Aaron T. Setterdahl

Signal transduction by the global regulator RegB is mediated by a redox-active cysteine

All living organisms alter their physiology in response to changes in oxygen tension. The photosynthetic bacterium uses the RegB–RegA signal transduction cascade to control a wide variety of oxygen‐responding processes such as respiration, photosynthesis, carbon fixation and nitrogen fixation. We demonstrate that a highly conserved cysteine has a role in controlling the activity of the sensor kinase, RegB. In vitro studies indicate that exposure of RegB to oxidizing conditions results in the formation of an intermolecular disulfide bond and that disulfide bond formation is metal‐dependent, with the metal fulfilling a structural role. Formation of a disulfide bond in vitro is also shown to convert the kinase from an active dimer into an inactive tetramer state. Mutational analysis indicates that a cysteine residue flanked by cationic amino acids is involved in redox sensing in vitro and in vivo. These residues appear to constitute a novel ‘redox‐box’ that is present in sensor kinases from diverse species of bacteria.

Repression of photosynthesis gene expression by formation of a disulfide bond in CrtJ

Many species of purple photosynthetic bacteria repress synthesis of their photosystem in the presence of molecular oxygen. The bacterium Rhodobacter capsulatus mediates this process by repressing expression of bacteriochlorophyll, carotenoid, and light-harvesting genes via the aerobic repressor, CrtJ. In this study, we demonstrate that CrtJ forms an intramolecular disulfide bond in vitro and in vivo when exposed to oxygen. Mutational and sulfhydryl-specific chemical modification studies indicate that formation of a disulfide bond is critical for CrtJ binding to its target promoters. Analysis of the redox states of aerobically and anaerobically grown cells indicates that they have similar redox states of approximately −200 mV, thereby demonstrating that a change in midpoint potential is not responsible for disulfide bond formation. In vivo and in vitro analyses indicate that disulfide bond formation in CrtJ is insensitive to the addition of hydrogen peroxide but is sensitive to molecular oxygen. These results suggest that disulfide bond formation in CrtJ may differ from the mechanism of disulfide bond formation used by OxyR.

Involvement of SenC in Assembly of Cytochrome c Oxidase in Rhodobacter capsulatus

SenC, a Sco1 homolog found in the purple photosynthetic bacteria, has been implicated in affecting photosynthesis and respiratory gene expression, as well as assembly of cytochrome c oxidase. In this study, we show that SenC from Rhodobacter capsulatus is involved in the assembly of a fully functional cbb(3)-type cytochrome c oxidase, as revealed by decreased cytochrome c oxidase activity in a senC mutant. We also show that a putative copper-binding site in SenC is required for activity and that a SenC deletion phenotype can be rescued by the addition of exogenous copper to the growth medium. In addition, we demonstrate that a SenC mutation has an indirect effect on gene expression caused by a reduction in cytochrome c oxidase activity. A model is proposed whereby a reduction in cytochrome c oxidase activity impedes the flow of electrons through the respiratory pathway, thereby affecting the oxidation/reduction state of the ubiquinone pool, leading to alterations of photosystem and respiratory gene expression.

Cytosolic, Mitochondrial Thioredoxins and Thioredoxin Reductases in Arabidopsis Thaliana

Thioredoxins, by reducing disulfide bridges are one of the main participants that regulate cellular redox balance. In plants, the thioredoxin system is particularly complex. The most well-known thioredoxins are the chloroplastic ones, that participate in the regulation of enzymatic activities during the transition between light and dark phases. The mitochondrial system composed of NADPH-dependent thioredoxin reductase and type o thioredoxin has only recently been described. The type h thioredoxin group is better known. Yeast complementation experiments demonstrated that Arabidopsis thaliana thioredoxins h have divergent functions, at least in Saccharomyces cerevisiae. They have diverse affinities for different target proteins, most probably because of structural differences. However, plant thioredoxin h functions still have to be defined.

Activity of the tetrapyrrole regulator CrtJ is controlled by oxidation of a redox active cysteine located in the DNA binding domain

CrtJ from Rhodobacter capsulatus is a regulator of genes involved in the biosynthesis of haem, bacteriochlorophyll, carotenoids as well as structural proteins of the light harvesting‐II complex. Fluorescence anisotropy‐based DNA‐binding analysis demonstrates that oxidized CrtJ exhibits ∼ 20‐fold increase in binding affinity over that of reduced CrtJ. Liquid chromatography electrospray tandem ionization mass spectrometric analysis using DAz‐2, a sulfenic acid (–SOH)‐specific probe, demonstrates that exposure of CrtJ to oxygen or to hydrogen peroxide leads to significant accumulation of a sulfenic acid derivative of Cys420 which is located in the helix–turn–helix (HTH) motif. In vivo labelling with 4‐(3‐azidopropyl)cyclohexane‐1,3‐dione (DAz‐2) shows that Cys420 also forms a sulfenic acid modification in vivo when cells are exposed to oxygen. Moreover, a Cys420 to Ala mutation leads to a ∼ 60‐fold reduction of DNA binding activity while a Cys to Ser substitution at position 420 that mimics a cysteine sulfenic acid results in a ∼ 4‐fold increase in DNA binding activity. These results provide the first example where sulfenic acid oxidation of a cysteine in a HTH‐motif leads to differential effects on gene expression.

Regulation of Gene Expression in Response to Oxygen Tension

Purple photosynthetic bacteria control numerous energy-generating and energy-utilizing processes in response to alterations in cellular redox, which is affected by environmental oxygen tension. The list of redox-regulated events includes synthesis of the pigmented and cytochrome components of the photosystem, enzymes for fixation of carbon and nitrogen, the synthesis of several terminal respiratory electron transport complexes, and synthesis of the energy-generating hydrogenase complex. Regulating synthesis of these components involves several well-characterized transcription factors including the sensor kinase RegB and its cognate response regulator RegA. Other redox-responding regulators include CrtJ and Fnr. Mechanisms of redox sensing by these transcription factors are discussed.

The role of tryptophan in the ferredoxin-dependent nitrite reductase of spinach

A system has been developed for expressing a His-tagged form of the ferredoxin-dependent nitrite reductase of spinach in Escherichia coli. The catalytic and spectral properties of the His-tagged, recombinant enzyme are similar, but not identical, to those previously observed for nitrite reductase isolated directly from spinach leaf. A detailed comparison of the spectral, catalytic and fluorescence properties of nitrite reductase variants, in which each of the enzyme’s eight tryptophan residues has been replaced using site-directed mutagenesis by either aromatic or non-aromatic amino acids, has been used to examine possible roles for tryptophan residues in the reduction of nitrite to ammonia catalyzed by the enzyme.