Ultrafast 2D-IR spectroscopy of nitrosylated haem-proteins using ULTRA Contact [email protected] K. Adamczyk, N. Simpson, N.T. Hunt Department of Physics, SUPA University of Strathclyde C. Bellota-Anton, P.A. Hoskisson, N.P. Tucker Strathclyde Institute of Pharmacy and Biomedical Sciences University of Strathclyde G.M. Greetham, M. Towrie, A.W. Parker Central Laser Facility STFC Rutherford Appleton Laboratory A. Gumiero, M.A. Walsh Diamond Light Source Introduction Understanding the function of biological molecules at the level of movements of atoms or the making/breaking of chemical bonds offers considerable potential for downstream benefits. These range from advanced drug design strategies to the production of synthetic, biology-inspired molecules for technological or medical applications. The concept of the structure-function relationship is well-established in biology but this does not offer a complete picture of the intimate ‘chemical’ processes occurring in the active sites of biological molecules because it neglects the role of solvent-induced and thermal fluctuations of the protein architecture as well as the effect of local vibrational modes. Indeed, the influence of fast protein structural dynamics on biological processes that take place many orders of magnitude more slowly is one of the key questions yet to be conclusively addressed.1 structural elements in the individual steps remain the topic of debate14,15 and enquiry. In particular, the distal side of the haem pocket includes a histidine residue located in close proximity to the haem centre.16-18 This residue is widely implicated in the catalase mechanism and mutation studies have shown that its presence is crucial to CpdI formation.19-21 The subtle way that biomolecule structure influences function is evidenced very clearly by the haemoprotein family. This group of proteins are responsible for a large number of biological roles ranging from reversible ligand binding to enzymatic activity but, to a first approximation, some of the major structural features located near the haem centre appear to be very similar, raising the question of exactly how the molecular architecture influences function. An example of this can be seen in studies showing that mutations at just four positions or fewer can engender nitric oxide reductase or peroxidase activity upon the ligand binding protein myoglobin. This flexibility of function within a relatively restricted structure has led to the haemoproteins becoming attractive templates for synthetic systems but for this to be successful, we must first fully understand the detailed roles of each of the main structural elements.2-5 Ultrafast 2D-IR spectroscopy has shown great potential for measuring the structural dynamics of biological molecules both at the global, whole molecule level and in terms of a single bond within the macromolecular structure by employing vibrational probes.6-8 The purpose of this report is to summarise recent advances of 2D-IR spectroscopy of haem proteins using the ULTRA laser system and to demonstrate how this technology can influence our view of the structure-function relationship. This will be done by reference to studies of two haem proteins: the ligand transport protein myoglobin 9 and the catalase enzyme.10 The catalases, common to almost all aerobically-respiring organisms, are responsible for the disproportionation of hydrogen peroxide in a reaction that is often represented as: 11-13 catalase-Fe(III) + H2O2 → O=Fe(IV)Por+. + H2O (1) O=Fe(IV)Por+. + H2O2 → catalase-Fe(III) + H2O + O2 (2) 2H2O2 → +. 