| Summary: | <p>Hydrogenase-2 from E. coli is a multi-faceted [NiFe]-hydrogenase that is important because of its dual role as a model enzyme capable of reversible proton reduction and its prominence in microbe metabolism, yet previous investigations of Hyd-2 have been limited by low yield. In this investigation, a novel overexpression system for the catalytic core of Hyd-2 (termed Hyd-2 NOP) was designed and used to increase the yield of Hyd-2. Hyd-2 NOP was thoroughly characterized to ensure it remained nearly identical to Hyd-2 purified by previous strategies. The increased yield enabled structural characterization of the enzyme, and the structure allowed for a more accurate prediction of the full quaternary structure of Hyd-2. The structure also confirmed the positioning of three amino acid residues that are theorized to play an important, yet ambiguous, role in catalysis.</p> <p>These residues were mutated, and the resulting protein variants were electrochemically and structurally characterized to better understand the role of these amino acid residues. Two of these residues, Asp103 and Arg479, are located directly above the bimetallic cofactor in the active site, and when mutated, confirmed the significance of the secondary coordination sphere in H<sub>2</sub> activation. Fortunately, the R479K mutation trapped intermediates in the inactivation pathway that have never been isolated. The third residue, Glu14, has been shown to be important for the proton pathway in [NiFe]-hydrogenase, yet recent evidence from Hyd-1 challenged that finding. Comparative studies between the Hyd-1 and Hyd-2 variants with a Glu14 mutation showed the unique role of the proximal Fe-S cluster in Hyd-1 of repairing the proton pathway.</p> <p>Collaborations with Guangyu Liu (Sir William Dunn School of Pathology, University of Oxford) and Dr. Liyun Zhang (Armstrong Group, Inorganic Chemistry Laboratory, University of Oxford) also explored the role of Hyd-2 in pathology and as a crucial component of biotechnology, respectively.</p>
|
|---|