The O2-stable [FeFe]-hydrogenase CbA5H reveals high resilience against organic solvents

Abstract

[FeFe]-Hydrogenases are highly efficient hydrogen-(H₂) converting enzymes which play pivotal roles for H₂ cycling in natural habitats, but which are also of interest for sustainable approaches to generate or employ H₂ gas. [FeFe]-Hydrogenases harbor a unique active site metal cofactor, the H-cluster, whose di-iron site by itself is nearly inactive but, as part of the protein, allows high turnover rates. Understanding this essential interplay of protein and co-factor might help to install [FeFe]-hydrogenases in biotechnological applications. The catalytic unit of the H-Cluster can be synthesized chemically and incorporated into [FeFe]-hydrogenase precursors, which allows to introduce non-natural metals or ligands and study their impact on catalytic activity. However, these compounds are often not water-soluble and have to be added to the proteins in solvents known to destabilize polypeptides. The resilience of [FeFe]-hydrogenases against organic solvents has hardly been investigated. To address this knowledge gap, we characterized the stability of the [FeFe]-hydrogenase CbA5H from Clostridium beijerinckii in several organic solvents (dimethylsulfoxide (DMSO), acetone, acetonitrile as well as water-miscible short-chain alcohols). These solvents are required to dissolve co-factor analogues and are also employed in chemical syntheses that might be combined with biocatalysts such as hydrogenases for more sustainable industrial processes. In the medium time-frame, CbA5H is remarkably stable in high concentrations of acetone and acetonitrile and also withstands intermediate concentrations of DMSO, ethanol and methanol. Combined with the unusual O₂ stability and high temperature and pressure tolerance, this makes CbA5H a candidate for its use in non-aqueous reaction environments.