Infrared Spectroscopy of Nicotinamide Adenine ... - ACS Publications

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Infrared Spectroscopy of Nicotinamide Adenine Dinucleotides in One and Two Dimensions Niall Simpson,† Daniel J. Shaw,†,‡ Pim W. J. M. Frederix,† Audrey H. Gillies,† Katrin Adamczyk,† Gregory M. Greetham,§ Michael Towrie,§ Anthony W. Parker,§ Paul A. Hoskisson,‡ and Neil T. Hunt*,† †

Department of Physics, University of Strathclyde, SUPA, 107 Rottenrow East, Glasgow G4 0NG, United Kingdom Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, 161 Cathedral Street, Glasgow G4 0RE, United Kingdom § Central Laser Facility, Research Complex at Harwell, STFC Rutherford Appleton Laboratory, Harwell Oxford, Didcot, Oxon, OX11 0QX, United Kingdom ‡

S Supporting Information *

ABSTRACT: The development of multidimensional spectroscopic tools capable of resolving site-specific information about proteins and enzymes in the solution phase is an important aid to our understanding of biomolecular mechanisms, structure, and dynamics. Nicotinamide adenine dinucleotide (NAD) is a common biological substrate and so offers significant potential as an intrinsic vibrational probe of protein−ligand interactions but its complex molecular structure and incompletely characterized infrared spectrum currently limit its usefulness. Here, we report the FTIR spectroscopy of the oxidized and reduced forms of NAD at a range of pD values that relate to the “folded” and “unfolded” forms of the molecules that exist in solution. Comparisons with structural analogs and the use of density functional theory simulations provide a full assignment of the observed modes and their complex pD dependencies. Finally, ultrafast two-dimensional infrared spectra of the oxidized and reduced forms of NAD are reported and their usefulness as biomolecular probes is discussed.

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INTRODUCTION Nicotinamide adenine dinucleotide (NAD) is a key component of many biological systems where it plays various roles including substrate, coenzyme, and redox partner. In the latter case, the transition between the oxidized (NAD+) and reduced (NADH) forms of NAD (Figure 1) provides the basis for numerous biochemical pathways. Despite this ubiquity, there remain gaps in our understanding of this important coenzyme. In particular, the molecular structure of NAD+ in solution has been the topic of much debate.1−12 Early nuclear magnetic resonance (NMR) spectroscopy studies,9−12 along with UV− visible spectroscopy experiments,13,14 suggested that a “folded” configuration is present in aqueous solution at neutral pH. This configuration was exemplified by a significant stacking interaction between the ring moieties of the nicotinamide and adenine bases. Later studies, while not in direct disagreement, indicated a more flexible structure with reduced interbase interaction and the existence of a dynamic equilibrium between folded and “unfolded” forms.4−8,15 More recently, the case for a largely folded structure in aqueous solution was further supported by molecular dynamics simulations with the dominant driving force being ascribed to the solvation properties of the nicotinamide unit.2 © 2013 American Chemical Society

Although the folded conformation predominates in solution, the unfolded structure is of particular biochemical relevance. This is because in the majority of systems for which a crystal structure is available the bound form of NAD has been found to adopt this structure. NMR studies as a function of pH have suggested a correlation between protonation of the adenine moiety and the transition to the unfolded structure, which was attributed to the presence of two charged ring systems on the adenine and nicotinamide units, respectively. It has also been shown that the incorporation of methanol or urea into an aqueous solvent leads to unfolding,3,10 but the biological driving force toward open-form ligation is far from clear. As well as the interest in NAD as a molecule in its own right, its biological ubiquity means that it has the potential to act as a powerful probe of binding sites of proteins. In particular, IR methods, including powerful difference-spectroscopy methodologies, can offer bond-level insights into protein structure and biomolecular interactions. However, such experiments rely heavily on component molecule spectra that are wellReceived: November 11, 2013 Revised: December 6, 2013 Published: December 6, 2013 16468

dx.doi.org/10.1021/jp411091f | J. Phys. Chem. B 2013, 117, 16468−16478

The Journal of Physical Chemistry B

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Figure 1. Molecular structures of the NAD derivatives and analogs studied. Labels associated with the NAD+ structure indicate the numbering system used in discussions and proton positions for NMR analysis (see text).

