Enhanced aerobic H2 production by engineering an [FeFe] hydrogenase from Clostridium pasteurianum

Abstract

Biological H2 production is one of the alternative technologies for producing H2 in a renewable and sustainable fashion. The technology often relies on the [FeFe] hydrogenase enzyme for catalyzing the H2 production reaction from protons and electrons as it is highly efficient. The high O2 sensitivity of the enzyme have been one of the key obstacles for implementing the technology, but recent findings showed the feasibility of improving O2 tolerance via protein engineering. In this study, we investigated the changes in the O2 tolerance and aerobic H2 production activity of an [FeFe] hydrogenase by replacing methionines on the surface with leucines. None of the mutations were detrimental to protein folding, and a few were able to exert either positive or negative influence on the O2 tolerance of the enzyme. We combined the mutation exhibiting the highest improvement in O2 tolerance with the rest of the Met – Leu replacements; however, the results indicated non-synergistic impact on the tolerance. Combinations with the other types of mutations that have been reported previously also failed to further improve the O2 tolerance of the hydrogenase. These results provide new insights on how mutagenesis affects the enzyme's functional properties, as well as the molecular mechanism(s) behind the tolerance.