Studies of enzyme dynamics are complicated by the high spatial and temporal heterogeneity of proteins. Infrared (IR) spectroscopy using vibrational probes, which provide an environment-sensitive absorbance distinct in frequency from intrinsic protein bond vibrations, has emerged as a method to address these challenges. In this application, a heme-bound nitrile probe has been used to characterize conformational changes in cytochrome P450cam (P450cam) upon binding of its redox partner, Putidaredoxin (Pdx). The mechanism of P450cam has received decades of attention, however, the effector role of Pdx is not yet fully understood. Pdx has recently been proposed to induce a conformational change in P450cam which promotes active site hydrogen bonding resulting in a proton relay. Using the nitrile probe, along with mutagenesis of Thr252 and solvent exchange, we have elucidated the effect of hydrogen bonding on the nitrile absorbance. This characterization was then used to interrogate the P450cam-Pdx complex in the solution state. Binding of Pdx was demonstrated to induce partial population of a state where the ligand participates in hydrogen bonding, aiding our understanding of its effector role. Mutation of Thr252 to alanine provides further insight into formation and control of the proton relay. This study additionally illustrates the utility of nitrile bonds as vibrational probes of local hydrogen bonding.

Effector-induced active site hydrogen bonding in cytochrome P450cam characterized by infrared spectroscopy

ACS Spring Meeting 2021

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