Analysis of Electrochemical Properties of S-Adenosyl-l-methionine and Implications for Its Role in Radical SAM Enzymes

Abstract

S-Adenosyl-l-methionine (SAM) is the central cofactor in the radical SAM enzyme superfamily, responsible for a vast number of transformations in primary and secondary metabolism. In nearly all of these reactions, the reductive cleavage of SAM is proposed to produce a reactive species, 5'-deoxyadenosyl radical, which initiates catalysis. While the mechanistic details in many cases are well-understood, the reductive cleavage of SAM remains elusive. In this manuscript, we have measured the solution peak potential of SAM to be ∼-1.4 V (v SHE) and show that under controlled potential conditions, it undergoes irreversible fragmentation to the 5'-deoxyadenosyl radical. While the radical intermediate is not directly observed, its presence as an initial intermediate is inferred by the formation of 8,5'-cycloadenosine and by H atom incorporation into 5'-deoxyadenosine from solvent exchangeable site. Similarly, 2-aminobutyrate is also observed under electrolysis conditions. The implications of these results in the context of the reductive cleavage of SAM by radical SAM enzymes are discussed.

Figure 1

Cyclic voltammograms of SAM in (a) water at 120 mV/s, (b) acetonitrile at 110 mV/s, (c) ethanol at 85 mV/s, and (d) tetrahydrofuran at 100 mV/s.

Figure 2

SAM is cleaved under reducing conditions to produce dAdo. (A) UHPLC trace of authentic dAdo standard (upper), extract ion chromatogram (EIC) of m/z 252.11 corresponding to dAdo from authentic standard (middle) and dAdo from electrolysis of SAM (lower). The small difference in retention times observed between UV and MS is the result of the distance between the UV–visible detector and the in-line MS analyzer. (B) Representative dAdo versus time plot at −1.5 V. dAdo standards were used to quantify dAdo. (C) Rate of dAdo as a function of cell potential reveals a midpoint of ∼−1.4 V vs SHE.

Figure 3

Isotope incorporation from bulk solvent into dAdo. Isotopic peak distribution of dAdo when electrochemically reduced in (A) H₂O or (B) D₂O.

Figure 4

Analysis of photolysis products of AdoCbl (A) UV–visible spectra of 0.1 mM AdoCbl solution during photolysis. (B) EIC traces at m/z 252.11 AdoCbl prior to (gray) and resulting from photolysis (black) (upper) and EIC trace at m/z 250.09 prior to (gray) and resulting from photolysis (black) (lower). (C) HCD fragmentation of authentic dAdo standard (upper; RT 12.2 min, m/z 252.11) and authentic cyc-dAdo (lower; RT 10.6 min, m/z 250.09). (D) HCD fragmentation of dAdo (upper; RT 12.2 min, m/z 252.11) and cyc-dAdo (lower; RT 10.6 min, m/z 250.09) from photolysis of AdoCbl.

Figure 5

Characterization of m/z 250 species from controlled potential electrolysis of SAM. (A) EIC at m/z 250.09 of electrolysis of SAM at −1.8 V. (B) Fragmentation of the species at m/z of 250.09 produces fragments that are identical to those observed with cyc-dAdo standards (Figure 4C).

Figure 6

Controlled potential electrolysis of SAM produces 2-AB. Controlled potential electrolysis experiments were carried out at −1.8 V. (A) The EIC traces correspond to 2-AB standard (upper trace, m/z = 104.07), electrolysis in H₂O (middle trace, m/z = 104.07), and electrolysis in D₂O (lower trace, m/z = 105.07). (B) Mass spectra corresponding to the peak at 5.7 min in the EIC traces are shown in panel A. (C) MS/MS analysis of the base peaks in spectra shown in B.

Figure 7

dAdo and 2-AB are produced with similar rates during controlled potential electrolysis of SAM. In these experiments, solutions were poised at −1.25 V (circle), −1.5 V (triangle), or −2.25 V (square). Samples were withdrawn at various times and (A) dAdo (black) and 2-AB (red) were quantified on the basis of the area of the EIC peak. (B) Rate of formation of dAdo (black) and 2-AB (red) versus the cell potential.