Emerging roles of low-molecular-weight thiols at the host–microbe interface

Low-molecular-weight (LMW) thiols are an abundant class of cysteine-derived small molecules found in all forms of life that maintain reducing conditions within cells. While their contributions to cellular redox homeostasis are well established, LMW thiols can also mediate other aspects of cellular physiology, including intercellular interactions between microbial and host cells. Here we discuss emerging roles for these redox-active metabolites at the host–microbe interface. We begin by providing an overview of chemical and computational approaches to LMW-thiol discovery. Next, we highlight mechanisms of virulence regulation by LMW thiols in infected cells. Finally, we describe how microbial metabolism of these compounds may influence host physiology.     

Protonation,alkylation, addition and redox reactions of 16, 17 and 18 valence electron [Mo(L)(NO)('S4')] complexes [L = SPh,PMe3, NO; 'S4'2– = 1,2-Bis-(2-mercaptophenylthio)ethane(2-)]

In the quest for complexes modelling functional characteristics of metal sulfur oxidoreductases, a series of molybdenum nitrosyl complexes with sulfur-dominated coordination sphere was synthesized. Treatment of the 16, 17 and 18 valence electron (VE) complexes [Mo(L)(NO)('S₄')] (1–3) [L?=?SPh (1), PMe₃ (2), NO (3), 'S₄'^2–?=?1,2-bis-(2-mercaptophenylthio) ethane(2-)] with the Brönsted acid HBF₄ resulted in formation of different types of products. 1 and 3 were reversibly protonated at one thiolate atom of the 'S₄'^2– ligand;2, however, yielded the phosphonium salt [HPMe₃]BF₄ and the dinuclear [Mo(NO)('S₄')]₂. Alkylation of 1, 2 and 3 by Me₃OBF₄ or Et₃OBF₄ uniformly resulted in high yields of [Mo(L)(NO)(R-'S₄')]BF₄ complexes [L?=?SPh: R?=?Me (5), Et (6); L?=?PMe₃: R?=?Me (7); L?=?NO: R?=?Me (8), Et (9)] in which one thiolate atom of the 'S₄'^2– ligand had become alkylated; the NMR spectra of 5, 6, 8 and 9 indicated that only one out of four theoretically possible diastereoisomers had formed. 5 and 6 were characterized also by single-crystal X-ray structure analyses. A comparison of ν(NO) bands and redox potentials (cyclic voltammetry) of parent complexes and alkylated derivatives showed that alkylation leads to a decrease in electron density at the molybdenum center and to a positive shift in redox potentials. The 16 VE complex 1 could be reduced, also chemically, to give the corresponding 17 VE anion [1]^–, and inserted elemental sulfur into the Mo-SPh bond, forming the 18 VE phenylperthio complex [Mo(η²–SSPh)(NO)('S₄')] (11) which, upon reaction with PPh₃, gave SPPh₃ and regenerated the parent complex 1. These results are discussed with regard to the sequence of proton and electron transfer steps occurring in substrate conversions catalyzed by metal sulfur oxidoreductases.     

Use of Plant Strategy Ordination,DCA and ANOVA to elucidate relationships among habitats of Salix planifolia and Salix monticola

Abstract. Detrended Correspondence Analysis (DCA) was used on a data set of 112 species from 14 stands dominated by Salix planifolia and/or S. monticola. Environmental variables were determined which might be important to explain differences in stand vegetation composition, and willow stature (height), in willow carrs in the Rocky Mountain National Park. Correlation of environmental variables with DCA stand scores indicate that peat depth and soil redox potential are highly related to the primary DCA axis. Soils in stands of S. planifolia are more reduced in the early summer and have greater peat depths, than soils in stands of S. monticola. The second DCA axis is related to a water chemistry gradient of hydrogen ion, sulphate, and ammonium concentration. Four environmental variables selected by correlation analysis were regressed on stand scores from the first two DCA axes. Regression coefficients of August soil redox potential and soil water pH were significantly non-zero on the primary DCA axis. In addition to overall vegetation relationships elucidated by DCA, the relationships between willow stature and environmental variables were tested by simultaneous ANOVAs. ANOVA results and DCA ordinations indicate that S. planifolia and S. monticola plants achieve the greatest stature in the least reduced soils, and in shallow peat. Competitive, stress tolerant, and ruderal plant strategy theory applied to the vegetation data produced an arrangement of stands highly correlated to the DCA Axis 1 stand arrangement. These correlations indicate that the primary axis of vegetation structure can be extracted either by species composition, or species morphology/physiology relationships. Concurrent use of DCA and plant strategy theory is suggested as a tool for the prediction of community composition.     

