The potential for femtosecond to picosecond time-scale motions to influence the rate of the intrinsic chemical step in enzymecatalyzed reactions is a source of significant controversy. Among the central challenges in resolving this controversy is the difficulty of experimentally characterizing thermally activated motions at this time scale in functionally relevant enzyme complexes. We report a series of measurements to address this problem using twodimensional infrared spectroscopy to characterize the time scales of active-site motions in complexes of formate dehydrogenase with the transition-state-analog inhibitor azide (N − 3 ). We observe that the frequency-frequency time correlation functions (FFCF) for the ternary complexes with NAD þ and NADH decay completely with slow time constants of 3.2 ps and 4.6 ps, respectively. This result suggests that in the vicinity of the transition state, the active-site enzyme structure samples a narrow and relatively rigid conformational distribution indicating that the transition-state structure is well organized for the reaction. In contrast, for the binary complex, we observe a significant static contribution to the FFCF similar to what is seen in other enzymes, indicating the presence of the slow motions that occur on time scales longer than our measurement window. 2D IR spectroscopy | enzyme dynamicsT he functional role of protein motions at the femtosecond to picosecond time scale is a hotly debated topic in enzymology. Although such a role could be general in nature, many experimental (1-10) and theoretical (11-23) studies of enzymecatalyzed hydrogen transfers have invoked protein motions at this time scale to explain anomalous kinetic isotope effects (KIE) and their temperature dependence. These studies result in the development of theoretical models, often referred to as Marcus-like models, in which the environmental reorganization that precedes the hydrogen-tunneling event has evolved to optimize the conformation of the transition state for tunneling (1,7,24). Fig. 1 illustrates the physical picture underlying such models. Heavy atom motions along the reorganization coordinate carry the system to a point where the donor and acceptor wells in the double-well hydrogen atom potential are degenerate and tunneling can proceed. At this position, the donor-acceptor distance and its fluctuations determine the tunneling probability. Mathematically, the rate constant for hydrogen transfer in these models is given by expressions of the form where C is the fraction of reactive complexes, the first exponential, in analogy with the Marcus theory for electron transfer, reflects the reorganization of the heavy atoms that modulates the relative energies of the reactants and the products to minimize the energy defect between the zero-point levels of the donor and acceptor wells. ΔG°is the driving force for the reaction, and λ is the reorganization energy. The second exponential gives the overlap between the donor and acceptor wave functions as a function of the donor-acceptor distan...

The lack of general spectroscopic probes that can be used in a range of systems to probe kinetics and dynamics is a major obstacle to the widespread application of two-dimensional infrared (2D IR) spectroscopy. We have studied 3-azidopyridine to characterize its potential as a probe of the protonation state of the pyridine ring. We find that the azido-stretching vibration is split by accidental Fermi resonance interactions with one or more overtones and combination states. Using 2D IR spectroscopy, we determine the state structure of the resulting eigenstates for complexes of 3-azidopyridine with formic acid and trifluoroacetic acid in which the pyridine ring is unprotonated and protonated, respectively. Based on the measurements, we develop a two-oscillator depurturbation model to determine the energies and couplings of the zeroth-order azido-stretching state and the perturbing dark state that couples to it. Based on these results, we conclude that the azido-stretching vibration is, in fact, sensitive to the protonation state of the pyridine shifting up in frequency by 8 cm(-1) in the complex with trifluoroacetic acid relative to the formic acid complex. These results suggest that, although 3-azidopyridine is not suitable as a spectroscopic probe, the approach of employing an organic azide as a remote probe of protonation state holds significant promise.

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We report transient grating and 2D IR spectra of the C-D stretching vibration of deuterated formic acid dimer. The C-D stretching transition is perturbed by an accidental Fermi resonance interaction that gives rise to a second transition. The transient grating results show that the population lifetime of these states, which are in rapid equilibrium, is 11 ps. 2D IR spectroscopy reveals the energies of the eigenstates in the regions of one quantum and two quanta of C-D stretching excitation. Using these eigenstate energies, we construct a simplified model for the zeroth-order states that we then use to simulate the 2D IR spectrum. The results of this simulation suggest that the model captures the essential features of the vibrational spectroscopy in the region of the C-D stretching transition and compares well with previous gas-phase spectroscopy of the C-D stretch of deuterated formic acid dimer.

We present infrared transient grating measurements of complexes of formic acid with pyridine and pyrazine at four excitation frequencies within the broad proton-stretching band. These experiments investigate the mechanism of the line broadening of the O-H stretching vibration. The transients show coherent oscillations that decay within a few hundred femtoseconds and population relaxation on two time scales. We fit the data using a simple model of three coupled oscillators that relax via sequential kinetics through an intermediate state. Based on this model, we conclude that the coherent oscillations result from superpositions of Fermi-resonance-coupled states involving formic acid overtone and combination states.