2H2O + O2 (1)+(2) where O=Fe(IV)Por is referred to as Compound 1 (CpdI). This mechanism is widely accepted but the precise roles of catalase Interestingly, a similarly-located and conserved distal histidine residue is found in myoglobin. The fact that this residue is apparently central to the functioning of two different proteins begs questions about its role. For example, it seems reasonable that it could be responsible for ligand binding in both cases. This then suggests that it is the rest of the haem pocket that controls specific functionality. Other residues in the active sites of these proteins do differ and so must contribute to the behaviour of the biomolecule. Most notably, the proximal residues that coordinate with the Fe atom of the haem moiety are different and this change could play a role in the chemical lability of the haem ligand.22 However, it is also instructive to ask whether the presence of the distal histidine in both myoglobin and catalase means that the haem ligand is subject to a similar chemical environment in both cases and it is this question that we address here. In each of the articles featured, the ferric form of the protein was considered with nitric oxide bound to the haem centre acting as a probe of the local dynamic environment.9,10 The choice of NO arose because it binds effectively to the haem site of Mb while the catalase enzyme is inhibited by NO binding, meaning that it provided a stable and effective probe in both cases. In addition, NO itself plays a fundamental role in biology, participating in processes such as signalling and immune responses,23-25 while higher concentrations can lead to the deleterious effects associated with nitrosative stress. The NO radical is also highly reactive with transition metals and metalloproteins, such as those containing haem groups and well-known examples include components of the respiratory chain such as cytochrome C oxidase and key enzymes of the tricarboxylic acid cycle such as fumarase and aconitase.26,27 Experimental For all 2D-IR experiments, catalase and myoglobin were contained in a pD7 deuterated phosphate buffer solution with care taken to ensure complete H/D exchange in all cases. MAHMA NONOate was used to nitrosylate the ferric proteins.10 For all 2D-IR experiments, the samples were held between two CaF2 windows separated by a 100 µm thickness spacer. The method for obtaining IR pump-probe and 2D-IR spectra has been described previously; briefly, 2D-IR spectra were acquired using the FT-2D-IR method described in Ref 10 using a sequence of three mid-infrared (IR) laser pulses arranged in a pseudo pump-probe beam geometry.28,29 The pulses were generated by the ULTRA Ti:sapphire laser system pumping a white-light seeded optical parametric amplifier (OPA) equipped Fig 1: 2D-IR spectra of the NO stretching vibrational moode of nitrosylated caatalase (a) and myoglobin H64Q (c). Figs (bb) and (d) are fits of tthe data in (a) and a (c) respectively to 2D Gauussian lineshape funcctions.9,10 m of the siignal and idler.. Mid with differencce frequency mixing IR pulses wiith a temporall duration of ~100 fs; a ceentral frequency of 1900 cm-1 withh a bandwidth of >300 cm-1 were employed. Results and D Discussion Representativee 2D-IR spectrra for catalase and the myogglobin (Mb) H64Q m mutant are show wn in Fig 1(a-d d). In the case oof the catalase proteiin, a single negative (red) peeak on the specctrum diagonal wass observed annd assigned to t the bleachh and stimulated em mission from the v=0-1 transition of thee NO stretching modde of the nitrosylated protein n (Fig 1 (a)). 100 This was accompannied by a positivve (blue) peak shifted s by arounnd 30 cm-1 to lowerr probe frequeency, which was w assigned too the accompanyingg v=1-2 excitedd state absorptio on.10 The 2D-IR speectrum of wild type (wt) Mb reported r was siimilar to that of cattalase in that it too featured d a single diaagonal infrared transittion in the NO stretching regio on. 9 By contrasst, the 2D-IR spectruum of Mb-H644Q shows two diagonal peakss (Fig 1(c)), one of w which was locatted at the same frequency as thhat of the wild-type protein peak and a one that was w shifted to llower The structure of Mb is well-known and it is frequency.9 T accepted thaat wt-Mb feaatures a direect H-bondingg-type interaction bettween the distaal histidine residue side chainn and the haem ligannd. 