(508b) Towards a Fully Defined In Vitro Maturation of [FeFe] Hydrogenases

The sustainable production of hydrogen for renewable energy technologies positions it as a promising alternative to current fossil fuel based technologies. By replacing fossil fuel based H₂ production with a biological process, we can exploit nature’s biological catalysts, hydrogenases. The [FeFe] hydrogenases are specific and productive in hydrogen evolution, but application of these complex enzymes is limited by inefficient and expensive production methods. The development of a fully defined in vitro hydrogenase maturation system will elucidate the assembly requirements for producing [FeFe] hydrogenases so they can be used commercially and enable technologies for carbon neutral hydrogen production. These enzymes are characterized by complex active sites, and knowledge of how the complex “H-cluster” active site is assembled and installed into the polypeptide remains unclear. The goal of this project is to identify the substrates and overall chemical mechanisms required for active site assembly and installation.

In particular, the in vitro [FeFe] hydrogenase maturation system seeks to activate purified Clostridium pasteurianum CpI apoprotein using E. coli cell extract containing recombinant Shewanella oneidensis HydE, HydF, and HydG maturases and exogenous small molecules. CpI is an [FeFe] hydrogenase containing a complex active site, the “H-cluster,” and four accessory Fe-S clusters involved in the delivery of electrons to the active site. Our group has previously developed an in vitro activation for CpI. However, activation can only be achieved in the presence of a crude E. coli cell extract, and its composition is undefined. Because previous results have demonstrated the installation of Fe-S centers in limiting the maturation of hydrogenases, the proteins involved in Fe-S assembly were explored in this context. The isc operon is a well-characterized biosynthetic system involved in the assembly of cellular Fe-S proteins, and is believed to maintain a general housekeeping biosynthetic function. To determine if these were the unknown components in the extract responsible for incurring full hydrogenase maturation, the isc proteins in E. coli were cloned, expressed, and purified with N- and C- terminal StrepII tags in order to use a streptavidin/biotin purification procedure. The activity of each purified proteins was confirmed with an assay specific to its purported function, and their role in the apoCpI activation was evaluated.

Because the isc proteins only demonstrated the ability to stimulate 1% activation versus full activation with a crude lysate, work is in progress to fractionate the cell extract using chromatography to allow further evaluation and identification the substrates required to fully define an in vitro [FeFe] hydrogenase activation. These findings will provide a deeper understanding of the mechanism for assembly and installation of the active site. This knowledge will also enable E. coli genetic engineering for high-level hydrogenase production. Finally, a fully functional in vitro hydrogenase maturation system can be used as a platform for high throughput evolution projects.