Oxidation–Reduction Properties of Two Engineered Redox-Sensitive Mutant Escherichia coli Malate Dehydrogenases

Setterdahl, Aaron T.; Hirasawa, Masakazu; Bucher, Laura M.; Dholakia, Chirag; Jacquot, Pierre; Yards, Heather; Miller, Fredric; Stevens, Fred J.; Knaff, David B.; Anderson, Louise E.

doi:10.1006/abbi.2000.1981

Cited by 3 publications

(3 citation statements)

References 23 publications

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“…Kinase activity of copper‐replete RegB s was measured at defined ambient redox potentials to determine the oxidation‐reduction midpoint potential of the ‘redox switch’ in RegB s . Individual autophosphorylation assays were performed using samples equilibrated at different defined ambient redox potentials ranging from −230 to −410 mV, in 10 mV increments, prior to kinase activity measurements (Hirasawa et al ., 1998; Krimm et al ., 1998; Setterdahl et al ., 2000). As shown in the plot of percentage activity against ambient redox potential (Figure 2C), the data give an excellent fit to the Nernst equation for a two‐electron process.…”

Section: Resultsmentioning

confidence: 99%

“…Although mBBr is itself weakly fluorescent, a covalent adduct of mBBr with cysteine thiols shows significant fluorescence. Thus, disulfide bond breakage/formation can be directly assayed by measuring the amount of mBBr that covalently binds to RegB s under defined redox conditions (Hirasawa et al ., 1998; Krimm et al ., 1998; Setterdahl et al ., 2000). To measure free thiols in copper‐replete RegB s , aliquots of the protein were first incubated for 2 h at potentials ranging from −230 to −400 mV, in 10 mV increments, and then allowed to react with mBBr.…”

Section: Resultsmentioning

confidence: 99%

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

Swem

2003

The EMBO Journal

117

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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 ®xation and nitrogen ®xation. 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 disul®de bond and that disul®de bond formation is metaldependent, with the metal ful®lling a structural role. Formation of a disul®de 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¯anked 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.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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

Swem

2003

The EMBO Journal

117

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“…Measurement of Disulfide Bond Redox Midpoint Potentials. The redox midpoint potential (E m ) titrations for Yap1-RD, Orp1, Trx1, and Trx2 were carried out by incubation of the protein samples at defined ambient redox potential (E h ) values as described previously (27). Briefly, solutions with defined E h values were prepared using mixtures of either oxidized and reduced glutathione (GSH) or oxidized and reduced dithiothreitol (DTT) (depending upon the E h range), at a total combined concentration of 2 mM.…”

Section: Methodsmentioning

confidence: 99%

Thermodynamic Basis for Redox Regulation of the Yap1 Signal Transduction Pathway

et al. 2006

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The Yap1 oxidative stress signal transduction pathway found in Saccharomyces cerevisiae is redox-regulated. We have examined the thermodynamic basis of the disulfide/dithiol couples that are involved in the regulation of this pathway. The oxidized form of the Yap1 redox domain (Yap1-RD) fragment, derived from the Yap1 transcription factor, contains two disulfide bonds, one between Cys303 and Cys598 and one between Cys310 and Cys629. Oxidation-reduction titrations reveal the presence of two separate two-electron redox couples in Yap1-RD, with redox midpoint potentials (E(m)) of -155 and -330 mV, respectively, at pH 7.0. We measured E(m) values of -275 and -265 mV for the two cytoplasmic S. cerevisiae thioredoxins, Trx1 and Trx2, respectively, both at pH 7.0. Last, we measured an E(m) value of -255 mV for the Cys36-Cys82 disulfide bond at pH 6.0 in the glutathione peroxidase-like enzyme, oxidant receptor protein (Orp1). We were unable to obtain satisfactory redox titration data for Orp1 at pH 7.0, but if the redox-active disulfide of Orp1 exhibits the -59 mV per pH unit dependence for E(m) typical of protein disulfides in this pH region, an E(m) value of -315 mV can be estimated for Orp1 at pH 7.0 by extrapolation. Together, these data suggest that, at physiological ratios of Trx(ox)/Trx(red), the reduction of both the E(m) = -315 mV disulfide of Orp1 and the E(m) = -330 mV disulfide of Yap1 by either Trx1 or Trx2 would be thermodynamically possible.

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Spectroscopic and oxidation–reduction properties of Rhodobacter capsulatus cytochrome c1 and its M183K and M183H variants

Darrouzet

Dhawan

et al. 2002

Biochimica et Biophysica Acta (BBA) - Bioenergetics

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Two variants of the cytochrome c1 component of the Rhodobacter capsulatus cytochrome bc1 complex, in which Met183 (an axial heme ligand) was replaced by lysine (M183K) or histidine (M183H), have been analyzed. Electron paramagnetic resonance (EPR) and magnetic circular dichroism (MCD) spectra of the intact complex indicate that the histidine/methionine heme ligation of the wild-type cytochrome is replaced by histidine/lysine ligation in M183K and histidine/histidine ligation in M183H. Variable amounts of histidine/histidine axial heme ligation were also detected in purified wild-type cytochrome c1 and its M183K variant, suggesting that a histidine outside the CSACH heme-binding domain can be recruited as an alternative ligand. Oxidation-reduction titrations of the heme in purified cytochrome c1 revealed multiple redox forms. Titrations of the purified cytochrome carried out in the oxidative or reductive direction differ. In contrast, titrations of cytochrome c1 in the intact bc1 complex and in a subcomplex missing the Rieske iron-sulfur protein were fully reversible. An Em7 value of -330 mV was measured for the single disulfide bond in cytochrome c1. The origins of heme redox heterogeneity, and of the differences between reductive and oxidative heme titrations, are discussed in terms of conformational changes and the role of the disulfide in maintaining the native structure of cytochrome c1.

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Oxidation–Reduction Properties of Two Engineered Redox-Sensitive Mutant Escherichia coli Malate Dehydrogenases

Cited by 3 publications

References 23 publications

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

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

Thermodynamic Basis for Redox Regulation of the Yap1 Signal Transduction Pathway

Spectroscopic and oxidation–reduction properties of Rhodobacter capsulatus cytochrome c1 and its M183K and M183H variants

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