Biosynthesis of the Catalytic H-Cluster of [FeFe] Hydrogenase
[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. With the James Swartz laboratory (Stanford University) 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 bound to a C-terminal Fe-S cluster (2). Electron and proton transfer to this 4OB. radical forms p-cresol with the conversion of this dehydroglycine ligand to Fe-bound CO and CN-, a key intermediate in the assembly of the [2Fe] subunit of the H-cluster. We apply stopped-flow Fourier transform infrared (SF-FTIR) and electron-nuclear double resonance (ENDOR) spectroscopies to explore in detail the formation such species which are used to build the H-cluster (3). New X-ray crystallography and EPR studies reveal a unique site-differentiated structure for this C-terminal Fe-S moiety that clarifies its role in H-cluster synthesis. Many open issues remained to be explored in this unique facet of biological catalytic cluster synthesis, including the roles of the additional Fe-S proteins HydE and HydF (4).
1. William K. Myers, Troy A. Stich, Daniel L. M. Suess, Jon M. Kuchenreuther, James R. Swartz, and R. David Britt. “The Cyanide Ligands of [FeFe] Hydrogenase: Pulse EPR Studies of 13C and 15N-Labeled H-Cluster” J. Am. Chem. Soc. (2014) 136:12237-12240.
2. Jon M. Kuchenreuther, William K. Myers, Troy A. Stich, Simon J. George , Yaser NejatyJahromy, James R. Swartz, and R. David Britt. “A Radical Intermediate in Tyrosine Scission to the CO and CN- Ligands of [FeFe] Hydrogenase” Science (2013) 342:472-475
3. Jon M. Kuchenreuther, William K. Myers, Daniel L. M. Suess, Troy A. Stich, Vladimir Pelmenschikov, Stacey A. Shiigi, Stephen P. Cramer, James R. Swartz, R. David Britt, and Simon J. George. “The HydG Enzyme Generates an Fe(CO)2(CN) Synthon in Assembly of the FeFe Hydrogenase H-Cluster” Science (2014) 343:424-427
4. Christopher J. Pickett. “Making the H-Cluster from Scratch” Science (2014) 343:378-379