Redox thermodynamics, acid-base equilibria and salt-induced effects for the cucumber basic protein. General implications for blue-copper proteins

 The reduction potential of the basic blue-copper protein from cucumber peels (CBP) was determined through voltammetric techniques in different conditions of temperature, pH and ionic composition of the medium. The most notable properties of CBP include a positive entropy change upon reduction, a low-pH protonation and detachment of a metal-binding histidine in the reduced protein, and specific binding interactions with a number of anions present in common laboratory buffers, which influence to some extent the redox thermodynamics. The enthalpy and entropy changes accompanying reduction of the Cu(II) center were compared with those for other blue-copper proteins and correlated with spectroscopic data, structural properties and theoretical calculations. This allows some general considerations to be offered regarding the determinants of the reduction potential in this protein class. It emerges, in line with previous studies of the electronic structure of blue-copper sites, that the enthalpic contribution to the reduction potential is mainly modulated by the metal-binding interactions in the trigonal N₂S ligand set, and particularly by the Cu-cysteinate bond, while the entropy term is mainly affected by solvation properties and possibly by the weak axial bond to copper. The role of solvent exposure of the metal site in the active-site protonations in reduced blue-copper proteins is discussed. Finally, it is shown that the Nernst-Debye-Huckel model qualitatively accounts for the ionic strength dependence of the reduction potential. Received: 20 December 1996 / Accepted: 26 March 1997     

Depleted mucosal antioxidant defences in inflammatory bowel disease

Experimental approaches designed to define the role of reactive oxygen and nitrogen species generated by inflammatory cells in the tissue injury seen in inflammatory bowel disease rarely consider the chemical antioxidant defences against such increased oxidant stress in the mucosa. In this investigation, we have analysed components of the aqueous and lipid phase antioxidant mucosal defences by measuring the total peroxyl radical scavenging capacity and the levels of urate, glutathione, -tocopherol, and ubiquinol-10 in paired noninflamed and inflamed mucosal biopsies from inflammatory bowel disease patients. Compared to paired noninflamed mucosa, decreases were observed in inflamed mucosa for total peroxyl radical scavenging capacity (55%, p = 0.0031), urate [Crohn's disease (CD), 62.2%, p = 0.066; ulcerative colitis (UC), 47.3%, p = 0.031], glutathione (UC, 59%, 7/8 patients, ns), total glutathione (UC 65.2%, 6/8 patients, ns), ubiquinol-10 (CD, 75.7%, p = 0.03; UC, 90.5%, p = 0.005). The mean -tocopherol content was unchanged. These observations support our earlier findings of decreased reduced and total ascorbic acid in inflamed IBD mucosa and demonstrate that the loss of chemical antioxidant defences affects almost all the major components. The decreased antioxidant defences may severely compromise the inflamed mucosa, rendering it more susceptible to oxidative tissue damage, hindering recovery of the mucosa and return of epithelial cell layer integrity. The loss of chemical antioxidant components provides a strong rationale for developing novel antioxidant therapies for the treatment of inflammatory bowel disease.     

Unusual redox behaviour of cytochrome b-561 from bovine chromaffin granule membranes

(1) Redox titrations of cytochrome b-561 have been performed with the purified cytochrome and with intact and detergent-solubilized chromaffin-granule membranes. (2) The midpoint redox potential of the cytochrome is 100–130 mV; this depends upon the composition of the buffer, but is independent of pH in the range 5.5–7.5; partial proteolysis of the cytochrome raises the midpoint potential to 160 mV. (3) The Nernst plots of titration data have slopes of 75–115 mV, and are in some cases sigmoid in shape. This may be explained by negative cooperativity during redox transitions in oligomeric cytochrome b-561. (4) Measurements of the haem and cytochrome content of chromaffin granule membrane suggest a haem content of 1 mol/mol protein. (5) Chemical crosslinking of cytochrome b-561 suggests that it may exist as an oligomer of 4–6 polypeptide chains within the chromaffin granule membrane. Aggregation of purified cytochrome b-561 was shown by gel filtration studies and by immunological methods in SDS-polyacrylamide gels. Studies of the molecular weight of the aggregates suggest that the monomer has a molecular weight close to 22 000, but migrates anomalously slowly during electrophoresis.     

