A screen for potential ferredoxin electron transfer partners uncovers new, redox dependent interactions
ABSTRACT Ferredoxin (Fd) is the primary soluble acceptor at the end of the photosynthetic electron transport chain, and is known to directly transfer electrons to a wide range of proteins for use in metabolism and regulatory processes. We have conducted a screen to identify new putative Fd interaction partners in the cyanobacteria Synechocystis sp. PCC 6803 using Fd-chromatography in combination with MALDI-TOF mass spectrometry. Many novel interactions were detected, including several redox enzymes, which are now candidates for further experiments to investigate electron transfer with Fd. In addition, some proteins with regulatory activity related to photosynthesis were identified. We cloned and expressed one such protein, known as RpaA, which is a specific regulator of energy transfer between phycobilisomes and PSI. Using the recombinant protein we confirmed direct interaction with Fd, and discovered that this was dependent on redox state. The screen for putative Fd-binding proteins was repeated, comparing oxidizing and reducing conditions, identifying many proteins whose interaction with Fd is redox dependent. These include several additional signaling molecules, among them the LexA repressor, Ycf53 and NII, which are all involved in interpreting the redox state of the cell.
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ABSTRACT: In marine systems, nitrate is the major reservoir of inorganic fixed nitrogen. The only known biological nitrate-forming reaction is nitrite oxidation, but despite its importance, our knowledge of the organisms catalyzing this key process in the marine N-cycle is very limited. The most frequently encountered marine NOB are related to Nitrospina gracilis, an aerobic chemolithoautotrophic bacterium isolated from ocean surface waters. To date, limited physiological and genomic data for this organism were available and its phylogenetic affiliation was uncertain. In this study, the draft genome sequence of N. gracilis strain 3/211 was obtained. Unexpectedly for an aerobic organism, N. gracilis lacks classical reactive oxygen defense mechanisms and uses the reductive tricarboxylic acid cycle for carbon fixation. These features indicate microaerophilic ancestry and are consistent with the presence of Nitrospina in marine oxygen minimum zones. Fixed carbon is stored intracellularly as glycogen, but genes for utilizing external organic carbon sources were not identified. N. gracilis also contains a full gene set for oxidative phosphorylation with oxygen as terminal electron acceptor and for reverse electron transport from nitrite to NADH. A novel variation of complex I may catalyze the required reverse electron flow to low-potential ferredoxin. Interestingly, comparative genomics indicated a strong evolutionary link between Nitrospina, the nitrite-oxidizing genus Nitrospira, and anaerobic ammonium oxidizers, apparently including the horizontal transfer of a periplasmically oriented nitrite oxidoreductase and other key genes for nitrite oxidation at an early evolutionary stage. Further, detailed phylogenetic analyses using concatenated marker genes provided evidence that Nitrospina forms a novel bacterial phylum, for which we propose the name Nitrospinae.Frontiers in Microbiology 02/2013; 4:27. DOI:10.3389/fmicb.2013.00027 · 3.94 Impact Factor
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ABSTRACT: Ferredoxins are electron shuttles harboring iron-sulfur clusters which participate in oxido-reductive pathways in organisms displaying very different lifestyles. Ferredoxin levels decline in plants and cyanobacteria exposed to environmental stress and iron starvation. Flavodoxin is an isofunctional flavoprotein present in cyanobacteria and algae (not plants) which is induced and replaces ferredoxin under stress. Expression of a chloroplast-targeted flavodoxin in plants confers tolerance to multiple stresses and iron deficit. We discuss herein the bases for functional equivalence between the two proteins, the reasons for ferredoxin conservation despite its susceptibility to aerobic stress and for the loss of flavodoxin as an adaptive trait in higher eukaryotes. We also propose a mechanism to explain the tolerance conferred by flavodoxin when expressed in plants.FEBS letters 07/2012; 586(18):2917-24. DOI:10.1016/j.febslet.2012.07.026 · 3.34 Impact Factor
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ABSTRACT: Rapid responses of chloroplast metabolism and adjustments to photosynthetic machinery are of utmost importance for plants' survival in a fluctuating environment. These changes may be achieved through post-translational modifications of proteins, which are known to affect the activity, interactions and localization of proteins. Recent studies have accumulated evidence about the crucial role of a multitude of modifications, including acetylation, methylation and glycosylation, in the regulation of chloroplast proteins. Both of the Arabidopsis (Arabidopsis thaliana) leaf-type ferredoxin-NADP(+) oxidoreductase (FNR) isoforms, the key enzymes linking the light reactions of photosynthesis to carbon assimilation, exist as two distinct forms with different isoelectric points. We show that both At FNR isoforms contain multiple alternative N-termini, and undergo light-responsive N(α)-acetylation that causes the change in pI. Both isoforms were also found to contain acetylation of a conserved lysine residue near the active site, while no evidence for in vivo phosphorylation or glycosylation was detected. The dynamic, multilayer regulation of At FNR exemplifies the complex regulatory network systems controlling chloroplast proteins by a range of PTMs, which continues to emerge as a novel area within photosynthesis research.Plant physiology 10/2014; DOI:10.1104/pp.114.249094 · 7.39 Impact Factor