Physiologie und Biotechnologie der pflanzlichen Zelle

Cyanobacterial hydrogenases

Our research focuses on the physiology, regulation, and structure of hydrogenases in cyanobacteria. We work on Synechocystis sp. PCC 6803 which is a unicellular model organism that is completely sequenced and naturally transformable. It is capable to take up DNA from its medium and to integrate it via homologous recombination into its genome. Synechocystis habours a bidirectional NiFe-hydrogenase. Hydrogenases are oxidoreductases that are able to catalyze both the oxidation of hydrogen (H2) as well as the reduction of protons to hydrogen.

H2 ↔ 2H+ +2e-

It is anticipated that primitive hydrogenases might have been important devices to provide energy to primordial life in the early atmosphere.


Synechocstis is able to produce hydrogen both under fermentative conditions in the dark and transiently as a byproduct of photosynthesis in the light. As the enzyme is oxygen sensitive, its activity is inhibited by the evolution of oxygen from photosystem II. Dark adapted cells that are illuminated show a short burst of hydrogen however. It was suggested that the hydrogenase acts in this case as a valve for low potential electrons generated in the photosynthetic electron chain (Appel et al., 2000; Cournac et al., 2004; Gutthann et al., 2007). The hydrogen thus stores surplus electrons in order to protect the photosystems from photodamage.
Although cyanobacteria are oxygenic phototrophs they periodically face anaerobic conditions e.g. in microbial mats or surface waterblooms (Hoehler et al., 2001). Whereas these communities can be even oxygen supersaturated during daytime, they may turn anoxic during the night as their members shift to respiration, consuming the oxygen. In order to survive under such conditions some cyanobacteria switch to fermentation. The hydrogen produced in the dark is thought of being important to regenerate reducing equivalents.
We study the electron flow to the hydrogenase both under photosynthetic and fermentative conditions. As hydrogen is not the only electron sink, we constructed several mutants, in which the electron flow was redirected from other cellular electron sinks to the hydrogenase. These mutants show an elevated hydrogen production both under phototrophic and fermentative conditions (Gutthann et al. 2007).


The NiFe-hydrogenase of Synechocystis is encoded by five genes (hoxEFUYH) that are all organized in one cluster. A promoter situated upstream of hoxE was shown of being competent to initiate the transcription of all five hox genes. It was however observed that the transcription of the different hox genes can also be activated distinctly pointing to additional promoters within the gene cluster that have not been characterized yet (Kiss et al., 2009). Two transcription factors, LexA and Sll0359, were shown to bind to the promoter and to influence its activity (Gutekunst et al., 2005; Oliveira and Lindblad, 2007). The detailed signal transduction pathway activating the transcription of the hox genes is however far from being understood and is studied in our group.


NiFe-hydrogenases have been classified on the basis of their sequence into four different groups (Vignais and Billoud, 2007). Cyanobacteria contain only two different hydrogenases, the uptake hydrogenase and the bidirectional hydrogenase (Ludwig et al., 2006). Up to now structural information on the NiFe-hydrogenases is limited to the group 1 hydrogenases (Fontecilla-Camps et al., 2007; Volbeda et al., 1995).
Investigations in our group indicate that the active site of the bidirectional hydrogenase is similar to the group 1 NiFe hydrogenases (Germer et al., 2009) but in contrast to these enzymes it is very quickly reactivted under anaerobic conditions and able to work in the presence of low oxygen concentrations.


