Detecting Vibrational Energy Transfer into an Enzyme Active Site via a Transition State Analog

Vibrational energy transfer (VET) in enzymes has been discussed in the context of enzyme catalysis. A key point for experimentally investigating VET is to position suitable vibrational energy sensors in the delicate active sites of enzymes without disturbing their native state. Ideally, native substrates and/or inhibitors can be repurposed as vibrational energy sensors. Here, we demonstrate that the azide anion (N₃^-), bound as an inhibitor to the active site of formate dehydrogenase (FDH), sensitively detects the vibrational energy, even though it is not covalently bound. Vibrational energy has been site-specifically injected into the enzyme via exciting an azulenylalanine (AzAla) energy donor, which has been introduced by genetic code expansion in a 19 Å distance. This proof-of-principle experiment opens the field toward the investigation of VET on a picosecond time scale in a range of enzymes by exploiting their natural affinity toward the infrared-absorbing small ligand azide.