A novel overproduction system for the structural determination of a proton-pumping hydrogen-producing...

The recent novel overproduction system for the membrane-bound oxygen-sensitive [NiFe]-hydrogenase-2 (Hyd-2) from Escherichia coli is detailed. Hyd-2 is an efficient and reversible catalyst for the interconversion of H₂ and 2H⁺. Produced at low levels during anaerobic respiration, Hyd-2 is instrumental in the generation of proton-motive force (PMF), and likewise uses PMF to generate H₂. The structure of the Hyd-2 complex could yield immense information on the mechanism of proton pumping in this group of enzymes, which are of energetic and pathogenic importance. The overproduction of the soluble "catalytic core" where H₂ oxidation/H⁺ reduction takes place, relies on gene deletions and use of a synthetic operon in the overproduction strain to ensure metal processing and redox center formation and incorporation are not limiting. Based on previous evidence of a cytoplasmic excess of the active site-containing subunit (HybC), the Hyd-2 production bottleneck is relaxed by overproduction of the FeS cluster-containing subunit (HybO). The hybO gene is altered to prevent translocation of the HybOC catalytic core to the membrane. Protein yield is increased by an order of magnitude, allowing protein-intensive techniques such as X-ray crystallography to flourish. The structure of the entire Hyd-2 complex is inferred from the catalytic core and homology modeling of the ferredoxin and membrane integral partners, leading to the proposal that Hyd-2 is a dimer of tetramers.