Article Cite This: J. Phys. Chem. A XXXX, XXX, XXX−XXX
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Vibrational Relaxation in EDTA Is Ion-Dependent Sean C. Edington and Carlos R. Baiz* Department of Chemistry, University of Texas at Austin, Austin, Texas 78712, United States
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S Supporting Information *
ABSTRACT: Ion binding by carboxylate groups is common in biomolecules such as metalloproteins, but dynamical aspects of ion binding are not fully understood. We present ultrafast spectroscopic measurements of vibrational relaxation in the ion-coordinating carboxylate groups of EDTA, which we use as a model of carboxylate-mediated ion binding, as EDTA binds a series of divalent and trivalent metal ions with high affinity. The measurements are interpreted using a Redfieldbased anharmonic model of vibrational relaxation that rationalizes trends in vibrational lifetimes in terms of vibrational energy transfer between EDTA’s asymmetric carboxylate stretching vibrational modes and lower-lying modes. Results show ion-dependent changes in complex structure and dynamics well outside the temporal and spatial resolution of common structural methods and demonstrate how vibrational relaxation measurements may contribute to exploration of ion-binding dynamics on ultrashort length and time scales.
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INTRODUCTION Ion binding is central to nearly all biological processes,1,2 but the structural dynamics and energy exchange characteristics of ion coordination events in proteins remain incompletely understood.3,4 Similarly, the complete sequences of structural changes induced by ion binding in proteins and culminating in signal transduction or other biochemical action are, in most cases, not known.5−8 While it is unlikely that all processes occurring on picometer/picosecond scales are critical to biomolecular events, measurements in these regimes, beyond the resolution of widely used structural methods such as NMR spectroscopy and X-ray crystallography,9−11 nonetheless contain useful information about slower, unquestionably important processes. As we will show, vibrational relaxation which proceeds in hundreds of femtoseconds reflects miniscule ion-dependent changes in equilibrium binding structure that modulate the anharmonic couplings between vibrational modes. Thus, with accurate modeling, measurements of ultrafast processes can be exploited as sensitive probes of structures and processes which persist on much longer time scales. Similarly, ultrafast measurements are often needed to provide unambiguous descriptions of events which unfold over more extended periods.12 In this paper, we demonstrate how ultrafast measurements of vibrational relaxation may be used to probe small structural and dynamic changes attending ion binding. Over the past two decades, ultrafast spectroscopy has helped map fast protein dynamics that remain opaque to other experimental methods.12−22 Notably, recent work in this area has exploited time-resolved spectroscopy for the study of ion binding and recognition dynamics in proteins.23−25 Spectroscopy is often limited by the models used to extract structural insights from experimental data; complex lineshapes and dynamics are difficult to interpret without sophisticated © XXXX American Chemical Society
models. While the electrostatic mapping approaches commonly used to interpret spectroscopic results are versatile,26−33 they are unable to reliably describe important effects.34−36 For example, ultrafast vibrational spectra report on processes such as anharmonic coupling and relaxation to dark states.37,38 The complexity of modeling vibrational relaxation processes,39−41 which involves estimates of anharmonic coupling between states, accurate determininations of system−bath interactions, and anharmonic models that can only be applied to small numbers of vibrational modes, means that a specialized approach is required to extract information about ion binding from related aspects of spectroscopic data. In this paper, we employ ultrafast transient absorption spectroscopy to directly observe time-dependent vibrational relaxation in the asymmetric stretching modes of the ionbinding carboxylate groups in several ethylenediaminetetraacetic acid (EDTA) chelation complexes (Figure 1) dissolved in D2O. Carboxylate stretching vibrations are sensitive to changes in their molecular environments and report structural perturbations with picometer spatial sensitivity and subpicosecond temporal precision.42−44 Thus, infrared spectroscopy of these vibrations serves as a useful probe of molecular structure and dynamics.45,46 Our earlier measurements of EDTA FTIR spectroscopic shifts attending the coordination of different ions23 demonstrate its ability to detect changes in metal ion coordination radius of less than 10 pm. We use transient absorption spectroscopy to measure ion-dependent vibrational relaxation rates, and employ EDTA47−55 as a simple, computationally tractable model of ion binding. The present results reveal ion-dependent trends in vibrational lifetime even Received: June 25, 2018 Revised: July 12, 2018 Published: July 19, 2018 A
DOI: 10.1021/acs.jpca.8b06075 J. Phys. Chem. A XXXX, XXX, XXX−XXX
Article
The Journal of Physical Chemistry A
depend on coordination structure. Since this method probes molecular configurations similar to those that exist in biological ion-binding sites, we expect similar models to aid in the interpretation of more complex relationships between structure and energy exchange in many carboxylate-rich biological coordination motifs.