transitions of proteins and the fact that it is relatively uncluttered, featuring the kind of high frequency, localized, stretching vibrational modes that are most likely to act as effective local vibrational probes. Another driving force for understanding the IR spectra of “small” biological molecules, such as NAD, arises from the increasing use of multidimensional infrared spectroscopies, which seek to provide information on local structure and dynamics within biological systems.22−26 Such experiments are the IR analog of multidimensional NMR experiments and employ a sequence of IR laser pulses to spread the spectrum of a molecule over a second frequency axis; the method and its applications have been reviewed.22−27 Multidimensional spectroscopy of biomolecules is hampered by the complex and

characterized under all conditions. Vibrational spectroscopy, largely in the form of Raman measurements, has been applied to both oxidized and reduced NAD.1,3,16−21 Studies in both H2O and D2O solutions employing structural analogs such as nicotinamide, adenine, and adenosine and including some pH(D)-dependent measurements have been performed but no detailed IR spectroscopy study has been reported matching the pH(D) range over which structural changes were observed in NMR experiments. This has resulted in open questions regarding the effect of molecular structure on the IR spectrum and despite a general consensus with regard to assignments of some IR bands several discrepancies still remain, primarily in the region near 1600 cm−1.1,3,16−21 This is an important spectral region due to its proximity to the sensitive amide I 16469



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time, between the second pump and probe pulses) were controlled using optical delay lines. All beams were polarized parallel with respect to each other. The signal, emitted in the direction of the residual probe beam, was recorded as a function of τ for a fixed Tw using a spectrometer and 128 channel mercury−cadmium−telluride (MCT) detector array to provide the probe frequency axis of the 2D-IR spectrum (resolution ∼2 cm−1). For laser power normalization purposes, the reference beam was also spectrally dispersed and imaged onto a 64 channel MCT detector array. The static arm of the interferometer was chopped at 5 kHz to enable removal of residual pump−probe signals and the pump frequency axis of the 2D-IR spectrum was recovered by Fourier transformation of the signal at each detector pixel as a function of τ. To accurately locate the time at which the pump pulse-pair were coincident on the sample the field autocorrelation was measured using the residual pulse pair from the interferometer on a single channel MCT detector. 1 H NMR. Spectra were acquired with an Oxford Instruments NMR AS400 operating at 400 MHz. Samples were prepared from the same solutions as used for FTIR measurements to ensure consistency. Density Functional Theory (DFT) Calculations. All geometry optimizations and subsequent frequency calculations were performed using the hybrid B3LYP functional and a tripleζ basis set with one added diffuse and one polarization function (6-311+G(d,p)) in Gaussian09 v.A02.47 Calculations in water were performed using the polarizable continuum model (PCM). All calculations were carried out in an unfolded molecular geometry for both NAD+ and NADH as no experimental effects on the spectrum were observed upon folding (see below). A correction factor of 0.9679 was applied to the frequency data of all calculated spectra.48

overcrowded IR spectral response of native proteins or enzymes and so a variety of strategies have been employed from isotopic labeling of amino acid residues28−35 to the incorporation of non-natural probe groups,36−39 to extract sitespecific information. Intrinsic probe modes, which naturally exist in the system and feature vibrational transitions that are both readily identifiable and separate from IR absorptions due to the rest of the protein scaffold, remove the need to isotopically modify the biological system and so offers considerable benefit in terms of efficiency and cost.40−43 NAD is an attractive candidate for two-dimensional IR (2D-IR) from this perspective and its spectrum features several potentially viable modes that could act as probes for ligandbinding studies. To further investigate NAD and to evaluate its use as a probe for 2D-IR applications, we report a series of experiments designed to determine the IR spectroscopic assignments of the vibrational modes of NAD in both oxidized and reduced forms in the region near 1600 cm−1. Through comparisons of spectra with structural analogs and monitoring of the spectra as a function of pD, we show that several modes exhibit significant changes arising from protonation of the adenine moiety. These changes assist in assignment of the spectrum and especially of the nicotinamide carbonyl stretching vibration, which has been a point of much discussion. Further, these results indicate no effect on the IR response that is solely attributable to any reported change in structure, consistent with a relatively weak interbase interaction in solution. Finally, we report the 2D-IR spectrum of NAD+ and NADH and discuss their suitability as biological probe molecules in light of the new assignments.

