Identification Of Amino Acid Sites Of NdhO That Bind With Fd And Their Function Analysis In Cyanobacteria

The photosynthetic NDH-1 complex is located on the thylakoid membrane,and is involved in cyclic electron transport around photosystem I(PSI CET)and CO₂uptake.In cyanobacteria,PSI CET mediated by NDH-1(NDH-CET)is an important antioxidant mechanism.Upon exposure of cyanobacterial cells to environmental conditions,the additional ATP produced by NDH-CET optimizes the ATP/NADPH ratio,thereby promoting carbon assimilation,decreasing reactive oxygen speicies(ROS)production and improving photosynthetic efficiency.Our structural and functional data indicated that NDH-1 regulatory subunit NdhO interacts with ferredoxin(Fd)and its absence increases NDH-CET activity.However,the negative mechanism underlying the effect of NdhO on PSI CET remains elusive.Collectively,in this thesis,the key amino acid sites of NdhO that bind with Fd were mutated and their function were analyzed.The main research results are summarized as follows:(1)To test the amino acid sites of NdhO that bind with Fd in Synechocystis sp.strain PCC 6803(hereafter Synechocystis 6803),we found that the NdhO subunit of Synechocystis 6803 is highly homologous with that in Thermosynechococcus elongatus using bioinformatics analysis.Through the analysis of surface electrostatic charge structure mapping,we found that the surface of cyanobacterial NdhO subunit has a positive potential,including two positively charged lysines.(2)To confirm whether the two lysines of NdhO interact with Fd,we constructed a yeast two-hybrid expression vector with point mutations of these two lysines.Our results of yeast two-hybrid experiment indicated that NdhO did not interact with Fd,after these two lysines(K6 and K7)of NdhO were replaced into aspartate(E6 and E7).This suggests that these two amino acids are necessary for the interaction of NdhO with Fd in cyanobacteria.(3)To investigate whether these two amino acid sites of NdhO that interact with Fd affect the function of NDH-1,we constructed their amino acid point mutants,including two single point mutants ndhO^?K6E and ndhO^?K7E and,a double-point mutation ndhO^?K/E.The results of DNA sequencing indicated that these two lysines(K6 and K7)of NdhO have been replaced into aspartate(E6 and E7).(4)To examine whether mutations of these two amino acids of NdhO influenced the function of NDH-1 further,the chlorophyll fluorescence method was used to analyze the NDH-CET activity of these ndhO^?K6E,ndhO^?K7E and ndhO^?K/E mutants.As expected,under conditions of growth and short-term high light,NDH-CET activities of ndhO^?K6E,ndhO^?K7E,and ndhO^?K/E mutants were increased when compared to the WT.These results indicated that mutations of these two amino acids of NdhO affect the function of NDH-1 in cyanobacteria.Collectively,this thesis uncovered the electrostatic interaction between NdhO and Fd and found that mutations of these two lysine to aspartate enhanced the NDH-CET activity,indicating the key amino acid sites for the interaction of NdhO with Fd.As a consequence,this thesis will provide relevant theoretical basis for uncovering the negative mechanism underlying the effect of NdhO on PSI CET.