30 The H64Q Q mutation feattures replacemeent of the distal histiidine residue with w glutamine. The latter featuures a more flexible side chain thaan histidine, allowing the term minal c to move away a from the haem functional grooup of the side chain ligand in a fraaction of the molecules m in the sample. As a rresult of this, the hiigher frequencyy peak observeed in the Mb-H H64Q spectrum was attributed to the t NO stretching vibration oof the sub-ensemble in which there was an interacttion between thhe NO t protein. The T lower frequuency ligand, similarr to the wild type mode correspoonded to the subb-ensemble of proteins p withouut this interaction.31 R spectra show wn in Fig 1 thaat the It is noticeablle in the 2D-IR lineshapes of the v=0-1 and v=1-2 transiitions are elonngated towards the diiagonal of the spectrum. The spectra for cattalase and Mb-H64Q Q shown were obtained o with a waiting time oof ~ 1 ps and this eloongation is duee to inhomogen neous broadeniing of the NO stretchhing vibrationall mode of both proteins. This eeffect has been widdely reported for fo haem proteiins and arises from fluctuations off the electrostattic environmentt of the ligand ddue to motion of thhe protein arcchitecture.9,10,32-34 As the prrotein fluctuates, the effect is to varry the NO stretcching frequencyy by a smalll amount leading to broadeninng of the transiition across thee ensemble. In a 2D-IIR experiment at waiting timees that are shortt in relation r to thee protein dynaamics that are causing thee broaadening, this results in a diagoonal elongation n of the 2D-IR R peak ks because the sample s maintaiins a ‘memory’’ of the state inn whicch it was exciteed; leading to a correlated 2D peakshape. Ass the waiting w time is allowed a to incre rease and becom mes comparablee to th he timescales of the underl rlying dynamiccs, the samplee flucttuations lead to a loss of this m memory and thee peak becomess moree circular. Thiis so-called sppectral diffusio on results in a chan nge in the profile of the 2D-IR R peak with waaiting time andd quan ntification of th he lineshape eevolution using g fitting to 2D D Gausssian lineshapes (eg Fig 1 (b&d))9,10 gives rise to ann expo onential-type decay d with thhe timescales reporting thee dynaamics of the frequency-freqquency correlation functionn (FFC CF) of the NO vibration, v which ch in turn reportt on the proteinn dynaamics influencin ng the ligand.9,335-39 The FFCFs extracted from the 2D D-IR data for catalase, c wt-Mbb and Mb-H64Q arre shown in FFig 2.9,10 In each case ann expo onential function was shown too represent the data d well (solidd liness) and it is interesting to note th the similarities and differencess in th he data across the three proteinns. Specifically y, both catalasee and the wt-Mb sh howed a fast ddynamic comp ponent (~3 ps)) o The stattic parameter was w assigned too alongside a static offset. w motions caussing broadeninng of the NO transition butt slow whicch were too slow w to be captureed by a 2D-IR experiment e thatt was temporally lim mited by the viibrational lifetiime of the NO O n. Although ccatalase and wt-Mb w showedd stretching vibration similar fast dynamics, the static ccomponent in Mb was large,, v small in ccatalase. This sh howed that thee wherreas this was very motiions causing brroadening are complete within 20-30 ps inn catallase while tho ose in Mb perrsist for much h longer. Thiss obseervation was ussed to infer a ddynamically more m constrictedd struccture in catalasee. 