Identification And Functional Analysis Of An NDH-PSI Supercomplex In Cyanobacteria

Light is required to drive oxygenic photosynthesis, and provides energy for all photosynthetic organisms. However, the change of natural environment and the interference of human activities cause a series of stresses, such as high light, high temperature and drought. These stress environmental factors, directly or indirectly, suppress the ability of CO2 assimilation, and consequently, decrease the efficiency of photosynthesis and damage the photosynthetic apparatus. To adapt to these stress conditions and to improve the efficiency of light energy utilization and the rate of photosynthetic electron transport, numerous mechanisms have been developed. Cyclic electron transfer(CET) plays a particularly important role in coping with various environmental stresses by building transmembrane proton gradient(Δp H) and generating additional ATP, thereby improving photosynthesis.NADPH dehydrogenase(NDH-1)-dependent CET(NDH-CET) has an important role in resisting environmental stresses, such as high light, in cyanobacteria. NDH-1 complex is involved in a variety of bioenergetic reactions, including respiration, NDH-CET and CO2 uptake. Structurally, the cyanobacterial NDH-1 complex closely resembles energy-converting complex I in eubacteria and the mitochondrial respiratory chain regardless of absence of homologues of three subunits in cyanobacterial genomes that constitute the catalytically active core of complex I. Recent studies showed that the C-terminal region of Ndh S subunit may form a tertiary structure similar to that of Psa E and is reinforced by the Src homology 3 domain that is involved in protein-protein interactions. It appears plausible that NDH-1 may interact with PSI, thereby assisting in the transfer of electons from Fd to NDH-1 complex. Therefore, Fd may be a potential electron donor for NDH-1 complex, although NADPH is suggested to be also electron donor for NDH-1 complex. Nonetheless, the interaction of NDH-1 with PSI has not been identified in cyanobacteria and whether Fd is an electron donor for cyanobacterial NDH-1 complex remains a subject of debate.In this study, we successfully identified a novel NDH-PSI supercomplex with a molecular mass of more than 1,000 k Da in Synechocystis sp. strain PCC 6803(Synechocystis 6803) by using blue native(BN)-PAGE, which gives the biochemical evidence for Fd as an electron donor of cyanobacterial NDH-1 enzyme. Major results are summarized as follows:(1) Two slow growth mutants at high light conditions were isolated and identified from Synechocystis 6803 transformed with a transposon-bearing library. Both mutants had a tag in fg1 by PCR analysis. Deletion of fg1 impaired NDH-CET activity and destablized the NDH-1 complexes. Therefore, we successfully obtained an NDH-CET activity impaired mutant.(2) Biochemical analyses of WT and mutants. A green band above 1,000 k Da(band I) was found in WT but was absent when fg1 was deleted. Further researches showed that band I was absent in M55 and PSI-less mutant strains. These results suggested that NDH-1 and PSI form an NDH-PSI supercomplex(band I) via Fg1 protein. To test this hypothesis, we analyzed the components of the band I by using immunoblot and mass spectrometry analyses. The results showed that band I contains the components of NDH-1, Fg1 and PSI complexes that may exist as a ratio of 1:2:8. These results indicated that the absence of Fg1 destabilized the supercomplex, thereby impairing the NDH-CET activity.(3) NDH-PSI supercomplex involved in NDH-CET. Band I was not visible or very faint in the mutants defective in NDH-CET, such as M55, ?ndh D1/D2, M9, ?ndh S and OX-ndh O. This suggested that formation of NDH-PSI supercomplex may be essential for the activity of NDH-1 complex.In conclusion, we successfully identified a novel NDH-PSI supercomplex in the cyanobacterium Synechocystis 6803 and Fg1 protein is required to form the supercomplex involved in NDH-1CET. Therefore, we give the biochemical evidences that Fd may be an electron donor of cyanobacterial NDH-1 enzyme. The results suggested that cyanobacterial NDH-1 accepts electrons from Fd and the formation of NDH-PSI supercomplex facilitates the transfer of electrons from PSI to NDH-1 via reduced Fd, thereby ensuring a high catalytic activity of the enzyme in light.