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EXPERIMENTAL SECTION All chemicals were purchased from Sigma Aldrich and used without further purification. Deuterated solvents were employed throughout as this is the norm for infrared and ultrafast multidimensional spectroscopic measurements near 1600 cm−1. In pD-dependence studies, the solution pD was corrected using deuterium chloride and sodium deuteroxide to prevent H-D exchange and associated spectral changes. Fourier Transform (FT) IR Spectroscopy. All FTIR absorption spectra were acquired at room temperature using a Bruker Vertex 70 spectrometer. Samples were housed between two CaF2 windows separated by a polytetrafluoroethylene (PTFE) spacer of 50 μm thickness. Solute concentrations were 100 mM throughout. Two-Dimensional-IR Spectroscopy. Samples were prepared as for FTIR spectroscopy above. The 2D-IR spectra were measured using the Fourier transform methodology in a pseudo pump−probe geometry that has been described elsewhere.40,44,45 Briefly, a white light seeded optical parametric amplifier (OPA) pumped by the ULTRA amplified Ti:Sapphire laser system46 and featuring difference frequency mixing of signal and idler beams was used to produce 100 fs-duration pulses (2 μJ per pulse, 10 kHz repetition rate) with a central frequency near 1550 cm−1 and a bandwidth in excess of 300 cm−1. This IR output was split to produce two low intensity beams that were used as probe and reference beams while the remainder was directed into a Michelson-type interferometer to generate two pump pulses with a variable time-delay between them. The latter were recombined collinearly and focused into the sample to overlap with the probe beam. Interpulse timings (denoted τ between the two pump pulses and Tw, the waiting

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RESULTS AND DISCUSSION NAD+. The FTIR spectrum of NAD+ at neutral pD is presented in Figure 2a. In the region between 1400 and 1750 cm−1, which is the principal region of interest in mid-IR biomolecular spectroscopy, five clear peaks are observable at

Figure 2. FTIR spectra of NAD+ and structural analogs near neutral pD. (a) NAD+ at pD7; (b) adenine at pD5; (c) adenosine at pD5, and (d) nicotinamide mononucleotide (NMN+) at pD7. Vertical lines indicate mode coincidences described in the text. 16470



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Figure 3. pD dependence of FTIR spectra of (a) NAD+ and structural analogs, (b) adenine, (c) adenosine, and (d) NMN+. Arrows show changes in intensity or position of peaks caused by reduction in pD.

Figure 4. (a) Ratio of intensities of pairs of peaks in the NAD+ and adenosine FTIR spectra as a function of pD. (b) NMR chemical shift values for each of the NAD+ protons labeled as in Figure 1 as a function of pD.

1666, 1625, 1577, 1482, and 1428 cm−1. These are consistent with recently reported data recorded under similar conditions.1 There is also an indication of a weak, broad feature at 1457 cm−1. Preliminary separation of these peaks across the nicotinamide and adenine moieties is made possible by comparison of this data to the spectra of the individual bases, which are shown in Figure 2b−d. It is noted that the displayed adenine and adenosine spectra were obtained at pD 5. This is due to the gradually reducing solubility of the compound above pD 2.5, resulting in decreased signal-to-noise ratios approaching neutral pD. To combat this, spectra of adenine and adenosine as a function of pD displayed subsequently were normalized to the intensity of the 1428 cm−1 mode in the pD2 sample. No scaling factor was applied to the data in Figure 2. Crucially, it was

determined that the spectral characteristics of adenine and adenosine are unchanged between pD values of 5 and 7. Comparing Figure 2a−d, it can be seen that the 1457 and 1428 cm−1 modes contain contributions observed in both adenine and nicotinamide mononucleotide (NMN+, black dashed lines) spectra. The modes at 1625, 1577, and 1482 cm−1 are due to adenine (red dashed lines); the 1666 cm−1 mode and a weak band at 1633 cm−1 that is obscured in the NAD+ spectrum are assignable to the nicotinamide moiety (blue dashed lines). In addition, NMN+ exhibits a band at 1589 cm−1 that is not observed in NAD+. However, the relative magnitude of this peak in relation to the 1666 cm−1 mode of NMN+ suggests that it would be very weak in the NAD+ spectrum. It can be seen from comparison of Figure 2b,c that addition of the ribose group to adenine results in only minor changes to 16471



