HEROS

H2 is regarded as an attractive, clean energy carrier, but to date it cannot be produced using any “green” method. An option is to produce this gas by means of biological approaches, e.g using photosynthetic organisms that couple H2 production and water photolysis thanks to the biological catalysts of H2 production: hydrogenases. They are complex metalloenzymes which catalyze the reversible oxidation of H2 at a bimetallic (FeFe or NiFe) active site. The intense competition in the field of hydrogenase research is motivated in part by the potential use of these enzymes in biotechnological systems; therefore, research is more specifically focused on the inactivation of hydrogenases by O2, which greatly limits their applications. It can be envisaged to use rational protein engineering to render O2 tolerant the hydrogenases from photosynthetic organisms, on condition that the mechanism of O2-inactivation is understood. The spectacular results recently published by the partners of this project have stressed the need for maintaining efforts in studying the mechanism underlying O2-inactivation and O2-tolerance of NiFe hydrogenases. First, in Nature Chemical Biology in 2013, we challenged the commonly accepted mechanism of O2-inactivation, leaving open questions regarding the chemical structure of the inactive states. In PNAS and JACS in 2012, we have described a series of mutants of a sensitive enzyme whose tolerance to O2 was greatly increased by a mechanism which must be investigated further. Here, our goal is to use new biophysical methods and new biological systems to understand the mechanism O2-inactivation and O2-tolerance of NiFe hydrogenases, in order to eventually design O2-resistant NiFe hydrogenases that will be used for biological H2 production. The four teams of the two French labs and the three foreign participants involved in the project have already collaborated in a very prolific manner. They are experts in the field of metalloenzymes (particularly hydrogenases) and master the biochemical, spectroscopic, electrochemical and theoretical tools which they use for studying these enzymes. We will use the genetic system that has shown indispensable for routinely producing the sensitive NiFe enzyme from Desulfovibrio fructosovorans and tolerant mutants. We will develop the production, the purification and the physico-chemical studies of O2-tolerant hydrogenase from Salmonella enterica Typhimurium and E. coli. We will also capitalize on this expertise to develop an original and innovative field of research: the design and production of artificial, chimeric enzymes in which large structural motifs that are believed to be specific to naturally tolerant enzymes are transferred into the protein framework of the sensitive enzyme. We will develop and use new electrochemical methods, theoretical chemistry, and high resolution spectroscopic techniques to characterize and optimize the properties of these enzymes using the interdisciplinary approach that is the hallmark of the laboratory of bioenergetics and engineering of proteins in Marseille. Three distinct teams from this lab will collaborate on the project with their own approach (molecular biology, electrochemistry, EPR spectroscopy), and three foreign partners have been recruited to contribute their expertise (IR spectroscopy and theoretical chemistry). The large-scale production of the enzymes that are most promising in terms of applications will be optimized and carried out in the MIO laboratory, and then used in a ANR-funded network of researchers whose aim is to design biofuel cells (CAROUCELL ANR 2013 BIOME). The information obtained from these studies will benefit the rational engineering of the NiFe hydrogenases from photosynthetic organisms such as cyanobacteria, making it possible to design biological systems of production of H2 using light and water as source of energy.