Diversity of the hydrogenases and their reactivity to oxygen
Towards new hydrogen-producing and oxygen-tolerant enzymes

National program of Research on Bioenergies ( PNRB) 2006

Coordinator: Laurent Cournac, CEA of Cadarache

The project associates the laboratories of the CEA (Cadarache and Grenoble), of the CNRS (Cadarache and Marseille) and the IRD ( Marseille).
The project began in December, 2006 and lasted 36 months. It benefited, within the framework of the PNRB, of a 587 706€ ANR's help for a global cost around 2 240 000€.

Identify new hydrogenases from bacterial diversity, select the most oxygen-tolerant ones and understand their reactivity

Several green algae and cyanobacteria are able to produce hydrogen at the expense of solar energy. However, H2 production is transient due to the sensitivity of hydrogenases (H2 synthesizing enzymes) to the oxygen which is produced by photosynthesis. Oxygen sensitivity of hydrogenases therefore constitutes a major impediment to their use in H2 production bioprocesses. This sensitivity shows however a great variability in nature.

The aim of the project is to better understand molecular bases of oxygen sensitivity, in order to overcome or at least alleviate it. For this, we study O2 tolerant enzymes which are brought by microbial strains screening.
These data will help us to propose hydrogenase optimization strategies for projects aiming at H2 production by photosynthetic microorganisms. Stable and oxygen-tolerant hydrogenases could also be used to produce electricity in bio-fuel cells.

Biodiversity screening, functional and structural characterization of isolated hydrogenases, applications

We have established setups for screening, selecting and culturing new mesophilic and thermophilic bacterial strains possessing hydrogenases. More than 1300 strains were screened so far and more than 150 identified as possessing hydrogenase activity, axenically cultured and conserved.
In the mean time, a bioinformatic analysis of sequenced genomes and metagenomes allowed us to get an overview of the diversity of hydrogenase-coding genes, of key elements in their sequences, and of their relationships with the environments in which the strains were found. A few strains selected during the project, from the screens or on the basis of former knowledge of participants, were produced in large amounts and their hydrogenases were extracted and purified in order to achieve their biochemical, spectroscopic and crystallographic characterization in order to better understand the interaction of these enzymes with O2. For this, innovative spectroscopic and electrochemical approaches were developed.
Finally, site-directed mutagenesis of cyanobacterial hydrogenase was undertaken in order to test whether the identified characteristics might be transposed to H2 (photo)producing organisms.

Main results

Hydrogenases from 4 enterobacteria close to Enterobacter radicincitans, particularly active and showing O2 tolerance, were analyzed, sequenced, and are under purification.
Setups for high temperature bacterial growth were used to characterize and mass-produce a thermophilic strain (Hydrogenobacter hydrogenophilus) for the purification of its highly O2-tolerant hydrogenase.
Owing to the new methodologies which were developed, we could quantify in a totally new way gas diffusion parameters within hydrogenases and their influence on O2 reactivity.
Mutagenesis of hydrogenase of the cyanobacterium Synechocystis PCC6803 was undertaken on the basis of these results and induced a slight but encouraging improvement of its tolerance.


Ten papers dealing with enzyme functional analysis or characterization of hydrogenotrophic strains were published or submitted. For instance, we showed that Clostridium acetobutylicum hydrogenase was inhibited by O2 in a slow and partially reversible manner, a new finding among FeFe enzymes (Baffert et al. 2008).
Relationships between gaz diffusion, nature of aminoacid at vicinity of active site and O2 tolerance were particularly studied on the NiFe enzyme of Desulfovibrio fructosovorans (Leroux et al. 2008, Dementin et al. 2009, Liebgott et al. 2009), giving conceptual bases for a patent applied in 2007.
In NiFeSe hydrogenase from Desulfomicrobium baculatum, a new O2 tolerance mechanism was identified during H2 production (Parkin et al. 2008), likely participating to its efficiency within hybrid photoproduction devices based on nanoparticles (Reisner et al. 2009).


structure of the active site of a hydrogenase

Compared structure of the active site of the O2-tolerant hydrogenase of the bacteria Desulfomicrobium baculatum in reduced state (A) and in oxidized state (B) : the oxygen interaction (in red) with the selenium (Se) is one of the keys of the remarkable properties of the enzyme.
This type of information can be used to the conception of new O2-tolerant hydrogenases in photosynthetic organisms.