoglobin in solution using TFY L-edge softX-ray absorption spectroscopy at the iron active centre. Basedon experimental observation, we could qualitatively show thatthe strength of the interaction between the ligand and theporphyrin active centre can be sorted in the following order: CNE CO > O2 E NO. More detailed investigations into this issueare currently in preparation using high resolution resonantinelastic X-ray scattering (RIXS) which has been introducedrecently in the liquid micro-jet technique.31This work was supported by the European Research Councilstarting grant no. 279344 and the Helmholtz-Gemeinschaft viathe young investigator fund VH-NG-635. SB acknowledges theUniversity of Bordeaux and the CNRS for financial supportsthrough his chair of excellence.Notes and references1 A. H. T. Theorell, Biochem. Z., 1934, 268, 73.2 J. B. Wittenberg, Phys. Rev., 1970, 50, 559–636.3 X. H. Huang and S. G. Boxer, Nat. Struct. Biol., 1994, 1, 226–229.4 J. Z. Ruscio, D. Kumar, M. Shukla, M. G. Prisant, T. M. Murali andA. V. Onufriev, Proc. Natl. Acad. Sci. U. S. A., 2008, 105, 9204–9209.5 J. P. Collman and L. Fu, Acc. Chem. Res., 1999, 32, 455–463.6 E. Antonini and M. Brunori, Hemoglobin and myoglobin in theirreactions with ligands, North-Holland Pub. Co., Amsterdam, 1971.7 E. F. Aziz, N. Ottosson, S. Bonhommeau, N. Bergmann, W. Eberhardtand M. Chergui, Phys. Rev. Lett., 2009, 102, 068103.8 N. Bergmann, S. Bonhommeau, K. M. Lange, S. M. Greil, S. Eisebitt,F. de Groot, M. Chergui and E. F. Aziz, Phys. Chem. Chem. Phys.,2010, 12, 4827–4832.9 E. F. Aziz, J. Phys. Chem. Lett., 2011, 2, 320–326.10 S. Eisebitt, T. Boske, J. E. Rubensson and W. Eberhardt, Phys. Rev. B,1993, 47, 14103–14109.11 E. F. Aziz, J. Electron Spectrosc. Relat. Phenom., 2010, 177,168–180.12 E. F. Aziz, S. Eisebitt, F. de Groot, J. W. Chiou, C. G. Dong, J. H. Guoand W. Eberhardt, J. Phys. Chem. B, 2007, 111, 4440–4445.13 H. X. Wang, G. Peng, L. M. Miller, E. M. Scheuring, S. J. George,M. R. Chance and S. P. Cramer, J. Am. Chem. Soc., 1997, 119,4921–4928.14 S. Bonhommeau, N. Ottosson, W. Pokapanich, S. Svensson,W. Eberhardt, O. Bjorneholm and E. F. Aziz, J. Phys. Chem. B,2008, 112, 12571–12574.15 S. Kim, G. Jin and M. Lim, J. Phys. Chem. B, 2004, 108, 20366–20375.16 S. Della Longa, S. Pin, R. Cortes, A. V. Soldatov and B. Alpert,Biophys. J., 1998, 75, 3154–3162.17 F. A. Lima, in Institut des sciences et inge´nierie chimiques, The`se E´colepolytechnique fe´de´rale de Lausanne EPFL, EPFL tous droitsre´serve´s, 2011, p. 66.18 Y. Harada, M. Taguchi, Y. Miyajima, T. Tokushima, Y. Horikawa,A. Chainani, Y. Shiro, Y. Senba, H. Ohashi, H. Fukuyama andS. Shin, J. Phys. Soc. Jpn., 2009, 78, 044802.19 J. Yi, A. M. Orville, J. M. Skinner, M. J. Skinner and G. B. Richter-Addo, Biochemistry, 2010, 49, 5969–5971.20 Y. Sugawara, A. Matsuoka, A. Kaino and K. Shikama, Biophys. J.,1995, 69, 583–592.21 M. A. Soldatov, K. M. Lange, M. D. Gotz, N. Engel, R. Golnak,A. Kothe and E. F. Aziz, Chem. Phys. Lett., 2012, 546, 164–167.22 M. D. Gotz, M. A. Soldatov, K. M. Lange, N. Engel, R. Golnak,R. Ko¨nnecke, K. Atak, W. Eberhardt and E. F. Aziz, J. Phys. Chem.Lett., 2012, 3, 1619–1623.23 E. F. Aziz, M. H. Rittmann-Frank, K. M. Lange, S. Bonhommeau andM. Chergui, Nat. Chem., 2010, 2, 853–857.24 M. Bauer, T. Stalinski and E. F. Aziz, ChemPhysChem, 2011, 12,2088–2091.25 R. Seidel, S. Ghadimi, K. M. Lange, S. Bonhommeau, M. A. Soldatov,R. Golnak, A. Kothe, R. Ko¨nnecke, A. Soldatov, S. Thu¨rmer, B. Winterand E. F. Aziz, J. Am. Chem. Soc., 2012, 134, 1600–1605.26 E. F. Aziz, K. M. Lange, S. Bonhommeau and M. Chergui, Nat.Chem., 2012, 4, 767–768.27 S. Carniato, L. Journel, R. Guillemin, M. N. Piancastelli, W. C. Stolte,D. W. Lindle and M. Simon, J. Chem. Phys., 2012, 137, 144303.28 T. Z. Regier, A. J. Achkar, D. Peak, J. S. Tse and D. G. Hawthorn, Nat.Chem., 2012, 4, 765–766.29 R. K. Hocking, E. C. Wasinger, Y. L. Yang, F. M. F. deGroot, F. AnnWalker, K. O. Hodgson, B. Hedman and E. I. Solomon, J. Am. Chem.Soc., 2007, 129, 113–125.30 J. J. Weiss, Nature, 1964, 202, 83–84.31 K. M. Lange, R. Ko¨nnecke, S. Ghadimi, R. Golnak, M. A. Soldatov,K. F. Hodeck, A. Soldatov and E. F. Aziz, Chem. Phys., 2010, 377, 1–5.ChemComm CommunicationPublished on 05 February 2013. Downloaded by Freie Universitaet Berlin on 27/01/2015 10:16:55. View Article Online

an iron L-edge X-ray absorption study of the active centre

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an iron L-edge X-ray absorption study of the active centre

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