10 In asssigning the fasst dynamics, it was noted thaat while the wt-Mb and the H64Q Q mutation both th showed sim milar slow/staticc c in th the wt-Mb dataa was replicatedd dynaamics, the fast component only y for the sub-ensemble of the H H64Q mutation n with a similarr vibraational frequency to the wt prrotein. 9 This fast f componentt was absent in the su ub-ensemble off Mb-H64Q thaat had no directt d side chaain and the NO O ligand. Thiss link between the distal allow wed assignmen nt of the faast dynamics of wt-Mb too interraction between n the distal histid idine and the haaem ligand. 9 Giveen the similaritiies in both distaal pocket archittecture and fastt dynaamics between n wt-Mb andd catalase, an a anaologouss assig gnment of the fast dynamics of catalase to an interactionn betw ween the distal histidine aand the NO would seem m apprropriate. Howev ver, it was repoorted that the crystal c structuree of the t nitrosylated protein was as more consiistent with ann interraction of NO with w a conservedd bound water molecule m in thee distaal pocket. Whille this would seeem contradicttory, the boundd wateer molecule wass also hydrogenn-bonded to the distal histidinee in su uch a way as to t communicatee the fast dynaamics from thee proteein scaffold to the haem lligand. Thus, the dynamicss obseerved in catalasee were comparaable to those off wt-Mb but thee mech hanism by which they were oobserved was different d for thee two proteins. It was further hypotthesised that th his bound waterr moleecule, in conjun nction with a nnetwork of otheers observed inn the crystal c structure, were the oriigins of the strructurally moree conffined active sitee and so the smaall size of the static s parameterr in th he catalase FFCF F. 10 In su ummary, by comparing these sets of results,, it can be seenn that 2D-IR providess insight into thhe local chemiccal environmentt of th he haem ligand d in both a liggand transport protein and ann enzy yme. Furtherm more, cross-diisciplinary intteraction withh strucctural biologistts enables this data to be in nterpreted in a physsically-meaning gful manner forr biological applications. Thee concclusions suggest that the ligannd transport pro otein features a moree flexible struccture with an iinteraction betw ween the distall pock ket and ligand that presumaably serves to aid reversiblee binding of a diiatomic ligand. In contrast, thee enzyme locatees the ligand in a moore constrained geometry conssistent with thee need to access a pparticular transiition state as part p of the reaaction mechanism. T Thus, although the t structures may m seem similaar, the natures of the active sites of these t two proteiins differ markeedly. Acknowledgeements Support for thhis work is acknnowledged from m the STFC Ceentral Laser Facilityy, the Europeean Research Council (2022706), EPSRC (EP/J000975X/1) and the Leverhulme Trust (RPG-2248). Fig 2: FFCF data for nitrossylated catalasee (a) and myogglobin m Ref9 (b) extracted ffrom 2D-IR datta.9,10 Figure reeproduced from - Reproduced by permission of o the PCCP Ow wner Societies References (1) Antoniou, D.; Schwartz, S. S D. J Phys Ch hem B 2011, 1155, 15147. (2) Bagchi, S.;; Nebgen, B. T..; Loring, R. F.;; Fayer, M. D. J Am Chem Soc 20110, 132 18367. (3) Ozaki, S. II.; Roach, M. P..; Matsui, T.; Watanabe, W Y. Accounts Chem m. Res. 2001, 34, 3 818. (4) Zhuang, J.; Amoroso, J. H.; H Kinloch, R.; Dawson, J. H. ; Baldwin, M. JJ.; Gibney, B. R. R Inorg. Chem. 2004, 43, 82188. (5) Yeung, N.;; Lin, Y.-W.; Gao, G Y.