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CO stretching mode. That the 1666 cm−1 mode in the protonated NAD+ spectrum contains contributions from both adenine and nicotinamide can also be observed because the latter remains when the former is reduced approaching neutral pDs. It is instructive to compare the IR spectral changes with those observed via NMR spectroscopy of NAD+ at a similar range of pD values. NMR peak shift data is displayed in Figure 4b and the results are entirely consistent with those reported previously;10 downfield shifts occur for both adenine protons as the solution pD decreases. This was attributed to protonation of adenine at the nitrogen in the A1 position (see Figure 1) and is consistent with the most significant variation in chemical shift occurring for the adjacent A2 proton. Smaller shifts are also observed for the nicotinamide protons arising from unfolding at low pH.10 It can be seen from comparison of Figure 4a,b that the changes in NMR peak positions assignable to adenine protonation correlate extremely closely with changes in the IR data and thus the latter can be confidently assigned to this process. It is noteworthy that these results are also in agreement with significant changes to the adenine components of the Raman spectrum of NAD that were reported at pDs below 4 with a connection to protonation proposed but a quantitative analysis of the effect was not undertaken.3 Given that all of the spectral changes associated with a reduced pD can be assigned to protonation of the adenine moiety, there are no indications from the IR spectrum that a conformational change of the NAD+ molecule is taking place. This would have been expected via a shift or change in line width of a nicotinamide mode correlated with the adenine protonation but this was not observed. A similar lack of sensitivity to changes in structure was also reported using Raman spectroscopy.3 In part, in the FTIR spectra this can be attributed to the relative lack of spectral contributions from the nicotinamide unit in this spectral region. Further investigations of NAD+ spectra were carried out as a function of temperature and methanol-d4 concentration. Despite reported NMR9−12 and UV−vis13,14 evidence that a change to the unfolded state occurs under these conditions, no evidence of this was seen in the FTIR data, suggesting that the folded state does not lead to sufficiently strong interaction of the bases to result in spectral shifts in this region. The results are shown in the Supporting Information. Density Functional Theory Calculations. DFT calculations were carried out to enable definitive assignments of each of the modes of NAD+. The results are shown in Figure 5a for NAD+ in both protonated and unprotonated forms. The agreement between calculation and experiment is generally good; the band positions themselves are somewhat underestimated following application of a standard correction factor, though errors of the magnitude observed are not unreasonable when taking into account the application of a polarizable continuum solvent water rather than a higher-level treatment. In particular, the changes observed upon protonation are wellreproduced and these are shown in the figure with arrows that correspond to those in Figure 3a. Most notably, it can be seen that protonation displaces the intense band at 1588 to 1634 cm−1 which mirrors the 1625/1666 cm−1 peak pair in the experimental data. Analysis of the calculated normal modes reveals this mode features a C−N/NH2 stretching motion between the A6 position carbon atom on the adenine ring and the nitrogen atom of the NH2 group with contributions from the NH2 scissor motion. This is consistent with the

the IR spectrum. This is consistent with previous studies, which show that the ribose group has no significant vibrational modes in this region1 and hence, the free ribose molecule has not been included in this study. Further insight into the origins of these modes was obtained by variation of the pD of the solution. The results of this are shown in Figure 3a for NAD+ and in Figures 3b−d for the component bases. The pD range was chosen to mimic that of NMR experiments,10 which concluded that NAD+ is in a folded conformation at neutral pH while under acidic conditions the molecule changes to its extended form. It is notable from Figure 3a that the IR spectrum of NAD+ is extremely sensitive to pD in this region. Arrows mark the changes in peak amplitude or position as the pD is decreased and all modes except those at 1428 and 1455 cm−1 are affected. Specifically, the 1666 cm−1 mode increases dramatically in amplitude and a new peak is observed at 1508 cm−1 while those at 1625 and 1482 cm−1 decrease. The 1577 cm−1 mode shifts slightly to higher wavenumber. Comparison with the data for the corresponding bases (Figure 3b−d) shows that, qualitatively, these changes can be attributed solely to the adenine unit of NAD+, which displays the same pD dependence of its spectrum: reduction in the pD exchanges the modes at 1625 and 1482 cm−1 for those at 1666 and 1508 cm−1 in the adenine and adenosine spectra. Figure 4a displays the ratios of the intensities of the pairs of modes at 1666/1625 and 1508/1482 for NAD+ and adenosine as a function of pD and from this it can be seen quantitatively that these changes are correlated, demonstrating that exchange of one species for another occurs between pD values of ∼3−4. Furthermore, the changes observed in the NAD+ spectrum can be attributed directly to the adenine moiety. The choice of mode pairs shown in Figure 4 is somewhat arbitrarily based upon spectral region but due to the similarity of the responses it is clear that all spectral changes due to reduced pD are correlated. The results of density functional theory simulations discussed below however do show that these pairs of modes feature the same assignment in the protonated and unprotonated forms of the molecule. In contrast to the adenine/adenosine spectra, the NMN+ spectrum (Figure 3d) demonstrates only minor pD dependence via a shift in frequency of the 1666 cm−1 mode and a slight narrowing and loss of intensity of the 1589 cm−1 peak. The latter is consistent with previous studies,1 which showed a splitting of this peak under D/H exchange suggesting that two weak modes are in fact overlapped, one belonging to a ring vibration centered at 1589 cm−1 alongside a transition originating from the NH2 group; the narrowing of the peak with decreasing pD is attributed to a slight change in the position of the latter similar to that observed for the 1666 cm−1 mode, which is assignable to the CO stretching mode of the nicotinamide moiety (see DFT results below). Overall however, the magnitude of this NMN+ pD response is negligible compared to that of NAD+ as a whole allowing for unambiguous assignment of the NAD+ peaks that originate from either nicotinamide or adenine vibrations. Thus, bands at 1428 and 1455 cm−1 feature clear contributions from NMN+ while the 1633 cm−1 band that was not clearly resolved at neutral pD, becomes clear as the adenine peak at 1625 cm−1 diminishes in size at low pD. Although the intense adenine band no longer overlaps the nicotinamide 1633 cm−1 vibration under these conditions, the new peak attributable to protonated adenine at 1666 cm−1 is now coincident with the nicotinamide 16472



