This study examines the impact of reactive oxygen species on enzymatic activity of the chloroplast ATP synthase in situ and in vitro. Isolated spinach thylakoids were shown to be less catalytically active upon treatment with singlet oxygen. It was demonstrated that exposed thylakoids lost their capacity to synthesize and hydrolyze ATP and to acidify the thylakoid lumen in an ATP-dependent manner. A central element of the study was the identification of specific target sites which could be solved in the course of the project. Structural predictions upon enzymatic redox-regulation served as a first platform for this approach. Purification of soluble catalytic CF1 protein revealed that ATPase activity in vitro attenuated by singlet oxygen in a comparable manner to the reaction in situ. With the help of a stepwise disassembly approach it could be shown that initially suggested inter-subunit cross-links between regulatory subunits were not responsible for singlet oxygen-induced loss of activity. Instead, the CF1 gamma subunit seemed to be a promising candidate that harbored functional targets responsible for activity attenuation. In silico analysis proposed a gamma subunit methionine-cysteine cluster to form a highly conserved set of potential targets of various reactive oxygen species. Further mass spectrometry analysis revealed that these residues were susceptible to singlet oxygen and hydrogen peroxide. Several point mutations within in the cluster were analyzed using a recombinant photosynthetic F-ATP synthase assembly system. In an extensive biochemical mutant characterization screen it could be demonstrated that some mutants displayed an aberration of catalytic properties, such as MgADP binding propensity and activity regulation by the gamma subunit redox state. The cluster residues were ascribed to mutually interact while having an effect on remote functional domains within the enzyme. Finally, it could be shown that oxidation of the cluster was responsible for hydrogen peroxide-induced activity attenuation. It is very likely that additional residues participate in singlet oxygen-dependent loss of activity.
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