Hydrogen is a promising carrier of renewable energy. It has the highest energy density of any known fuel and can be used in fuelcells reacting with oxygen to pure water. In Synechocystis both the production of hydrogen in the light and in the dark rely on solar energy. In the first case, protons are directly delivered from the photosynthetic electron chain to produce hydrogen, whereas in the dark, glycogen is metabolized that was previously accumulated during phototrophic growth.
Natural microbial strains produce only small amounts of hydrogen. In order to increase their hydrogen evolution, investigations on the physiological function, the transcriptional regulation and the structure of the hydrogenase are neccessary.

example of a photobioreactor                                                          © AG Schulz

Three different options are envisaged in our group:
We constructed physiological mutants with a modified electron flow to the hydrogenase, resulting in cyanobacteria with an elevated hydrogen production. Further studies in this direction are on the way.
Additionally a mutant with a strong promoter (psbAII) inserted upstream of the hox cluster resulted in elevated enzyme concentrations and enzyme activity in vivo. Further investigations on the signal transduction pathway to the hydrogenase are undertaken in order to gain the ability to influence its activity on the transcriptional level.
In a third approach the structure of the hydrogenase is investiagted in order to get insights into the process of the oxygen inhibition of the enzyme. Modifying the hydrogenase in a way that would leave the enzyme oxygen resistant would open the way to produce larger amounts of hydrogen under phototrophic conditions in the light.


Appel, J., Phunpruch, S., Steinmüller, K., and Schulz, R. (2000) The bidirectional hydrogenase of Synechocystis sp. PCC 6803 works as an electron valve during photosynthesis. Archives of Microbiology 173: 333-338.

Cournac, L., Guedeney, G., Peltier, G., and Vignais, P.M. (2004) Sustained Photoevolution of Molecular Hydrogen in a Mutant of Synechocystis sp. Strain PCC 6803 Deficient in the Type I NADPH-Dehydrogenase Complex. J. Bacteriol. 186: 1737-1746.

Fontecilla-Camps, J.C., Volbeda, A., Cavazza, C., and Nicolet, Y. (2007) Structure/Function Relationships of [NiFe]- and [FeFe]-Hydrogenases. Chemical Reviews 107: 4273-4303.

Germer, F., Zebger, I., Saggu, M., Lendzian, F., Schulz, R.d., and Appel, J. (2009) Overexpression, Isolation, and Spectroscopic Characterization of the Bidirectional [NiFe] Hydrogenase from Synechocystis sp. PCC 6803. Journal of Biological Chemistry 284: 36462-36472.

Gutekunst, K., Phunpruch, S., Schwarz, C., Schuchardt, S., Schulz-Friedrich, R., and Appel, J. (2005) LexA regulates the bidirectional hydrogenase in the cyanobacterium Synechocystis sp. PCC 6803 as a transcription activator. Molecular Microbiology 58: 810-823.

Gutthann, F., Egert, M., Marques, A., and Appel, J. (2007) Inhibition of respiration and nitrate assimilation enhances photohydrogen evolution under low oxygen concentrations in Synechocystis sp. PCC 6803. Biochimica et Biophysica Acta (BBA) - Bioenergetics 1767: 161-169.

Hoehler, T.M., Bebout, B.M., and Des Marais, D.J. (2001) The role of microbial mats in the production of reduced gases on the early Earth. Nature 412: 324-327.

Kiss, É., Kós, P.B., and Vass, I. (2009) Transcriptional regulation of the bidirectional hydrogenase in the cyanobacterium Synechocystis 6803. Journal of Biotechnology 142: 31-37.

Ludwig, M., Schulz-Friedrich, R., and Appel, J. (2006) Occurrence of Hydrogenases in Cyanobacteria and Anoxygenic Photosynthetic Bacteria: Implications for the Phylogenetic Origin of Cyanobacterial and Algal Hydrogenases. Journal of Molecular Evolution 63: 758-768.

Oliveira, P., and Lindblad, P. (2007) An AbrB-like protein regulates the expression of the bidirectional hydrogenase in Synechocystis sp strain PCC 6803. Journal of Bacteriology 190: 1011-1019.

Vignais, P.M., and Billoud, B. (2007) Occurrence, Classification, and Biological Function of Hydrogenases: An Overview. Chemical Reviews 107: 4206-4272.

Volbeda, A., Charon, M.-H., Piras, C., Hatchikian, E.C., Frey, M., and Fontecilla-Camps, J.C. (1995) Crystal structure of the nickel-iron hydrogenase from Desulfovibrio gigas. Nature 373: 580-587.