Specific interactions between substrates and the protein binding pocket determine the molecular mechanisms of enzymatic reactions, but exactly which interactions are most important and what role the fluctuations of the local environment play in carrying reactants to products remain unclear. Proton-transfer reactions are important in the catalytic mechanisms of many enzymes, and interactions with the local environment are crucial in controlling those reactions. I study proton-transfer reactions of model compounds and proteins using time-resolved vibrational spectroscopy, including both novel two-dimensional infrared experiments and more conventional infrared pump-probe measurements, to probe the intermolecular interactions and how those interactions control the chemistry of enzyme catalysis.

Hydrogen-bonded complexes of substituted phenols with various bases such as tertiary amines or amides in nonpolar solvents exist in equilibrium with their proton-transferred tautomers. These complexes are similar in composition to the donor-acceptor complexes involved in the catalytic mechanism of many enzymes and have interesting infrared absorption spectra that provide a convenient spectroscopic window into their chemical structure and dynamics. Studies of the infrared spectra of these complexes as a function of acid, base, and solvent suggest that the complexes are highly polarizable and that solvent fluctuations shift the equilibrium between hydrogen-bonded and proton transferred states. I use a variety of experimental approaches to explore the structures and proton-transfer dynamics of various hydrogen bonded complexes in solution including femtosecond infrared pump-probe spectroscopy, infrared three-pulse photon-echo peak-shift measurements, and two-dimensional infrared spectroscopy.

The protein environment of enzyme active sites is important in facilitating enzymatic reactions. The protein binds the substrate holding it in a particular geometry so that nearby functional groups are oriented to stabilize the transition state. These functional groups are involved in the reaction mechanism acting as hydrogen-bond partners, providing an electrostatic environment favorable for the reaction, and taking part as acidic or basic groups in proton-transfer reactions. The reaction kinetics are controlled by these local interactions with the protein. This static picture of the protein/substrate interactions, however, is an incomplete description of the catalytic process because fluctuations of these protein functional groups can also be important in the reaction mechanism. In enzymatic proton-transfer reactions, for example, fluctuations of the protein can cause a time-dependent variation in the donor-acceptor separation resulting in large changes in the proton-transfer barrier height.

To explore the role of the protein environment in controlling chemical reaction dynamics I also study proton-transfer reactions in proteins, like the excited-state proton-transfer reaction in green fluorescent protein (GFP), where the structured interior of the protein constrains the fluctuations of the chromophore and specific interactions with the protein determine the course and outcome of the chemical reaction. These systems exhibit much of the richness and complexity of enzyme reactions, and they are easy to photoinitiate making them an attractive starting point for studies of chemical reaction dynamics in proteins.

Structural studies of enzyme-substrate or substrate analog complexes under various conditions provide pictures of enzyme active sites with molecular detail. Although structural measurements are tremendously valuable in understanding the chemistry of enzymes, the static picture that they provide can be misleading. Enzyme active sites are dynamic environments, and the fluctuations modulate the specific interactions at the active site and can mediate the chemistry.

To study the fluctuations of the enzyme active site and the specific interactions that control the reaction dynamics, I use isotopically labelled NAD, a cofactor that is used by a variety of enzymes. Replacing selected CH hydrogens with deuterium shifts the vibrational frequency of those bonds into a convenient window in the infrared absorption spectrum of water. Nonlinear vibrational spectroscopy can quantify the electrostatic interactions between the CD oscillators and neighboring groups within the enzyme active site and identify changes in the vibrational spectroscopy induced by complexation with the enzyme or with the enzyme and substrate-analog that reflect chemical changes in the CD bonds. In addition, two-dimensional infrared lineshapes probe the fluctuations of the local environment that are important in mediating soem enzymatic reactions. These measurements also probe the equilibrium barrier crossing events of transition-state analogs that are representative of the actual chemical changes that take place in the functioning enzyme.