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| - Deuterium Magic Angle Spinning Studies of Substrates Bound toCytochrome P450
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| Abstract
| - We report solid-state deuterium magic angle spinning NMR spectra of perdeuterated adamantanebound to the active site of microcrystalline cytochrome P450cam (CP450cam) in its resting state. CP450camcontains a high-spin ferric (Fe3+) heme in the resting state; the isotropic shift was displaced from thediamagnetic value and varied with temperature consistent with Curie-law dependence. A nondeuteratedcompetitive tighter binding ligand, camphor, was used to displace the adamantane-bound species. Thisaddition resulted in the disappearance of the hyperfine-shifted signal associated with a perdeuteratedadamantane bound to CP450cam, while signals presumably associated with adamantane bound to othercavities persisted. We simulated the deuterium spinning side-band intensities for the enzyme-bound speciesusing dipolar hyperfine coupling as the only anisotropic interaction; the deuterium quadrupolar interactionwas apparently averaged due to a fast high-symmetry motion. These data provide direct support for previousproposals that substrates are conformationally mobile on the time scale of enzymatic turnover. Thesimulations suggested that the adamantane binds with an average metal-deuterium distance of 6.2 (±0.2)Å, corresponding to a dipolar coupling constant of 6.5 (±0.5) kHz.
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