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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...

Anion exchange at positively charged interfaces plays an important role in a variety of physical and chemical processes. However, the molecular scale details of these processes, especially with heavy and large anionic complexes, are not well-understood. We studied the adsorption of PtCl6 2anionic complexes to floating DPTAP monolayers in the presence of excess Clas a function of the bulk chlorometalate concentration. In situ x-ray scattering and fluorescence measurements, which are element and depth sensitive, show that the chlorometalate ions only adsorb in the diffuse layer at lower concentrations, while they adsorb predominantly in the Stern layer at higher concentrations. The response of DPTAP molecules to the adsorbed ions is determined independently by grazing incidence x-ray diffraction, and supports this picture. Molecular dynamics simulations further elucidate the nanoscale structure of the interfacial complexes. The results suggest that ion hydration and ion-ion correlations play a key role in the competitive adsorption process. TOC GRAPHICSKEYWORDS Air/water interface, anion recognition, Hofmeister effects, specific ion effects, solvent extraction.

Ion hydration and interfacial water play crucial roles in numerous phenomena ranging from biological to industrial systems. Although biologically relevant (and mostly smaller) ions have been studied extensively in this context, very little experimental data exist about molecular scale behavior of heavy ions and their complexes at interfaces, especially under technologically significant conditions. It has recently been shown that PtCl6 2complexes adsorb at positively charged interfaces in a two-step process that cannot fit into well-known empirical trends, such as Hofmeister series. Here, a combined vibrational sum frequency generation and molecular dynamics study reveals that a unique interfacial water structure is connected to this peculiar adsorption behavior. A novel sub-ensemble analysis of MD simulation results show that after adsorption, PtCl6 2complexes partially retain their first and second hydration spheres, and it is possible to identify three different types of water molecules around them based on their orientational structures and hydrogen bonding strengths. These results have important implications for relating interfacial water structure and hydration enthalpy to the general understanding of specific ion effects. This in turn influences interpretation of heavy metal ion distribution across and reactivity within, liquid interfaces.

Recent technological advances have led to major changes in the apparatuses used to collect 2D IR spectra. Pulse shaping offers several advantages including rapid data collection, inherent phase stability, and phase cycling capabilities. Visible array detection via upconversion allows the use of visible detectors that are cheaper, faster, more sensitive, and less noisy than IR detectors. However, despite these advantages, many researchers are reluctant to implement these technologies. Here we present a quantitative study of the S/N of 2D IR spectra collected with a traditional four-wave mixing (FWM) apparatus, with a pulse shaping apparatus, and with visible detection via upconversion to address the question of whether or not weak chromophores at low concentrations are still accessible with such an apparatus. We find that the enhanced averaging capability of the pulse shaping apparatus enables the detection of small signals that would be challenging to measure even with the traditional FWM apparatus, and we demonstrate this ability on a sample of cyanylated dihydrofolate reductase (DHFR).