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METHODS
EDTA Preparation. EDTA was used as received and prepared at 5 mg/mL (17 mM) concentration in D2O at an uncorrected pH reading of 11. This corresponds to a corrected pH reading of ca. 11.2−11.4.56,57 The pH was adjusted with DCl (Sigma-Aldrich, 99 atom % D) and NaOD (SigmaAldrich, 99 atom % D). Complete deprotonation of EDTA’s carboxylate groups, as expected at the given pH values,58 was verified via FTIR spectroscopy. Solutions equimolar in EDTA and the chloride salt of the given ion were used for FTIR and pump−probe measurements, sufficient to saturate EDTA’s one-to-one binding58 of the metal ions under study. Ka values for all of the EDTA complexes used are given in Table 1. The following salts were used: SrCl2 (Alfa Aesar, ultradry, 99.995%), CaCl2 (Sigma-Aldrich, anhydrous, >97%), MgCl2 (Sigma-Aldrich, anhydrous, >98%), LaCl3 (Alfa Aesar, anhydrous, 99.9%), TbCl3 (Alfa Aesar, anhydrous, 99.9%), and LuCl3 (Alfa Aesar, anhydrous, 99.9%). FTIR Spectroscopy. FTIR measurements were taken using a Bruker Vertex 70 spectrometer with a DTGS detector at 2 cm−1 resolution and averaging 32 scans per spectrum. The 50 μL sample was held between two CaF2 windows separated by a 50 μm Teflon spacer. The sample area was purged with dry (−100 °F dew point) air until no water vapor absorption lines were visible in the spectrum. Transient Absorption Spectroscopy. Transient absorption measurements of EDTA’s carboxylate asymmetric stretching mode were collected using a custom-built 2D IR spectrometer, which is described in detail in our previous work23 and in the Supporting Information (Section S1 and Figure S1). In short, 100 fs mid-infrared laser pulses probed the time-dependent IR absorption spectrum of the sample following excitation by a pump pulse. A four-frame phase cycle generated using a pulse shaper is used to suppress pump
Figure 1. Schematic representation of EDTA molecular structure bound to Ca2+ ion. Cartoon depictions of calculated vibrational normal modes referred to in this work are included at the bottom. Representative vibrational frequencies for each mode, taken from calculations of the Ca2+ complex, are included for each normal mode (see Table 1).
though corresponding structural changes are small. For example, EDTA vibrational lifetimes increase by nearly 30% when the bound ion is changed from La3+ to Lu3+ even though this substitution blue-shifts the carboxylate asymmetric stretching frequency by only 16 cm−1 and decreases the computed ion coordination radius by only 22 pm. We combine transient absorption spectroscopy with a Redfield-based model of vibrational relaxation to probe changes to vibrational dynamics which accompany ion binding by carboxylate groups. This approach provides a direct microscopic description of vibrational energy exchange within the carboxylate groups and allows the rationalization of vibrational relaxation in terms of anharmonic couplings that
Table 1. EDTA Structural and Spectroscopic Parameters Extracted from Density Functional Theory and Compared to Experimental Measurementsa ri (pm) rc (pm) log(Kf) νa1 (cm−1) νa2 (cm−1) νa3 (cm−1) νa4 (cm−1) νprincipal (cm−1) νshoulder (cm−1) t1/e (fs) scaled t1/e (fs)
source
Mg2+
Ca2+
Sr2+
Lu3+
expt comp expt comp comp comp comp expt expt expt comp
72 206 8.79 1616 1620 1625 1639 1600 1628 861 ± 10 879
100 236 10.65 1611 1613 1618 1628 1588 1612 843 ± 5 815
118 245 8.72 1614 1616 1620 1629 1586 1608 840 ± 9 850
86 220 19.74 1631 1635 1632 1654 1608 1638 1091 ± 5 1478
Tb3+ 92 17.87
1604 1634 952 ± 25
La3+ 103 242 15.36 1603 1604 1615 1622 1592 1625 853 ± 23 1087
a ri, six-coordinate ionic radius;65rc, average oxygen−ion coordination radius (all values have error
