Environment- and Conformation-Induced Frequency Shifts of C–D Vibrational Stark Probes in NAD(P)H Cofactors

NAD(P)H cofactors are found in all forms of life and are essential for electron and hydrogen atom transfer. The linear response of a carbon-deuterium (C-D) vibration based on the vibrational Stark effect can facilitate measurements of electric fields for critical biological reactions including cofactor-mediated hydride transfer. We find both inter- and intramolecular electric fields influence the C-D frequency in NAD(P)H and nicotinamide-like models where the reactive C4-hydrogen has been deuterated. Hence, the C-D frequency can report both environmental electrostatics and conformational changes of the nicotinamide ring. Conformation-dependent effects are mediated through space as electrostatic effects, rather than through-bond. A Stark tuning rate of ∼0.57 cm^-1/(MV/cm) was determined using both experimental and computational approaches, including vibrational solvatochromism, molecular dynamics simulations, and <i>in silico</i> Stark calculations. The vibrational probe's Stark tuning rate is shown to be robust and suitable for measuring fields along hydride transfer reaction coordinates in enzymes.