Abstract
Ferredoxin:thioredoxin reductase (FTR) is a key regulatory enzyme of oxygenic photosynthetic cells involved in the reductive regulation of important target enzymes. It catalyzes the two-electron reduction of the disulfide of thioredoxins with electrons from ferredoxin involving a 4Fe-4S cluster and an adjacent active-site disulfide. We replaced Cys-57, Cys-87, and His-86 in the active site of Synechocystis FTR by site-directed mutagenesis and studied the properties of the mutated proteins. Mutation of either of the active-site cysteines yields inactive enzymes, which have different spectral properties, indicating a reduced Fe-S cluster when the inaccessible Cys-87 is replaced and an oxidized cluster when the accessible Cys-57 is replaced. The oxidized cluster in the latter mutant can be reversibly reduced with dithionite showing that it is functional. The C57S mutant is a very stable protein, whereas the C87A mutant is more labile because of the missing interaction with the cluster. The replacement of His-86 greatly reduces its catalytic activity supporting the proposal that His-86 increases the nucleophilicity of the neighboring cysteine. Ferredoxin forms non-covalent complexes with wild type (WT) and mutant FTRs, which are stable except with the C87A mutant. WT and mutant FTRs form stable covalent heteroduplexes with active-site modified thioredoxins. In particular, heteroduplexes formed with WT FTR represent interesting one-electron-reduced reaction intermediates, which can be split by reduction of the Fe-S cluster. Heteroduplexes form non-covalent complexes with ferredoxin demonstrating the ability of FTR to simultaneously dock thioredoxin and ferredoxin, which is in accord with the proposed reaction mechanism and the structural analyses.
Highlights
Mechanism, which enables oxygenic photosynthetic cells to switch between light and dark metabolism and to adapt to changes in light intensity [1, 2]
Production and Purification—The wild type (WT) and mutant Synechocystis Ferredoxin:thioredoxin reductase (FTR) were well expressed in E. coli and could be purified by our standard procedure except for the C87S mutant
Spectral Characterization—WT FTR has a typical UV-visible absorbency spectrum, which is characteristic of the 4Fe-4S cluster in its 2ϩ redox state and its interactions with the redox-active disulfide bridge [16]
Summary
Mechanism, which enables oxygenic photosynthetic cells to switch between light and dark metabolism and to adapt to changes in light intensity [1, 2]. WT and mutant FTRs form stable covalent heteroduplexes with active-site modified thioredoxins. Heteroduplexes formed with WT FTR represent interesting one-electron-reduced reaction intermediates, which can be split by reduction of the Fe-S cluster.
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