Experimental guidelines for trapping paramagnetic reaction intermediates in radical S-adenosylmethionine enzymes

• Published in: Methods in enzymology Vol. 666; p. 451
• Main Authors: Balo, Aidin R, Britt, R David
• Format: Journal Article
• Language: English
• Published: United States 2022
• Subjects: Electron Spin Resonance Spectroscopy; Iron - metabolism; Iron-Sulfur Proteins - metabolism; S-Adenosylmethionine - metabolism; Sulfur - metabolism; Biochemistry; Radical SAM enzymes; Enzymology; Metalloenzymes; EPR spectroscopy
• ISSN: 1557-7988
• DOI: 10.1016/bs.mie.2022.02.015

Due to their biological importance and functional diversity, radical S-adenosylmethionine (rSAM) enzymes have become popular targets for electron paramagnetic resonance (EPR) spectroscopic studies. EPR spectroscopy is a powerful tool that allows for the observation of the iron-sulfur clusters as well as paramagnetic reaction intermediates, thus providing insight into their catalytic mechanisms. While the iron-sulfur clusters may be readily observable by EPR spectroscopy in the enzymes' resting states, radical intermediates are often elusive and must be trapped. Here, we describe a protocol for trapping and analyzing the Lys-Trp intermediate of the Lys-Trp-crosslinking rSAM enzyme SuiB, including modified expression and purification steps. This protocol is also intended to serve as a primer for trapping paramagnetic intermediates in other rSAM enzymes for studying by EPR spectroscopy.