Carbon-deuterium bonds as reporters of electric fields in solvent and protein environments

Use of vibrational probes to measure electric fields inside proteins has illuminated the role of electrostatics in biological applications ranging from binding interactions to enzyme catalysis. While prior work focused on strongly absorbing carbonyl and nitrile probes, more recent work has begun exploring weakly absorbing carbon-hydrogen bonds, beginning with measurements of electric fields inside oxidoreductases using the vibrational Stark effect (VSE). Originally, an alcohol dehydrogenase inhibitor containing a deuterated aldehyde was used to measure the field in two directions—on carbonyl and carbon-deuterium (C-D) bonds—in the enzyme active site. Now, several more distinct cases demonstrate how C-D probes map electric fields to vibrational frequency shifts, with new implications for our understanding of the VSE. To probe the field along a different dimension in oxidoreductases, we consider singly deuterated NAD(P)H which reports fields along the hydride transfer axis and whose frequency detection is facilitated by advancements in quantum cascade laser (QCL) technology for infrared spectroscopy. Unusually, the solvatochromic shift for this C-D probe appears opposite to that observed for carbonyls and nitriles; that is, the probe blueshifts in more polar solvents. In another example, the chromophore of green fluorescent protein (GFP) with a deuterated bridge carbon demonstrates similarly unusual frequency shifts in solvent and protein environments, with stronger fields parallel to the C-D dipole causing blueshifts. Observed Stark tuning rates for these molecules are recapitulated by in silico Stark calculations at the B3LYP or ωB97X-D/6-31+G(d) level. Stark tuning rate comparison with other C-D containing molecules including deuterated aldehydes and chloroform-d indicates a large sensitivity of the Stark tuning rate to molecular structure. We demonstrate how properly calibrated C-D probes can be effective reporters of electric fields in applications such as enzyme-catalyzed hydride transfer, biological photophysics and beyond.