-G.; Zhao o, X.; Russell, B B. S.; Lei, L.; Minerr, K. D.; Robinsson, H.; Lu, Y. Nature N 2009, 4462, 1079. (6) Adamczykk, K.; Candelareesi, M.; Robb, K.; K Gumiero, A A.; Walsh, M. A.; Parker, A. W.;; Hoskisson, P. A.; Tucker, N. P.; Hunt, N. T. M Meas Sci Tech 20012, 23, 062001 1. (7) Hunt, N. T T. Chem Soc Revv 2009, 38, 183 37. (8) Hamm, P.;; Zanni, M. T. Concepts C and Method M of 2D Infrared Specttroscopy; Cambbridge University Press: Cambridge, 20011. (9) Adamczykk, K.; Candelareesi, M.; Kania, R.; R Robb, K.; Bellota-Antónn, C.; Greetham, G. M.; Pollard d, M. R.; Towriie, M.; Parker, A.. W.; Hoskissonn, P. A.; Tuckerr, N. P.; Hunt, N N. T. PCCP 2012, 114, 7411. (10) Candelareesi, M.; Gumierro, A.; Adamczzyk, K.; Robb, K K.; Bellota-Antónn, C.; Sangul, V.; V Munnoch, J. T.; Greetham, G G. M.; Towrie, M M.; Hoskisson, P. P A.; Parker, A. A W.; Tucker, N N. P.; Walsh, M. A.; Hunt, N. T. Orrg Biomol Chem m 2013, 11, 77778. H Kazzaz, J. A.; A Koo, H.; Josseph, (11) Arita, Y.;; Harkness, S. H.; A. Am J Physiiol Lung Cell Mol M Physiol 2006, 290, L978. (12) Isobe, K.;; Inoue, N.; Takkamatsu, Y.; Kaamada, K.; Wakkao, N. J Biosci Biooeng 2006, 1011, 73. G J. M M. C. Free radiccals in biology (13) Halliwell, B.; Gutteridge, and medicine,xxxvi Oxford Univerrsity Press: Oxfford ; New York k, 2007. (14) Jones, P.; Dunfford, H. B. Jourrnal of Inorgan nic Biocchemistry 2005, 99, 2292. (15) Jones, P. J Biol Chem 2001, 2276, 13791. (16) Chelikani, P.; Fita, F I.; Loewenn, P. C. Cellular and Moleecular Life Scieences 2004, 61, 192. (17) Fita, I.; Silva, A. A M.; Murthy,, M. R. N.; Rossmann, M. G. Acta a Crystallograph hica Section B--Structural Scieence 1986, 42, 497. mann, M. G. J M Mol Bio 1985, 18 85, 21. (18) Fita, I.; Rossm (19) Nicholls, P.; Fiita, I.; Loewen,, P. C. Advancees in Inorganic mistry, Vol 51 2001, 2 51, 51. Chem (20) Kato, S.; Ueno o, T.; Fukuzumii, S.; Watanabe, Y. J Biol m 2004, 279, 52 2376. Chem (21) Matsui, T.; Ozaki, S.; Liong, E E.; Phillips, G. N.; Watanabe, 99, 274, 2838. Y. J Biol Chem 199 (22) Soper, J. D.; Kryatov, K S. V.; R Rybak-Akimov va, E. V.; Noceera, D. G. J Am m Chem Soc 20007, 129, 5069. (23) Alderton, W. K.; K Cooper, C. E E.; Knowles, R. G. Biochem. 001, 357, 593. J. 20 (24) Hill, B. G.; Draanka, B. P.; Bai ailey, S. M.; Lan ncaster, J. R., D V. M. J. Biol. C Chem. 2010, 28 85, 19699. Jr.; Darley-Usmar, (25) Kass, D. A.; Taakimoto, E.; Naagayama, T.; Champion, H. C Res. 2007, 75, 303 . C. Cardiovasc. (26) Jones-Carson, J.; Laughlin, J. ; Hamad, M. A.; A Stewart, A. V M. I.; Vazquez-Torres V s, A. Plos One 2008, 3, 1. L.; Voskuil, (27) Brunori, M. Trrends Biochem. Sci. 2001, 26, 21. R. A.; Tokmako off, A. Optics (28) DeFlores, L. P.; Nicodemus, R Lett. 2007, 32, 2966 6. Phys 2009, 11, (29) Shim, S.-H.; Zanni, M. T. PhyysChemChemP 748. (30) Brunori, M. Heemoglobin and myoglobin in their reactions with ligands; North Holland Publisshing Co: Amstterdamdon, 1971. Lond (31) Soldatova, A. V.; V Ibrahim, M..; Olson, J. S.; Czerrnuszewicz, R. S.; Spiro, T. G.. J Am Chem So oc 2010, 132, 4614 4. (32) Thielges, M. C.; C Chung, J. K. ; Fayer, M. D. J Am Chem 2 133, 3995. Soc 2011, (33) Bagchi, S.; Nebgen, B. T.; Looring, R. F.; Fay yer, M. D. J Am Chem C Soc 2010 0, 132, 18367. (34) Ishikawa, H.; Finkelstein, F I. J. J.; Kim, S.; Kwaak, K.; Chung, M.; Fayer, M. D. D Proc Nat J. K..; Wakasugi, K..; Massari, A. M Acad d Sci 2007, 104, 16116. (35) Finkelstein, I. J.; J Zheng, J. R. ; Ishikawa, H.; Kim, S.; Kwaak, K.; Fayer, M. M D. PCCP 20007, 9, 1533. (36) Roberts, S. T.; Loparo, J. J.; T Tokmakoff, A. J Chem Phys 2006 6, 125, 084502. (37) Ghosh, A.; Qiu u, J.; DeGrado, W. F.; Hochstrrasser, R. M. Procc Nat Acad Sci 2011, 2 108, 61155. (38) Kim, Y. S.; Liu u, L.; Axelsen, P. H.; Hochstraasser, R. M. Procc Nat Acad Sci 2009, 2 106, 177551. (39) Woys, A. M.; Lin, L Y.-S.; Redddy, A. S.; Xion ng, W.; de Pablo, J. J.; Skinnerr, J. L.; Zanni, M M. T. J Am Cheem Soc 2010, 132, 2832.
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