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Table 1. Vibrational Band Assignments for NAD+ and Its DFT Calculated Spectra, Including Observed and Predicted pD Dependences NAD+ (experiment, cm−1) pD 7

pD 2

1666 (br) 1633 (w)

1666 (br) 1633 (w)

1624 (st) 1577 1482 1457 1428

1666 (st) 1582 1508 1457 1428

NAD+ (DFT, cm−1) assignment Nic CO str Nic CONH2 NH2 bend Ad C−N/NH2 Ad NH2 + ring Ad ring Ad/Nic ring Ad/Nic ring

unprotonated

protonated

1655 1572

1642 1560

1588 1634 1557 1584 1444 1466 1393−1484 1393−1484

Figure 5. DFT predictions of infrared absorption spectra of (a) NAD+ and (b) NADH under protonated (adenine A1 position) and nonprotonated conditions. Arrows indicate peak changes upon protonation.

experimental data both in terms of the blueshift upon protonation and the fact that the high-frequency mode in the protonated state overlaps with a pD-independent peak near 1650 cm−1, assigned to the CO stretching mode of the nicotinamide moiety in the DFT result. The CO stretching mode of the amide group is often attributed to the most intense peak in the NAD+ spectrum but both the pD-dependent experimental data and simulations show that it is unexpectedly weak and obscured at low pD. It is noteworthy that DFT also predicts this mode to have a strong contribution from the NH2 group, which is consistent with the greater than expected dependence of this absorption upon H/D exchange that has been reported.1 The latter effect was reproduced by carrying out DFT simulations incorporating substitution of the nicotinamide amide protons by deuterons, which resulted in a subtle shift of the CO mode and maintained the good agreement between experiment and theory. By contrast, predictions following D/H exchange at the adenine NH2 moiety reduced the quality of agreement significantly, suggesting either that the contribution from this group to the C−N/NH2 mode is relatively insignificant or that exchange does not occur at this functional group to a significant degree. Finally, it can be seen from the DFT predictions that the loss of the experimental mode at 1482 cm−1 and appearance of the mode at 1508 cm−1 is also reproduced, though the frequency agreement of the calculations is less accurate at lower wavenumber. These peaks are both assigned to ring vibrations of the adenine system, which, as is to be expected, are affected by protonation at the A1 position. A complete assignment of the observed bands at both neutral and acidic pD is given in Table 1 based on the preceding text. NADH. Figure 6a shows the spectrum of NADH in direct comparison with that of NAD+ at neutral pD. NADH displays a greater number of bands in this region than the oxidized species with modes visible at 1688, 1624, 1575, 1555 (broad), 1482, 1455, and 1426 cm−1. In the case of the vibrations at 1624, 1575, 1482, 1455, and 1426 cm−1, these were previously assigned, at least in part, to the adenine moiety (Table 1) and these contributions persist as would be expected due to the structural similarities of the two forms of NAD (Figure 1). The peak at 1455 cm−1, attributed to both NMN+ and adenine also

Figure 6. (a) Comparison of FTIR spectra of NADH (black) and NAD+ (red) at neutral pD. (b) pD dependence of the FTIR spectrum of NADH. Arrows show changes in band positions and intensities as pD is reduced. Color scale runs from blue (neutral pD) to red (acidic pD). N indicates peaks lost due to disruption of the dihydronicotinamide moiety at pD < 5 (see text).

remains, suggesting that it is not wholly affected by reduction as would be expected. In contrast, the broad 1666 cm−1 mode that was assigned to the nicotinamide CO vibration in NAD+ at neutral pD can no longer be found at the same position while a new, narrower absorption band is located at 1688 cm−1 alongside an additional broad peak at 1555 cm−1. NADH and NAD+ differ structurally via the addition of a hydride to the H4 position (Figure 1) of the nicotinamide group and loss of the cyclic conjugation of the pyridine ring. It is therefore reasonable to hypothesize that the new absorptions at 1688 and 1555 cm−1 arise from this change in structure. This 16473



