APS reductase from Pseudomonas aeruginosa has been shown to contain a [4Fe-4S] cluster. Thiol determinations and site-directed mutagenesis studies indicate that the single [4Fe-4S] cluster contains only three cysteine ligands, instead of the more typical arrangement in which clusters are bound to the protein by four cysteines. Resonance Raman studies in the Fe-S stretching region are also consistent with the presence of a redox-inert [4Fe-4S](2+) cluster with three cysteinate ligands and indicate that the fourth ligand is likely to be an oxygen-containing species. This conclusion is supported by resonance Raman and electron paramagnetic resonance (EPR) evidence for near stoichiometric conversion of the cluster to a [3Fe-4S](+) form by treatment with a 3-fold excess of ferricyanide. Site-directed mutagenesis experiments have identified Cys139, Cys228, and Cys231 as ligands to the cluster. The remaining two cysteines present in the enzyme, Cys140 and Cys256, form a redox-active disulfide/dithiol couple (E(m) = -300 mV at pH 7.0) that appears to play a role in the catalytic mechanism of the enzyme.

Oxidation-reduction midpoint potential (E(m)) versus pH profiles were measured for wild-type thioredoxins from Escherichia coli and from the green alga Chlamydomonas reinhardtii and for a number of site-directed mutants of these two thioredoxins. These profiles all exhibit slopes of approximately -59 mV per pH unit, characteristic of the uptake of two protons per reduction of an active-site thioredoxin disulfide, at acidic, neutral, and moderately alkaline pH values. At higher pH values, these profiles exhibit slopes of either -29.5 mV per pH unit, characteristic of the uptake of one proton per disulfide reduced, or are pH-independent, indicating that neither proton uptake nor proton release is associated with reduction of the active-site disulfide. Reduction of the two wild-type thioredoxins is accompanied by the uptake of two protons even at pH values where the more acidic cysteine thiol group of the reduced proteins would be expected to be completely unprotonated. The effect of site-directed mutagenesis of two highly conserved aspartate residues that play important structural and/or catalytic roles in both thioredoxins, and which could in principle play a role in proton transfer, on the pK(a) values of redox-linked acid dissociations (deduced from changes in slope of the E(m) versus pH profiles) has also been determined for both E. coli thioredoxin and C. reinhardtii thioredoxin h.

APS reductase from Pseudomonas aeruginosa has been shown to form a disulfide-linked adduct with mono-cysteine variants of Escherichia coli thioredoxin and Chlamydomonas reinhardtii thioredoxin h1. These adducts presumably represent trapped versions of the intermediates formed during the catalytic cycle of this thioredoxin-dependent enzyme. The oxidation-reduction midpoint potential of the disulfide bond in the P. aeruginosa APS reductase/C. reinhardtii thioredoxin h1 adduct is -280 mV. Site-directed mutagenesis and mass spectrometry have identified Cys256 as the P. aeruginosa APS reductase residue that forms a disulfide bond with Cys36 of C. reinhardtii TRX h1 and Cys32 of E. coli thioredoxin in these adducts. Spectral perturbation measurements indicate that P. aeruginosa APS reductase can also form a non-covalent complex with E. coli thioredoxin and with C. reinhardtii thioredoxin h1. Perturbation of the resonance Raman and visible-region absorbance spectra of the APS reductase [4Fe-4S] center by either APS or the competitive inhibitor 5'-AMP indicates that both the substrate and product bind in close proximity to the cluster. These results have been interpreted in terms of a scheme in which one of the redox-active cysteine residues serves as the initial reductant for APS bound at or in close proximity to the [4Fe-4S] cluster.

Redox properties of the photosynthetic gene repressor PpsR and the blue-light photoreceptor/antirepressor AppA from Rhodobacter sphaeroides have been characterized. Redox titrations of PpsR reveal the presence of a two-electron couple, with an E (m) value of -320 mV at pH 7.0, which is likely to arise from the reversible conversion of two cysteine thiols to a disulfide. This E (m) value is very much more negative than the E (m) = -180 mV value measured previously at pH 7.0 for the disulfide/dithiol couple in CrtJ, the homolog for PpsR in the closely related bacterium Rhodobacter capsulatus. AppA, a flavin-containing blue-light receptor that is also involved in the regulation of gene expression in R. sphaeroides, contains multiple cysteines in its C-terminal region, two of which function as a redox-active dithiol/disulfide couple with an E (m) value of -325 mV at pH 7.0 in the dark. Titrations of this dithiol/disulfide couple in illuminated samples of AppA indicate that the E (m) value of this disulfide/dithiol couple is -315 mV at pH 7.0, identical to the value obtained for AppA in the dark within the combined experimental uncertainties of the two measurements. The E (m) values of AppA and PpsR demonstrate that these proteins are thermodynamically capable of electron transfer for their activity as an anti-repressor/repressor in R. sphaeroides.

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.

The 5'-adenylyl sulfate (APS) reductase from the marine macrophytic green alga Enteromorpha intestinalis uses reduced glutathione as the electron donor for the reduction of APS to 5'-AMP and sulfite. The E. intestinalis enzyme (EiAPR) is composed of a reductase domain and a glutaredoxin-like C-terminal domain. The enzyme contains a single [4Fe-4S] cluster as its sole prosthetic group. Three of the enzyme's eight cysteine residues (Cys166, Cys257, and Cys260) serve as ligands to the iron-sulfur cluster. Site-directed mutagenesis experiments and resonance Raman spectroscopy are consistent with the presence of a cluster in which only three of the four ligands to the cluster irons contributed by the protein are cysteine residues. Site-directed mutagenesis experiments suggest that the thiol group of Cys250, a residue found only in algal APS reductases, is not an absolute requirement for activity. The other four cysteines that do not serve as cluster ligands, all of which are required for activity, are involved in the formation of two redox-active disulfide/dithiol couples. The couple involving Cys342 and Cys345 has an E(m) value at pH 7.0 of -140 mV, and the one involving Cys165 and Cys285 has an E(m) value at pH 7.0 of -290 mV. The C-terminal portion of EiAPR, expressed separately, exhibits the cystine reductase activity characteristic of glutaredoxins. It is proposed that the Cys342-Cys345 disulfide provides the site for entry of electrons from reduced glutathione and that the Cys166-Cys285 disulfide may serve as a structural element that is essential for keeping the enzyme in the catalytically active conformation.