This blog describes the outcome of a scientific project related to the structure and function of FeFe hydrogenases, and funded by the French National Agency of Research (ANR) since 2007 (projects CAFE 2007-2010 and ECCHYMOSE 2012-2016)

FeFe-hydrogenases (H2ases) are large and complex metallo-enzymes which catalyse H2 oxidation and production at a conserved inorganic active-site, the so-called H-cluster. They are studied in various contexts, ranging from bioenergetics to inorganic catalysis, but the main motivation is certainly that both these enzymes and the knowledge we might acquire by studying them will prove useful for designing the catalysts we need to produce hydrogen from water in a clean process. Yet understanding why these enzymes cease to work under adverse conditions, and designing H2ases that are best suited for biotechnological applications, remain major challenges which we plan to tackle using a multidisciplinary approach that combines state-of-the-art electrochemistry, molecular biology, biochemistry and theoretical methods.

The current attempts to use the photosynthetic green alga Chlamydomonas reinhardtii to produce H2 from water and light are hampered by the inactivation of this enzyme. Several observation implie that it should be possible to use molecular biology to modify H2ases and make them more resistant, provided the molecular basis of damage is understood.

We combine direct electrochemistry and theoretical chemistry  to understand the inhibition of theses enzymes. Our approach is unique in that we learn about the molecular determinants of inhibition by studying and comparing the properties of a number of FeFe H2ases isolated from different microorganisms: the project involves two teams of biochemists and molecular biologists who use original methods to produce

  • distinct (but homologous) FeFe-H2ases from three different microorganisms,
  •  site-directed mutants of these enzymes, where a small number of amino-acids have been selectively replaced,
  • chimeric enzymes assembled from subunits of different origins, and
  •  enzymes that have been randomly modifed and then selected according to their functional properties using original strategies.

Combining genetic engineering and appropriate electrochemical and theoretical investigations will bring an entire set of new data that will increase greatly our understanding of the reactivity and vulnerability of FeFe-H2ases. It should also take us closer to the Graal of biohydrogen research: an H2-production biological catalyst that remains active both in the presence of O2.


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