Hydrogenases can be used in biotechnological devices but the O2-inactivation of theses enzymes limits their potential applications. The aim of the project was to decipher inhibition mechanisms and to increase dioxygen tolerance using enzyme modification.
Hydrogenases are complex metalloenzymes which catalyze the reversible oxidation of H2 at a bimetallic active site. They can potentially be used in biotechnological systems such as dihydrogen production by photosynthetic organism using solar energy. However, the inactivation of hydrogenases by O2 greatly limits their applications. Hydrogenases are produced in photosynthetic or non-photosynthetic bacteria. Depending the organism, the enzyme is extracted, their dioxygen tolerance varies. <br />Our aim was to use new biophysical methods and several biological systems to understand NiFe hydrogenase O2-inactivation mechanism and natural tolerance of certain hydrogenases. To this end, we produced, purified and characterized hydrogenases from two organisms, as well as a variant (by site-directed mutagenesis). Some of these enzymes are sensitive to dioxygen while other are naturally tolerant. <br />Comparison of the inactivation properties of these different hydrogenases gives information on the molecular mechanism of O2-inhibition of NiFe hydrogenase and on structural determinants of O2-tolerance.
For this interdisciplinary project, we used and developed various techniques. Molecular biology techniques were used, in particular SLIC method (sequence- and ligationindependent cloning), to delete genes and to construct plasmids for enzyme production in the bacteria Escherichia coli. Hydrogenases were purified with usual biochemical techniques, in particular affinity chromatography. Bacterial cultures were optimized thanks to fermentation platform in wellcontrolled conditions. Purified hydrogenases were characterized by protein film electrochemistry and by biophysical methods. Advanced EPR techniques were used to study the interaction between dioxygen and metallic centers of the enzymes. X Ray crystallography and site-directed mutagenesis were used to identify the cavities that transport O2 within the NiFeSe enzyme. Theoretical chemistry calculation, in agreement with experimental data gave information on reaction pathway during dioxygen inhibition of the NiFe hydrogenases, leading to the formation of inactive states (NiA and NiB). Most probable structures of these two states were proposed.
E. coli Hyd-1 enzyme was produced homologously from a plasmid but in low quantity in its soluble form (most of the enzyme are in inclusion bodies). We constructed, produced and purified several mutants of the cysteine ligand of the proximal FeS cluster that is important for O2-resistance , as well as the V78C mutant of the large subunit. The catalytic activity of the wild-type enzyme purified is 30 time higher than that reported in the same conditions (32 U/mg vs 1 U/mg). The V78C mutation induced an increase in dioxygen resistance of a naturally O2-tolerant hydrogenase.
The production and the purification of Hyd-1 and Hyd-2 hydrogenases and of their mutants were difficult because of the formation of inclusion bodies and post-traductionnal maturation processes. Various solutions were tested without success but the optimization of their production is essential for spectroscopic characterization in order to understand the molecular determinant of O2 resistance. The mutant enzyme V78C, the most O2-resistante enzyme tested so far in the lab, could be used in biofuel cell by another team of our lab.
Seven articles related to this project were published in peer-reviewed journals with impact factors between 2.95 (J. Biol. Inorg. Chem) and 20.95 (Account of chemical research) with an average of 7.8. One of these articles is the work of the Italian colla
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.
Madame Carole Baffert (Centre National de la Recherche Scientifique délégation Provence et Corse_Laboratoire de bioénergétique et ingénierie des protéines)
The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.
M.I.O. INSTITUT MEDITERRANEEN D’OCEANOLOGIE
University of Milan-Bicocca
CSIC Laboratory de Bioelectrocatalysis
University College London
CNRS DR12_UMR7251 Centre National de la Recherche Scientifique délégation Provence et Corse_Laboratoire de bioénergétique et ingénierie des protéines
Help of the ANR 399,464 euros
Beginning and duration of the scientific project: September 2014 - 48 Months