Protein dynamics in cytochrome P450 molecular recognition and substrate specificity using 2D IR vibrational echo spectroscopy

Abstract

Cytochrome (cyt) P450s hydroxylate a variety of substrates that can differ widely in their chemical structure. The importance of these enzymes in drug metabolism and other biological processes has motivated the study of the factors that enable their activity on diverse classes of molecules. Protein dynamics have been implicated in cyt P450 substrate specificity. Here, 2D IR vibrational echo spectroscopy is employed to measure the dynamics of cyt P450(cam) from Pseudomonas putida on fast time scales using CO bound at the active site as a vibrational probe. The substrate-free enzyme and the enzyme bound to both its natural substrate, camphor, and a series of related substrates are investigated to explicate the role of dynamics in molecular recognition in cyt P450(cam) and to delineate how the motions may contribute to hydroxylation specificity. In substrate-free cyt P450(cam), three conformational states are populated, and the structural fluctuations within a conformational state are relatively slow. Substrate binding selectively stabilizes one conformational state, and the dynamics become faster. Correlations in the observed dynamics with the specificity of hydroxylation of the substrates, the binding affinity, and the substrates' molecular volume suggest that motions on the hundreds of picosecond time scale contribute to the variation in activity of cyt P450(cam) toward different substrates.

Figure 1

Structures of cyt P450_cam and substrates. A) Structure of the cyt P450_cam-CO-camphor complex in the immediate vicinity of the CO ligand (PDB entry 1T87). Shown are the heme, the CO distal ligand, the Cys357 proximal ligand, the bound camphor substrate, and several residues of helix I, which forms one wall of the active site. B) Structures of substrates whose complexes with cyt P450_cam were investigated with 2D IR vibrational echo spectroscopy.

Figure 2

Linear FT IR spectra (solid lines), along with the Gaussian fits (dashed lines) to the spectra, of A) substrate-free cyt P450_cam-CO and B) its complex with camphor.

Figure 3

2D IR spectra of substrate-free cyt P450_cam-CO (left), the complex with camphor (middle), and the complex with norcamphor (right), at short (upper) and long (lower) T_w delay times. The spectral region around the 0–1 transition is shown, with the contour lines representing a 5 % change in amplitude. To illustrate the differences in lineshapes, lines are drawn over five contours along the diagonal and antidiagonal in the 2D IR spectra with T_w of 24 ps.

Figure 4

CLS decay curves and corresponding exponential fits for A) cyt P450_cam-CO bound with its natural substrate camphor. The two timescales seen in the decays are evident. B) the 1939 cm⁻¹ (red), 1952 cm⁻¹ (green), and 1963 cm⁻¹ (blue) bands of substrate-free cyt P450_cam-CO. The fits have been extended for two of the bands as an aid to the eye for comparing to the other curves. C) The different substrate complexes: camphor (black), camphane (blue), adamantane (green), norcamphor (purple), and norbornane (red).