Characterisation of ionisations that influence the redox potential of mitochondrial cytochrome c and photosynthetic bacterial cytochromes c2

Several cytochromes c₂ from the Rhodospirillaceae show a pH dependence of redox potential in the physiological pH range which can be described by equations involving an ionisation in the oxidised form (pK_o) and one in the reduced form (pK_r). These cytochromes fall into one of two groups according to the degree of separation of pK_o and pK_r. In group A, represented here by the Rhodomicrobium vannielii cytochrome c₂, the separation is approx. one pH unit and the ionisation is that of a haem propionic acid. Members of this group are unique among both cytochromes c₂ and mitochondrial cytochromes c in lacking the conserved residue Arg-38. We propose that the role of Arg-38 is to lower the pK of the nearby propionic acid, so that it lies out of the physiological pH range. Substitution of this residue by an uncharged amino acid leads to a raised pK for the propionic acid. In group B, represented here by Rhodopseudomonas viridis cytochrome c₂, the separation between pK_o and pK_r is approx. 0.4 pH unit and the ionisable group is a histidine at position 39. This was established by NMR spectroscopy and confirmed by chemical modification. Only a few other members of the cytochrome c₂/mitochondrial cytochrome c family have a histidine at this position and of these, both Crithidia cytochrome c-557 and yeast cytochrome c were found to have a pH-dependent redox potential similar to that of Rps. viridis cytochrome c₂. Using Coulomb's law, it was found that the energy required to separate pK_o and pK_r could be accounted for by simple electrostatic interactions between the haem iron and the ionisable group.     

Relationships between the effects of redox potential, alpha-thenoyltrifluoroacetone and malonate on O(2) and H2O2 generation by submitochondrial particles in the presence of succinate and antimycin

A successful approach has been developed for the sequencing of apolipoprotein B based upon the procedure of Cleveland et al. [(1977) J. Biol. Chem. 252, 1102-1106] involving limited proteolysis in the presence of sodium dodecyl sulfate. Staphylococcus aureus protease was employed to produce large peptides which were isolated in relatively pure form by preparative gel electrophoresis. Two peptides were partially sequenced using spinning-cup microsequencing techniques. The sequences are: Peptide R2-5, -Ala-Leu-Val-Gly-Ile-Asn- Gly-Glu-Ala-Asn-Leu-Asp-Phe-Leu-Asn-Ile-Pro-Leu-Arg-Ile-Pro-Pro- Met-Arg-(Arg)-; Peptide R3-1, -Leu-Val-Ala-Lys-Pro-Ser-Val-Ser-Val-Glu- Phe-Val-Thr-Asn-Met-Gly-Ile-Ile-Pro-Lys-Phe-Ala-Arg-. Several stretches of residues suitable for the construction of oligonucleotide probes have been identified.     

The influence of pH on the EPR and redox properties of cytochrome c oxidase in detergent solution and in phospholipid vesicles

The EPR signals of oxidized and partially reduced cytochrome oxidase have been studied at pH 6.4, 7.4, and 8.4. Isolated cytochrome oxidase in both non-ionic detergent solution and in phospholipid vesicles has been used in reductive titrations with ferrocytochrome c.The g values of the low- and high-field parts of the low-spin heme signal in oxidized cytochrome oxidase are shown to be pH dependent. In reductive titrations, low-spin heme signals at g 2.6 as well as rhombic and nearly axial high-spin heme signals are found at pH 8.4, while the only heme signals appearing at pH 6.4 are two nearly axial g 6 signals. This pH dependence is shifted in the vesicles.The g 2.6 signals formed in titrations with ferrocytochrome c at pH 8.4 correspond maximally to 0.25–0.35 heme per functional unit (aa₃) of cytochrome oxidase in detergent solution and to 0.22 heme in vesicle oxidase. The total amount of high-spin heme signals at g 6 found in partially reduced enzyme is 0.45–0.6 at pH 6.4 and 0.1–0.2 at pH 8.4. In titrations of cytochrome oxidase in detergent solution the g 1.45 and g 2 signals disappear with fewer equivalents of ferrocytochrome c added at pH 8.4 compared to pH 6.4.The results indicate that the environment of the hemes varies with the pH. One change is interpreted as cytochrome a₃ being converted from a high-spin to a low-spin form when the pH is increased. Possibly this transition is related to a change of a liganded H₂O to OH^? with a concomitant decrease of the redox potential. Oxidase in phosphatidylcholine vesicles is found to behave as if it experiences a pH, one unit lower than that of the medium.