[FeFe] hydrogenase enzymes rapidly evolve H2 at a 6-Fe catalytic site termed the H-cluster, which consists of a traditional [4Fe-4S] cluster linked via a cysteine bridge to a dinuclear Fe subcluster [2Fe]H that possesses unusual biological ligands: two terminal CN- ligands, two terminal CO ligands, and azadithiolate and CO bridges, all of which are thought to be synthesized and installed by a set of Fe-S proteins denoted HydE, HydF, and HydG. We can generate [FeFe] hydrogenase in high yield using cell free synthesis methods, allowing for specific isotope labelling of its components as needed for definitive spectroscopic studies (1). The radical S-adenosylmethionine (SAM) enzyme HydG lyses free L-tyrosine to produce CO and CN- for the assembly of the H-cluster. We use electron paramagnetic resonance (EPR) spectroscopy to detect and characterize HydG reaction intermediates generated with a set of 2H, 13C, and 15N nuclear spin labeled tyrosine substrates. 5’-deoxyadenosyl cleavage of tyrosine at the Calpha-Cbeta bond generates a transient 4-oxidobenzyl (4OB.) radical and a dehydroglycine which is converted to the CO and CN ligands of Fe in a second Fe-S cluster. 3). New X-ray crystallography and EPR studies reveal a unique site-differentiated structure for this second Fe-S cluster that clarifies its role in H-cluster synthesis (4,5), with the proposed product a Fe(II)(cysteine)(CO)2(CN) organometallic species. 1. Myers, William, et al., J. Am. Chem. Soc. (2014) 136:12237-12240. 2. Kuchenreuther, Jon, et al., Science (2013) 342:472-475 3. Kuchenreuther, Jon, et al., Science (2014) 343:424-427 4. Dinis, Pedro, et al., Proc. Natl. Acad. Sci. U.S.A. (2015) 112:1362-1367. 5. Suess, Daniel, et al., Proc. Natl. Acad. Sci. U.S.A. (2015) 112:11455-11460.

Enyzmatic chemistry with radicals and cyanide and carbon monoxide, be careful there!

251st National Meeting (2016)

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