LAUSR

Structural heterogeneity and the dynamics of the complexes of enzymes with substrates can determine the selectivity of catalysis; however, fully characterizing how remains challenging as heterogeneity and dynamics can vary at the spatial level of an amino acid residue and involve rapid timescales. We demonstrate the nascent approach of site-specific two-dimensional infrared (IR) spectroscopy to investigate the archetypical cytochrome P450, P450cam, to better delineate the mechanism of the lower regioselectivity of hydroxylation of the substrate norcamphor in comparison to the native substrate camphor. Specific locations are targeted throughout the enzyme by selectively introducing cyano groups that have frequencies in a spectrally isolated region of the protein IR spectrum as local vibrational probes. Linear and two-dimensional IR spectroscopy were applied to measure the heterogeneity and dynamics at each probe and investigate how they differentiate camphor and norcamphor recognition. The IR data indicate that the norcamphor complex does not fully induce a large-scale conformational change to a closed state of the enzyme adopted in the camphor complex. Additionally, a probe directed at the bound substrate experiences rapidly interconverting states in the norcamphor complex that explain the hydroxylation product distribution. Altogether, the study reveals large- and small-scale structural heterogeneity and dynamics that could contribute to selectivity of a cytochrome P450 and illustrates the approach of site-selective IR spectroscopy to elucidate protein dynamics.