Electron transfer and hydrogen production: identification of three new essential subunits
In the chloroplastic membranes of higher plants, the complex Ndh, an analogue of mitochondrial respiratory complex I, catalyses the entry of electrons in a cyclic transfer pathway around photosystem I. This pathway has proved to be most important in conditions of environmental stress. In cyanobacteria, the ancestors of the photosynthesis organelles (chloroplasts), the operation of this enzyme complex is closely correlated with hydrogen bioproduction capacity.
On the basis of analogies with the minimal respiratory complex I of bacteria, which has 14 subunits, 11 subunits have been characterised in the chloroplasts of Ndh, pointing to the probable existence of others, in particular subunits responsible for the catalytic activity of the complex NAD(P)H plastoquinone oxidoreductase.
Owing to their small quantity (0.2% of the total membrane proteins of the plasts) and very low stability, any characterisation of the complex Ndh is impossible without rapid and efficient methods of purification.
Drawing on its expertise in chloroplast transformation methods and its skills in membrane protein purification, the Photosynthesis Ecophysiology Laboratory (DEVM, CEA Cadarache) has successfully isolated the complex Ndh. To do this, the gene (located in the chloroplast) of one of the subunits of the complex, the subunit NDH-H, was labelled with a sequence favouring its purification by affinity (Tag-histidine). The subunits thereby obtained were characterised by mass spectrometry at the Protein Chemistry Laboratory (DRDC, CEA Grenoble). Three new subunits coded by the nuclear genome were identified in this way. These three subunits are essential for the functional integrity of the complex: mutants of Arabidopsis thaliana in which the genes coding for the three subunits are inhibited no longer possess a functional Ndh complex.
The presence of genes orthologous to respiratory complex I in cyanobacteria raises the question of the role of the complex Ndh in the process of hydrogen bioproduction, which in one of these photosynthetic bacteria (Synechocystis), has been shown to depend closely on its activity. In the photosynthetic electron transfer pathways in cyanobacteria, the NADPH produced can be re-oxidised either via the complex Ndh or via a Ni-Fe hydrogenase activity. These two activities compete for the same substrate, and it can be shown that mutants of cyanobacteria lacking complex Ndh produce more H2 than their wild counterparts.
(Figure 2 : from)