Photoinduced Electron Transfer in a Radical SAM Enzyme Generates an S-Adenosylmethionine Derived Methyl Radical

Abstract

Radical SAM (RS) enzymes use S-adenosyl-L-methionine (SAM) and a [4Fe-4S] cluster to initiate a broad spectrum of radical transformations throughout all kingdoms of life. We report here that low-temperature photoinduced electron transfer from the [4Fe-4S] 1+ cluster to bound SAM in the active site of the hydrogenase maturase RS enzyme, HydG, results in specific homolytic cleavage of the S-CH 3 bond of SAM, rather than the S-C5′ bond as in the enzymecatalyzed (thermal) HydG reaction. This result is in stark contrast to a recent report in which photoinduced ET in the RS enzyme pyruvate formate-lyase activating enzyme cleaved the S-C5′ bond to generate a 5′-deoxyadenosyl radical, and provides the first direct evidence for homolytic S-CH 3 bond cleavage in a RS enzyme. Photoinduced ET in HydG generates a trapped • CH 3 radical, as well as a small population of an organometallic species with an Fe-CH 3 bond, denoted Ω M. The • CH 3 radical is surprisingly found to exhibit rotational diffusion in the HydG active site at temperatures as low as 40 K, and is rapidly quenched: whereas 5′-dAdo • is stable indefinitely at 77 K, • CH 3 quenches with a half-time of ~2 min at this temperature. The rapid quenching and rotational/translational freedom of • CH 3 shows that enzymes would be unable to harness this radical as a regio-and stereospecific H atom abstractor during catalysis, in contrast to the exquisite control achieved with the enzymatically generated 5′-dAdo • .

S‐Adenosyl‐l‐ethionine is a Catalytically Competent Analog of S‐Adenosyl‐l‐methionine (SAM) in the Radical SAM Enzyme HydG

“…This generates a . CH 3 radical in the active site, as characterized by electron paramagnetic resonance (EPR) spectroscopy [29] . We found that this .…”

“…Reduced HydG with SAM bound to the [4Fe‐4S] + cluster furthermore undergoes cryogenic photoinduced electron transfer from the cluster to SAM, resulting in regiospecific cleavage of the S−CH 3 bond, rather than the S−C5′ bond (as in the case of PFL‐AE) [29, 30] . This generates a .…”

“…We found that this . CH 3 undergoes rapid rotational diffusion even at 40 K, and converts to a slower‐tumbling state at lower temperatures [29] . The .…”

“…The . CH 3 radical decays rapidly at 77 K with a half‐life of minutes, [29] in contrast to the 5′‐dAdo . radical formed by photolysis of [4Fe‐4S] + :SAM in PFL‐AE, which is indefinitely stable at 77 K [9] .…”

“…CH 3 is intrinsically more reactive than 5′‐dAdo . , primarily because of its small size and ability to undergo rapid translational diffusion in search of a reaction partner [29] …”