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Table 2. Vibrational Band Assignments for NADH and Its DFT Calculated Spectra, Including Observed and Predicted pD Dependences NADH (experiment, cm−1)

NADH (DFT, cm−1)

pD 7

pD 2

assignment

unprotonated

protonated

1688 1624 (br) 1624 (st) 1575 1555 (br) 1482 1455 1426

1688 1624 (br) 1666 (st) 1580 1555 (br) 1509 1455 1426

Nic dihydr ring sym(CC) Nic CO str Ad C−N/NH2 Ad NH2 + ring Nic dihydr ring antisym (CC) Ad ring Ad/Nic ring Ad/Nic ring

1674 1600 1589 1556 1558 1443

1674 1601 1645 1585 1558 1476 1401−1435 1401−1435

Figure 7. (a) The 2D-IR spectrum of NAD+ (pD 2.5) obtained with a waiting time (Tw) of 0.5 ps. (b) The 2D-IR spectrum of NADH (pD 7) obtained with a waiting time (Tw) of 0.5 ps. Vertical and horizontal dashed lines show positions of FTIR peaks for each sample, intersections show possible off-diagonal peak positions. The first contour line represents ±10% of the maximum positive and negative signal.

primary acid product, a tetrahydropyridine species, leading to removal of the unsaturated CC bond between positions 5 and 6 (H5 and H6 in Figure 1) of the 6-membered ring was characterized by 1H NMR signals at 7.24, 2.20, 2.05, 1.33, and 1.26 ppm.51 NMR spectra of the NADH samples for which IR spectra are displayed in Figure 6 were consistent with this reaction taking place (see Supporting Information). This is entirely consistent with loss of the 1688 and 1555 cm−1 modes at mildly acidic pD values if they are attributable to the nicotinamide ring structure. It is important to note that this process does not involve the adenine structure and hence, modes assigned to this base are unaffected until protonation of this moiety occurs at pD 3−4. Taken together, these data confirm an assignment of these new modes to the 1,4 dihydronicotinamide ring moiety of NADH and it is thus possible to separate the effects due to hydration of the nicotinamide ring50,51 from those due to protonation of adenine.3,10 Finally, the peak at 1428 cm−1 was assigned to a mixture of nicotinamide and adenine contributions, thus the reduction in intensity due to changes in the nicotinamide moiety are consistent with this assignment.

can be further supported via examination of FTIR spectra of NADH as a function of pD, which are shown in Figure 6b. NADH is less stable at low pD than NAD+ and some degradation of the sample over time was noted from a pD of ∼4. Despite this, it can clearly be seen from Figure 6b that the adenine-assigned vibrations of NADH demonstrate an identical pD dependence to those reported for NAD+ above. Specifically, protonation leads to a new peak arising at 1666 cm−1 at the expense of the 1624 cm−1 mode while a peak at 1509 cm−1 replaces that at 1482 cm−1. Once again these changes were noted between pD values of 3−4. In addition to these changes, the peaks observed at 1688, 1555, and 1428 cm−1 diminished in intensity at pD values of 5 and below (marked with “N” in Figure 6b). In the case of the modes at 1688 and 1555 cm−1, the fact that these modes are NADH-specific implies that their origin lies in the reduced nicotinamide ring structure present in NADH. This is also consistent with the fact that these changes occur at a higher pD than the changes ascribable to the adenine unit. It has been reported that instability of the nicotinamide group in NADH occurs under acidic conditions as the pD value approaches 4.49−52 In particular, formation of the NADH 16474



