Completing the Circuit: Direct-Observe 13C,15N Double-Quantum Spectroscopy Permits Sequential Resonance Assignments near a Paramagnetic Center in Acireductone Dioxygenase
Year: 2008
Type: article
Abstract: Acireductone dioxygenase (ARD) is a 179-residue enzyme containing a paramagnetic Ni +2 ion in the active site. Because of electron−nuclear spin interactions, 1 H resonances within ∼9 Å of the Ni +2 are broadened beyond detection. For this reason, 1 H-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 13 C detection NMR methods previously allowed correlation of carbonyl ( 13 C‘) carbons with directly bonded 13 C α and 15 N spins in ARD (Kostic, M.; Pochapsky, S. S.; Pochapsky, T. C. J. Am. Chem. Soc. 2002, 124, 9054−9055), but not 15 N with 13 C α, a critical connection for sequential assignment of backbone resonances. It is now shown that complete sample deuteration combined with direct 13 C detection using a cold probe/preamplifier permits the one-bond 13 C α − 15 N correlation to be made via a four-pulse double-quantum experiment CAN. Combined with data from other 13 C direct-observe 2D NMR experiments, CAN data permits sequential assignments to be made for many resonances in ARD close to the active-site metal.
DOI: 10.1021/ja710187x
Institution: Brandeis University
Language: English
Subfield: Spectroscopy
Field: Chemistry
Domain: Physical Sciences
Open Access status: green
OpenAlex ID: W1964498522