Study On The Photoprotection Mechanism Of The Chlorophyll A In The Cytochrome B₆f Complex

The Cytochrome b6f complex (cyt b6f) is one of the three key integral membraneprotein complexes. Cytochrome b6f complex（Cyt b6f）mediates electron transfer fromphotosystem II to photosystem I in the linear photosynthetic electron transfer pathwayfrom H2O to NADP+and contributes to the electrochemical proton gradient generatedacross the membrane that is used for ATP synthesis. With the exception of the eightprotein subunits, the complex also contains only one chlorophyll a (Chl a) and onecarotenoid molecule per monomer. It is reported that the Chl a in the complex had a stableenvironment. The singlet excited state life time and the fluorescence quantum yield of theChl a in the cytochrome b6f complex were～200ps and～1.8%, compared to the5～6nsand20%～30%fluorescence quantum yield for monomeric Chl a molecule in methanol. Itis proposed that the Chl a singlet excited state lifetime is quenched by the subtlemicro-environment, that shortens the singlet excited state lifetime of the Chl a and reducesthe formation of the Chl a triplet state and subsequent singlet oxygen. However, there isnot any evidence to support the mechanism still.The crystal structure of Cyt b6f at a2.7resolution[1]shows that the magnesium ionof the Chl a is coordinated by a water molecular which reacts with G136and T137of thesubunit IV by hydrogen bonds. Based the structure,1）the site-directed mutagenesis wasused to construct the mutants at G136and T137of subunit IV of Cyt b6f complex inSynechocystis sp. PCC6803;2) the Cyt b6f preparations from the wild and mutants wereprepared;3) the photostabilities of the Chl a in the preparations were investigated andcompared through the photodamage experiment and time resolved spectrometry (TRS).The results are as follows:1) The three mutants, two single mutants G136A and T137A, and a double mutantG136A/T137A, in subunit IV of Cyt b6f were screened and identified;2) The four Cyt b6f preparations, one from wild and three from mutants, wereseparated and purified by the means of biochemical methods;3) The results of photodamage experiment show not only the affinity weredecreased between the Chl a and their micro-environment, but also the stability of Chl aagainst photodamage also decreased for the preprarations from the mutant preparations.The Chl a in the Cyt b6f was wild>T137A>G136A>G136A/T137A in descending order of stability. And results from TRS show the fluorescence lifetime of Chl a from the wild was240ps, which was very consistent with the previous reports, while the lifetime from thedouble mutant was~470ps, which is2times longer than that of the wild.These results mentioned above showed disturbing the hydrogen network form by theamino acids G136, T137in subunit IV and the Chl a was important to maintain the highphotostability of the Chl a in the complex.