Electrochemical Investigation of the Radical SAM Enzyme, BtrN from Bacillus Circulans

Abstract

Radical SAM enzymes catalyze a variety of reactions involved in biological pathways including the biosynthesis of antibiotics, cofactors, and biosynthesis and repair of DNA. Measurement of the electrochemical characteristics of radical SAM enzymes has been limited due to the typically buried location of the radical SAM cluster within the enzyme. The midpoint potential of a radical SAM cluster has only been determined for lysine aminomutase using spectroelectrochemical titrations with the use of mediators. This study presents the first direct electrochemical measurement of a radical SAM enzyme using the technique of protein film voltammetry (PFV). BtrN from Bacillus circulans is an emerging class of radical SAM dehydrogenases, which catalyze the third step in the biosynthetic pathway of the antibiotic butirosin. BtrN has been shown to contain a second [4Fe-4S] in addition to the canonical radical SAM [4Fe-4S] cluster. Nonturnover PFV has characterized the electrochemical properties of these clusters and provided insight into the mechanism of electron transfer. Additionally, PFV has been used to characterize the effect of substrate binding on the clusters. These results provide insight into the catalytic mechanism of BtrN and the electrochemical characteristics of radical SAM enzymes in general.