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positive and shifted slightly to lower probe frequencies. This peak is assignable to the v = 1−2 transition with the shift arising from the anharmonicity of the mode in question. These features can be seen in the spectra of NAD+ and NADH in Figure 7 by following the vertical or horizontal dashed lines, which mark FTIR peak positions on each axis, to the intersection with the diagonal of the 2D IR spectrum. In addition to these diagonal features, the off-diagonal region of the 2D-IR plot shows peaks linking pairs of diagonal features arising from modes that are vibrationally coupled to one another. These are analogous to the off-diagonal peaks observed in 2D-NMR spectra, which link coupled nuclear resonances. These peaks can be observed by following the horizontal and vertical dashed lines in the plots. It is noted that not all intersections of these guide lines represent a peak, rather, the occurrence of a peak at these positions indicates intermode coupling. Figure 7a shows the 2D-IR spectrum of NAD+ under acidic conditions. It is clear from this spectrum that a large number of the modes exhibit coupling, as identified by spectral features lying away from the diagonal line. In particular, the intense mode at (pump, probe) = (1666 cm−1, 1666 cm−1) exhibits coupling to each of the other diagonal modes at 1633, 1577, 1508, and 1428 cm−1. Of these latter peaks, two are exclusively adenine vibrations (1508 and 1577 cm−1), one is assignable to both adenine and nicotinamide (1428 cm−1) and the peak at 1633 cm−1 is assignable to nicotinamide. In this respect, the coupling pattern seems to be inconsistent with expectations from the IR band assignment. However, at this pD the 1666 cm−1 mode of NAD+ itself contains contributions from both the CO stretching mode of the nicotinamide unit and from the C−N/NH2 mode of adenine, hence, excitation of these two modes can only occur simultaneously with the associated coupling patterns for both being observed. Similarly, this coincidence in the position of the major adenine and nicotinamide modes prevents potential observations of coupling effects arising from a folded structure. Couplings between the weaker, adenine-derived modes are also present in the spectrum and are consistent with the FTIR band assignments, though these are not all clearly shown in Figure 7a due to the large scale of the 1666 cm−1 signal. A similar situation arises in the NADH spectrum (Figure 7b) where the intense 1624 cm−1 mode couples to each of the other diagonal resonances. Once again, this mode has been shown to be a convolution of the CO stretching mode of the nicotinamide unit and from the C−N/NH2 mode of adenine and so this is expected. It is noticeable that the modes located at 1555 and 1575 cm−1, although of similar intensity, do not show coupling to each other. This is consistent with the assignments of them to different bases; 1555 cm−1 is a dihydronicotinamide ring mode while 1575 cm−1 is assigned to the adenine base system. Both peaks show coupling to the intense, convoluted mode at 1625 cm−1 as would be expected. There may also be expected to be an off-diagonal peak linking the 1688 and 1555 cm−1 peaks as these both originate on the nicotinamide base but the low intensity of the 1555 cm−1 mode and the large frequency separation will contribute to weak coupling and so small off-diagonal signals for this pair. As a result, the 1688 cm−1 mode of NADH couples only to the C O stretching mode of the nicotinamide base at 1625 cm−1, again consistent with the above assignments. Taking the data sets together Figure 7 suggests that the complex spectroscopy of NAD in either form will reduce its

In previous IR studies of NADH, it has been proposed that the 1688 cm−1 mode of NADH is the equivalent of the 1666 cm−1 band observed in NAD+ at neutral pD, which was itself assigned above to the carbonyl stretching mode of the nicotinamide moiety.1 However, it was also evident that the 1688 cm−1 mode of NADH differed from the NAD+ 1666 cm−1 mode in both line width and sensitivity to H-D exchange,1 implying a different chemical origin. The correlation of loss of the 1688 cm−1 peak to dihydronicotinamide ring-related effects at reduced pD above further shows that this is not the CO stretching mode as proposed; while a frequency shift may have been expected on disruption of the dihydronicotinamide ring moiety, loss of the CO peak would not occur. Assignment of the 1688 cm−1 mode to a nicotinamide ring vibration of NADH would also explain the narrower width of the 1688 cm−1 peak in comparison to the 1666 cm−1 mode of NAD+. The latter would be expected to be inhomogeneously broadened by hydrogen bonding to the solvent in contrast to the ring vibrations. It is proposed that the carbonyl stretching mode of NADH at neutral pD in fact lies underneath the C−N/NH2 vibration of uprotonated adenine located at 1624 cm−1, justifying the substantially more intense 1624 cm−1 band in NADH when compared to that of NAD+. However, the slight asymmetry of the NADH 1624 cm−1 peak indicates that the two modes do not have identical central frequencies. At pD 2.5, when the adenine peak is fully shifted to its protonated position of 1666 cm−1, a residual peak consistent with the carbonyl stretching mode remains, however the effects of dihydronicotinamide ring breakdown and the IR contributions from breakdown products mean that caution must be exercised in making this assignment at such low pD values. Density Functional Theory Calculations. DFT calculations were performed on NADH (Figure 5b) and its A1protonated analogue. It can be seen that the agreement with experiment, particularly near 1600 cm−1 is good and, once again, the changes observed upon adenine protonation reflect accurately those observed experimentally. These are marked with arrows in the figure, consistent with those used for NAD+ above. It is also noteworthy that the DFT-predicted spectra in Figure 5b provide support of an overlap of the C−N/NH2 and CO stretching bands at neutral pD as suggested above. The only changes not mimicked by DFT calculations are those attributed to disruption of the nicotinamide ring. These changes were not reflected in the DFT calculations due to the complexity arising from the range of intermediate products involved. Importantly however, the DFT simulations do support the assignment of the modes at 1688 and 1555 cm−1 to the symmetric and antisymmetric stretches of the two double bonds in the reduced nicotinamide ring, consistent with the discussion above. A comprehensive list of modes and assignments is given in Table 2. Two-Dimensional IR Spectroscopy. Example 2D-IR spectra of both NAD+ and NADH are shown in Figure 7 for a waiting time (pump−probe delay time) of 0.5 ps alongside FTIR spectra for comparison. In these spectra, the plot shows a correlation of excitation (pump) and detection (probe) frequency such that the one-dimensional IR absorption response (similar to that recovered from FTIR) is positioned on the diagonal of the spectrum. Each mode leads to a pair of peaks near the diagonal of the 2D-IR spectrum. One of these is a negative peak corresponding to the bleach of the v = 0−1 transition that is located on the spectrum diagonal (with equal pump and probe frequencies), while the second component is 16475



