Deuterium magic angle spinning studies of substrates bound to cytochrome P450.
We report solid-state deuterium magic angle spinning NMR spectra of perdeuterated adamantane bound to the active site of microcrystalline cytochrome P450cam (CP450cam) in its resting state. CP450cam contains a high-spin ferric (Fe3+) heme in the resting state; the isotropic shift was displaced from the diamagnetic value and varied with temperature consistent with Curie-law dependence. A nondeuterated competitive tighter binding ligand, camphor, was used to displace the adamantane-bound species. This addition resulted in the disappearance of the hyperfine-shifted signal associated with a perdeuterated adamantane bound to CP450cam, while signals presumably associated with adamantane bound to other cavities persisted. We simulated the deuterium spinning side-band intensities for the enzyme-bound species using dipolar hyperfine coupling as the only anisotropic interaction; the deuterium quadrupolar interaction was apparently averaged due to a fast high-symmetry motion. These data provide direct support for previous proposals that substrates are conformationally mobile on the time scale of enzymatic turnover. The simulations suggested that the adamantane binds with an average metal-deuterium distance of 6.2 (+/-0.2) A, corresponding to a dipolar coupling constant of 6.5 (+/-0.5) kHz.[1]References
- Deuterium magic angle spinning studies of substrates bound to cytochrome P450. Lee, H., Ortiz de Montellano, P.R., McDermott, A.E. Biochemistry (1999) [Pubmed]
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