Isotopically labeled flavoenzymes and their uses in probing reaction mechanisms

The incorporation of stable isotopes into proteins is beneficial or essential for a range of experiments, including NMR, neutron scattering and reflectometry, proteomic mass spectrometry, vibrational spectroscopy and "heavy" enzyme kinetic isotope effect (KIE) measurements. Here, we present detailed protocols for the stable isotopic labeling of pentaerythritol tetranitrate reductase (PETNR) via recombinant expression in E. coli. PETNR is an ene-reductase belonging to the Old Yellow Enzyme (OYE) family of flavoenzymes, and is regarded as a model system for studying hydride transfer reactions. Included is a discussion of how efficient back-exchange of amide protons in the protein core can be achieved and how the intrinsic flavin mononucleotide (FMN) cofactor can be exchanged, allowing the production of isotopologues with differentially labeled protein and cofactor. In addition to a thorough description of labeling strategies, we briefly exemplify how data analysis and interpretation of "heavy" enzyme KIEs can be performed.