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regarding the location of the CO stretching modes and peaks due to the dihydronicotinamide ring moiety of NADH have been addressed. No indications of a folded structure are observed in the IR spectrum, consistent with a loose, dynamic stacking arrangement that does not significantly affect the bonding within the rings of the two bases. The 2D-IR spectra of both forms of NAD have been recorded and their usefulness as potential biological probes assessed. In light of the complex spectroscopy and considerable overlap of modes due to nicotinamide and adenine bases, it has been concluded that such applications, while not impossible, must proceed with caution if location specific data is required.

usefulness as a probe species for 2D-IR spectroscopy. In order to provide an effective intrinsic probe of a biomolecule environment, it is desirable that the probe should exhibit IR signatures that are well-separated from the protein backbone absorption (typically characterized by the amide I′ band near 1650 cm−1) and sufficiently intense to be detectable at low concentrations. It is therefore unfortunate that the most intense absorptions from each of the bases, the CO and C−N/NH2 modes of nicotinamide and adenine, respectively, are convoluted under most protonation conditions, which prevents a clear signal being obtained from a single base. In the case of both NADH and NAD+ (especially at higher pD values), other candidate modes exist, specifically the 1666 cm−1 mode of NAD+ and the 1688 cm−1 mode of NADH. It is also desirable however that the modes in question have a clear relationship to the molecular structure of the probe in order that site-specific information is obtained rather than a response that is delocalized or originating from more than one functional group on the probe molecule. As previously discussed, this is not the case for the 1666 cm−1 mode seen in the spectrum of protonated NAD+. Increasing the pD value enables the nicotinamide mode to be selected in preference to the adenine, though the modes are not fully separable and this occurs at the expense of considerable absorption intensity, which would limit the usefulness of the mode as a probe. The NADH mode at 1688 cm−1 has the twin advantages of being well-separated from the amide I region and moderately intense and represents perhaps the best candidate mode in this respect. It is a single mode with no overlapping resonances, leading to the straightforward off-diagonal peak pattern associated with it. Conversely, this mode is attributable to a dihydro ring vibration and has been shown to be very insensitive to the effects of environment1 and so is likely to be of limited value in terms of relating dynamic information from the biomolecular environment. Finally, pump−probe data suggests that the lifetime of all of the modes observed in this region is on the order of 1−2 ps (see Supporting Information), which also acts to limit their usefulness for dynamics significantly longer than H-bond fluctuations. With the benefit of the assignment of the vibrational modes for NAD+ in Table 1, it is clear that alternative vibrational modes are few in number; several are restricted to the adenine base but are generally weak and attributable to ring vibrations, which are relatively insensitive to their chemical environment or H-bonding. For these reasons, straightforward 2D-IR spectroscopy studies employing unmodified NAD+ or NADH are rather impractical as the dynamics that occur within the waiting time could not be confidently assigned to effects relating to the nicotinamide or adenine modes alone. One approach to circumventing this is to employ chemical functionalization of the NAD molecule using IR chromophores such as azido groups providing that any effects due to the chemical insertion of such a chromophore are not deleterious to the biological system of interest.53

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ASSOCIATED CONTENT

S Supporting Information *

Supporting Information associated with this manuscript is available as outlined in the main text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Funding from The Leverhulme Trust (RPG248) for this work is gratefully acknowledged. REFERENCES

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CONCLUSIONS A combination of experimental FTIR, NMR, and DFT calculations was used to assign the vibrational bands of NAD in both oxidized and reduced forms at a range of pD values. The use of structural analogs and the effect of protonation on the spectrum allowed a clear assignment of the contributions to the modes to their component bases and discrepancies 16476



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