藍綠藻 Synechocystis PCC 6803中光系統二細胞色素b559的結構與功能研究

Abstract

本研究使用H22Kα以及Y18Sα 細胞色素b559的藍綠藻Synechocystis sp. PCC6803突變株研究細胞色素b559在光系統二的生理功能。 這兩個突變株皆保有正常的光合作用釋放氧氣的能力，且能合成穩定的光系統二進行光自營生長。然而，這兩突變株相較於野生株更容易受到光抑制。 EPR的結果顯示從H22Kα突變株所純化的光系統二反應中心細胞色素b559失去與血紅素軸心的一側鍵結。 這兩個突變株光系統二反應中心的細胞色素b559主要為低氧化還原電位型態，這個發現支持了血紅素的鍵結環境會強烈影響細胞色素b559氧化還原特性的概念。我們的研究結果支持了細胞色素b559具有在光抑制條件下保護光系統二的功能。此外，我們也進一步探討這兩個細胞色素b559突變株是否更需要光系統二的修復機制。 在高光照的生長條件下這兩突變株都累積了更多的FtsH-PSII super-complex，並且顯著的減少了光系統二的含量，且H22Kα/ΔFtsH2及Y18Sα/ΔFtsH2雙重突變株皆顯著的失去光合作用和光自營生長的能力。我們的研究結果顯示在這兩個細胞色素b559突變株細胞中，FtsH2扮演了重要的修復角色以維持這兩個細胞色素b559突變株細胞中光系統二的正常功能。

Part I: The functional role of Cyt b559 in PSII was investigated in H22Kα and Y18Sα Cyt b559 mutants of the cyanobacterium Synechocystis sp. PCC6803. H22Kα and Y18Sα Cyt b559 mutant carries one amino acid substitution on and near one of heme axial ligands of Cyt b559 in PSII, respectively. Both mutants grew photoautotrophically, assembled stable PSII, and exhibited the normal period-four oscillation in oxygen yield. However, both mutants showed several distinct chlorophyll a fluorescence properties and were more susceptible to photoinhibition than wild type. EPR results indicated the displacement of one of the two axial ligands to the heme of Cyt b559 in H22Kα mutant reaction centers, at least in isolated reaction centers. The maximum absorption of Cyt b559 in Y18Sα mutant PSII core complexes was shifted to 561 nm. Y18Sα and H22Kα mutant PSII core complexes contained predominately the low potential form of Cyt b559. The findings lend support to the concept that the redox properties of Cyt b559 are strongly influenced by the hydrophobicity and ligation environment of the heme. When the Cyt b559 mutations placed in a D1-D170A genetic background that prevents assembly of the manganese cluster, accumulation of PSII is almost completely abolished. Overall, our data support a functional role of Cyt b559 in protection of PSII under photoinhibition conditions in vivo. Part II: The physiological function of cytochrome (Cyt) b559 in photosystem II (PSII) was investigated using H22Kα and Y18Sα Cyt b559 mutants of the cyanobacterium Synechocystis sp. PCC6803. Both mutant cells grew photoautotrophically and assembled stable PSII-like wild-type cells under normal light conditions (light intensity about 30 μmol photons m-2 s-1). However, both mutant cells suffered from photoinhibition under high light conditions (light intensity about 200 μmol photons m-2 s-1). Western blot analysis showed that the FtsH-PSII super-complex was significantly increased but PSII sub-complexes (PSII/RC47 heterodimer, PSII monomer and RC47 sub-complexes) were largely decreased in both Cyt b559 mutant cells under high light conditions. Our results suggest that Cyt b559 is important for maintaining normal photosynthetic growth and PSII activity in Synechocystis sp. PCC6803 cells under light stress conditions. In addition, H22Kα/ΔftsH2 and Y18Sα/ΔftsH2 double mutant cells showed severe impairments in their photosynthetic growth and PSII activities. Thylakoid membranes of the H22Kα/ΔftsH2 double mutant showed significant reductions in D1, D2 and Cyt b559 α protein contents compared to those of H22Kα mutants. In contrast, thylakoid membranes of ΔftsH2 mutant showed significant increases in PSII protein contents compared to those of wild-types. Our results demonstrate that FtsH2 protease may plays an important role in repairing PSII and maintaining its function in Synechocystis Cyt b559 mutant cells.