A New Mechanism For Stabilizing The Structure Of NDH-1L Complex In Cyanobacteria

Cyanobacterial NDH-1 complex is located in the thylakoid membrane,which is involved in a series of important bioenergetic reactions,such as cyclic electron transport around photosysterm I(NDH-CET)and cellular respiration.The structure of NDH-1 complex is homologous to that of NADH quinone oxidoreductase(also known as Complex I)in Escherichia coli,both of them conserved a "L" shape skeleton,this skeleton included a hydrophilic arm and a hydrophobic arm.However,during the process of evolution,there are many oxygenic photosynthesis-specific subunits have been added in the membrane arm of Cyanobacterial NDH-1 L complex,such as NdhP and NdhQ,they jointly stabilize the NDH-1 L complex.In addition,the C-terminal segment of NuoL subunit of Escherichia coli NDH-1 complex was also identified as the key to stabilized the structure of this complex.In the case of new subunits occur in Cyanobacterial NDH-1 L,the function of NdhF1 subunit,which is homologous to the Escherichia coli NuoL subunit,might has been changed.According to previous studies,the carboxyl terminus of NdhP subunit is involving in stabilization of NDH-1 L complex.In this study,we found that the absence of C-terminal segment of NdhF1 subunit did not lead to the complete collapse of NDH-1 L complex,but the deletion of both the NdhF1 C-terminal segment and that of NdhP can severely disrupted the NDH-1 L complex.The results are summarized as follows:(1)We constructed the ndhF1 C-terminal knock-out mutant(ndhF1?C)of glucose tolerant strain Synechcoystis sp.PCC 6803,and separating the membrane protein complexes of ndhF1?C by blue native electrophoresis(BN-PAGE).Through western blot analysis,we found that the amount of NDH-1 L complex in thylakoid membrane was reduced more than half of the accumulation of NDH-1 L complex in WT,and similar to the result observed in ndhFl knock-out mutant.(2)We further deleted the C-terminal segment of NdhP in ndhF1?C,and the result of BN-PAGE and Western blot analysis show that the amount of NDH-1 L complex in ndhF1?C/ndhP?C significantly reduced to a very low level comparing with WT,and similar to the result observed in ndhFl/ndhP double knock-out mutant(OndhF1/?ndhP).(3)In order to confirm the effect of NdhF1 C-terminal deletion on the stability of NDH-1 L,we also detected the chlorophyll fluorescence,and the redox state of photosystem I reaction center P700,as well as cellular respiration activity of mutants.Results show that ndhF1 C-terminal deletion decreased the activity of cyclic electron transprot around photosystem I and cellular respiration,demonstrating that the absence of NdhF1 C-terminal segment reduced the stability of NDH-1Lthus impaired the function of NDH-1L.By the way,NuoL C-terminal deletion only affects cellular respiration activity,this result indicating that the functional role of NdhF1 C-terminal segment is different from that of NuoL C-terminal segment.In summary,this study found that the C-terminal segment of NdhFl is required for stabilizing the structure of NDH-1L complex,but the disassembly of NDH-1L complex in ndhF1?C was different from the result observed in bacterial NuoL C-terminal knock-out mutant,indicating that the C-terminal segment of NdhFl is not the only factor required for stabilizing NDH-1L complex.In addition to the impairment of cellular respiration,deleting the C-terminal segment of NdhFl also affects the activity of cyclic electron transport around photosystem I,which indicates that the structural and functional role of NdhF1 is different from that of NuoL.While deleting both the C-terminal segments of NdhF1 and NdhP lead to the result that the amount of NDH-1L complex has been significantly reduced,but there are still a few NDH-1L complex remained in the thylakoid membrane,it is possible that the NdhQ subunit still stabilized the NDH-1L in ndhF1?C/ndhP?C.Therefore,we suggest that both the C-terminal segments of NdhFl and NdhP are involved in the formation of a new mechanism for stabilizing the structure of cyanobacterial NDH-1L complex,which is different from that of bacteria.