Acireductone dioxygenase (ARD) is a 179-residue enzyme containing a paramagnetic Ni+2 ion in the active site. Because of electron-nuclear spin interactions, 1H resonances within ∼9 Å of the Ni+2 are broadened beyond detection. For this reason, 1H-detected multidimensional NMR experiments are not suitable for structural characterization of the active site of ARD, and no isostructural diamagnetic homologue is available. Rapid recycle two-dimensional direct 13C detection NMR methods previously allowed correlation of carbonyl (13C′) carbons with directly bonded 13Cα and 15N spins in ARD (Kostic, M.; Pochapsky, S. S.; Pochapsky, T. C. J. Am. Chem. Soc. 2002, 124, 9054-9055), but not 15N with 13Cα, a critical connection for sequential assignment of backbone resonances. It is now shown that complete sample deuteration combined with direct 13C detection using a cold probe/preamplifier permits the one-bond 13Cα-15N correlation to be made via a four-pulse double-quantum experiment CAN. Combined with data from other 13C direct-observe 2D NMR experiments, CAN data permits sequential assignments to be made for many resonances in ARD close to the active-site metal.
ASJC Scopus subject areas
- Colloid and Surface Chemistry