The enzyme dihydrofolate reductase (DHFR), which catalyzes hydride transfer from the cofactor nicotinamide adenine dinucleotide phosphate (NADPH) to 7, 8-dihydrofolate to produce tetrahydrofolate, has emerged as a paradigm for the study of enzyme catalysis.[1–3] It has been suggested that electrostatic complementarity between the enzyme and the transition state for hydride transfer contributes significantly to catalysis,[4–7] and computational studies have identified a number of residues that may mediate these interactions.[5, 7] One of the most important is Tyr100, which directly contacts the nicotinamide hydride donor (Figure 1) and is thought to stabilize the developing positive charge on the cofactor in the hydride-transfer transition state. However, protein dynamics have also been suggested to contribute to DHFR catalysis through the population of rare but reactive substrate conformations.[8–12]

Vibrational spectroscopy provides a direct and bondspecific approach to the characterization of the microenvironments and motions of molecules, but with proteins its application is limited by congestion in the spectra. Previous approaches to observe individual vibrations, such as those associated with the amide backbone, sulfhydryl or carboxyl side chains, or bound water molecules, have used heavy atom isotope labeling and difference Fourier transform infrared (FTIR) spectroscopy.[13, 14] In some cases, changes in the difference spectra have even been time-resolved.[14, 15] However, the linewidths and frequencies of the absorptions are often difficult to deconvolute, as they remain in a congested region of the spectrum, and they are even more difficult to interpret in terms of specific protein motions, because of coupling with other vibrations. As part of a program to develop general probes of protein microenvironments and dynamics we have developed the use of carbon–deuterium (CÀD) bonds as FTIR probes.[16–24] CÀD bonds are sensitive to their environment and may be incorporated anywhere throughout a protein. While they are weaker than the other endogenous chromophores, their detection and analysis are facilitated by their unique absorption in an otherwise transparent region (ca. 2100 cmÀ1) of the protein IR spectrum. In principle, the CÀD-based FTIR technique may be applied to a protein of any size. However, the available methods to site-selectively deuterate a protein are limited to synthesis or semisynthesis unless the amino acid of interest is present at only a single position. These limitations preclude the general application of the technique to many proteins, including DHFR, unless specific residues are made unique by site-directed mutagenesis. This latter approach has been applied to DHFR in a previous study, wherein all but one methionine residue was mutagenized to leucine to allow for site-specific labeling.[23] To examine a residue such as Tyr100 in DHFR without the introduction of potentially perturbative mutations we have used a biosynthetic method to siteselectively incorporate a photocaged, deuterated amino acid, which after photolysis yields the site-selectively deuterated, but otherwise natural, protein. In previous studies, o-nitrobenzyl-O-tyrosine (ONBY), a tyrosine derivative protected with a photolabile o-nitrobenzyl group, was genetically encoded in E. coli by using an orthogonal tRNATyr