Molecular hydrogen is a promising alternative to the limited fossil fuel resources. Hydrogenases, the enzymes involved in hydrogen metabolism, are divided in two major classes: [FeFe]-hydrogenases, which catalyze the reduction of protons to molecular hydrogen, and [NiFe]-hydrogenases, that can both catalyze the oxidation and the reduction of hydrogen. [FeFe]-hydrogenases show specific activity higher than [NiFe]-hydrogenases, nevertheless, in the last few years, the interest for the cyanobacterial [NiFe]-hydrogenase significantly increased. A multisubunits, bidirectional [NiFe]-hydrogenase is present in Synechocystis sp PCC 6803, the most studied cyanobacterial strain. This enzyme possesses five related subunits combined in two functional units: the hydrogenase moiety, i.e. HoxYH, which harbors the active site, and the diaphorase moiety, i.e. HoxEF, likely working as a redox partner for the catalytic subcomplex. Its physiological role is still under debate. Indeed, cyanobacteria lacking the Hox hydrogenase are viable when cultured under standard, physiological conditions, and a general consensus is emerged that this protein may play a role only under transient and/or specific growth conditions. Interestingly, although the enzyme is completely inactive in the presence of oxygen, the Hox protein is constitutively expressed in Synechocystis both in anaerobiosis and aerobiosis, suggesting an additional function, besides hydrogen metabolism, at least in selected conditions. In this work we generated several Synechocystis mutant strains, deleting individual or combinations of hox and hyp genes, encoding respectively for the hydrogenase subunits and for the proteins involved in the assembly of the active site, and analyzed their phenotype during prolonged, complete darkness in aerobiosis, a growth condition to which cyanobacteria are frequently exposed in nature. We found that the ΔHoxEFUYH deletion mutant strain, which lacks the entire hox operon, has a remarkable reduction in growth when compared to the WT strain. Since the hydrogenase function of the Hox protein is promptly inactivated by oxygen, the observed phenotype should not rely in its YH portion. We explored this hypothesis by knocking out the genes coding for i) the HypA and HypB proteins, involved in the last step of the hydrogenase maturation, ii) the HoxYH portion and iii) the HoxEFU diaphorase portion, and evaluated their behavior under this environmental stress condition. We found that a correctly folded, functional [NiFe]-hydrogenase active site is indeed not essential to confer to Synechocystis the capabilty to face a prolonged darkness. Moreover, preliminary data suggest that the diaphorase moiety could have a specific role in this phenotype, which could shed light on the the hydrogenase expression and function in (selected) aerobic conditions.

[NiFe]-hydrogenase is essential for the cyanobacterium Synechocystis sp PCC 6803 to survive long term darkness.

DE ROSA, EDITH;CHECCHETTO, VANESSA;COSTANTINI, PAOLA
2014

Abstract

Molecular hydrogen is a promising alternative to the limited fossil fuel resources. Hydrogenases, the enzymes involved in hydrogen metabolism, are divided in two major classes: [FeFe]-hydrogenases, which catalyze the reduction of protons to molecular hydrogen, and [NiFe]-hydrogenases, that can both catalyze the oxidation and the reduction of hydrogen. [FeFe]-hydrogenases show specific activity higher than [NiFe]-hydrogenases, nevertheless, in the last few years, the interest for the cyanobacterial [NiFe]-hydrogenase significantly increased. A multisubunits, bidirectional [NiFe]-hydrogenase is present in Synechocystis sp PCC 6803, the most studied cyanobacterial strain. This enzyme possesses five related subunits combined in two functional units: the hydrogenase moiety, i.e. HoxYH, which harbors the active site, and the diaphorase moiety, i.e. HoxEF, likely working as a redox partner for the catalytic subcomplex. Its physiological role is still under debate. Indeed, cyanobacteria lacking the Hox hydrogenase are viable when cultured under standard, physiological conditions, and a general consensus is emerged that this protein may play a role only under transient and/or specific growth conditions. Interestingly, although the enzyme is completely inactive in the presence of oxygen, the Hox protein is constitutively expressed in Synechocystis both in anaerobiosis and aerobiosis, suggesting an additional function, besides hydrogen metabolism, at least in selected conditions. In this work we generated several Synechocystis mutant strains, deleting individual or combinations of hox and hyp genes, encoding respectively for the hydrogenase subunits and for the proteins involved in the assembly of the active site, and analyzed their phenotype during prolonged, complete darkness in aerobiosis, a growth condition to which cyanobacteria are frequently exposed in nature. We found that the ΔHoxEFUYH deletion mutant strain, which lacks the entire hox operon, has a remarkable reduction in growth when compared to the WT strain. Since the hydrogenase function of the Hox protein is promptly inactivated by oxygen, the observed phenotype should not rely in its YH portion. We explored this hypothesis by knocking out the genes coding for i) the HypA and HypB proteins, involved in the last step of the hydrogenase maturation, ii) the HoxYH portion and iii) the HoxEFU diaphorase portion, and evaluated their behavior under this environmental stress condition. We found that a correctly folded, functional [NiFe]-hydrogenase active site is indeed not essential to confer to Synechocystis the capabilty to face a prolonged darkness. Moreover, preliminary data suggest that the diaphorase moiety could have a specific role in this phenotype, which could shed light on the the hydrogenase expression and function in (selected) aerobic conditions.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11577/3040971
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