Research On Ferredoxin/Cytochrome B₆f Pathway Of Cyclic Electron Transport Chain

There exists cyclic electron transport/flow（CET）in photosynthesis,whose function is to provide additional ATP to ensure plant resistance to adversity under adverse conditions.At present,it is widely believed that plants have two CETs:one dependent on NADPH dehydrogenase（NDH）for cyclic electron transfer（NDH-CET）and the other dependent on PGR5/PGRL1-CET for cyclic electron transfer（PGR5/PGRL1-CET）.However,recent studies have also suggested that the above two pathways are not the main pathways of CET and that Fd/Cyt b₆f-CET is likely to be the main pathway of CET.In order to study the Fd/Cyt b₆f-CET pathway,firstly,the Cyt b₆f（cytochrome b₆f,Cyt b₆f）protein complex was isolated and purified from spinach chloroplasts by an improved protocol;Secondly,the heterologous expression of spinach Ferredoxin（Ferredoxin,Fd）and Fd-NADP⁺reductase（Ferredoxin NADP⁺reductase,FNR）in Escherichia coli were by using molecular biology methods and plastocyanin（PC）was isolated and purified from spinach chloroplasts by using biochemistry means.And the electron transport activity of Cyt b₆f using Fd（or Fd-FNR complex）as substrate,namely the Fd-PC oxidoreductase activity of Cyt b₆f,was identified by spectroscopy under different enzymatic reaction conditions in vitro.Finaly,the interaction of Fd,FNR and Cyt b₆f were studied in vitro using cross-linking agents in an attempt to obtain a protein complex of the three interacting proteins.The main research results are as follows:（1）Optimisation of a method for the isolation and purification of the spinach Cyt b₆f protein complex by removing the sucrose density gradient centrifugation process,and obtaining a highly purified Cyt b₆f sample for follow-up studies by ion exchange chromatography.（2）Water-soluble electron transport proteins Fd,FNR and PC involved in photosynthetic electron transport chains were heterologous expressed by molecular biological methods or biochemistry means.Simulating the in vivo environment,it was determined in vitro that the FNR-mediated transfer of electrons from NADPH to Fd could be catalyzed.The electron transfer activity of Fd to Cyt b₆f（Cyt b _H）was verified.Also,it was confirmed that only when NADPH,FNR and Fd were present at the same time could Cyt b _H be reduced.Finally,it is further verified that PC can obtain electrons from Cyt b _H.According to the above results,the electron transport pathway of is deduced as follow:NADPH→FNR→Fd→（Heme ci→Cyt b _H）→Cyt b₆f internal PQ→ISP→Cyt f→PC.（3）Based on the above studies,an in vitro cross-linking approach was attempted to probe the interaction of Fd,FNR and Cyt b₆f.By EDC crosslinking,it was determined that a new band appeared at about 57 k D,which were crosslinked products of FNR and Fd proteins according to molecular weight analysis.Glutaraldehyde cross-linking showed that a new band appeared at 37 k D,suggesting that the subunit IV of Cyt b₆f was cross-linked with ISP.It was further found that under the same concentration of glutaraldehyde,the crosslinked bands of subunit IV and ISP became weaker after the addition of FNR and on this basis,the crosslinked bands became weaker after the addition of Fd,which suggests that FNR and Fd affected the structure of Cyt b₆f and changed the degree of tight binding of subunit IV and ISP.In summary,it was demonstrated in vitro that FNR could catalyze electron transfer from NADPH to Fd,and the electron transfer activity of Fd to Cyt b₆f was determined.It was verified that the reduction of Cyt b _H requires the participation of NADPH,FNR and Fd simultaneously.Further confirming that PC can obtain electrons from Cyt b _H,and the Fd/Cyt b₆f-CET pathway was verified in vitro.Through in vitro assembly,it was determined that FNR and Fd could cross-link and FNR and/or Fd can weaken the cross-linking effect between the Cyt b₆f subunit IV and ISP,and affect the structure of Cyt b₆f,indirectly indicating that FNR and Fd interact with Cyt b₆f.