Redox Turnover Research Articles

Internal Reorganization Energies for Copper Redox Couples: The Slow Electron-Transfer Reactions of the [CuII/I(bib)2]2+/+ Couple

[CuII(bib)2]2+ and [CuI(bib)2]+ (bib = 2,2‘-bis(2-imidazolyl)biphenyl) have similar geometries, with the Cu(II) complex best described as pseudo-square planar and the Cu(I) complex pseudo-tetrahedral. The cyclic voltammograms in acetonitrile are only quasi-reversible, with ΔEp/p ∼ 103 mV, which leads to uncertainty in E1/2. UV−vis measurements of the reaction [CuII(bib)2]2+ + [CoII((nox)3(BC6H5)2)] ⇌ [CuI(bib)2]+ + [CoIII((nox)3(BC6H5)2)]+ ((nox)3(BC6H5)2 = a clathrochelate ligand) yield K = 2.6 in acetonitrile at 25 °C with μ = 0.1 M (Et4NBF4). Using the known E° for the CoIII/II couple yields E° = 0.274 V vs Ag/AgCl for [CuII/I(bib)2]2+/+. This, in turn, leads to K = 72 for reaction with [CoII((nox)3(BC4H9)2)]. The reaction of [CuII(bib)2]2+ with [CoII((nox)3(BC4H9)2)] obeys simple bimolecular kinetics with k = 87 M-1 s-1. Similar behavior occurs with [CoII((nox)3(BC6H5)2)] and [CoII((dmg)3(BC4H9)2)] as reductants. A value of k11 = 0.16 M-1 s-1 is calculated for the [CuII(bib)2]2+/[CuI(bib)2]+ self-exchange reaction based on the Marcus cross-exchange relationship and the known electron-self-exchange rate constants for the Co(III)/Co(II) couples. This value of k11 is remarkably small for a Cu(II/I) system, especially in view of the small structural change at copper (Δ(Cu−N)ave = 0.07 Å) during redox turnover. However, molecular mechanics calculations with newly developed copper force fields demonstrate ΔG‡in to be the major factor that accounts for the small value of k11. Much of this large reorganization energy appears to arise from angular distortions around copper. Nevertheless, the structural changes for the [CuII/I(bib)2]2+/+ couple are small relative to those for systems that have much greater self-exchange rate constants. These considerations underscore the difficulties in applying the entatic hypothesis to cuproprotein active sites.

Molybdenum cofactor properties and [Fe-S] cluster coordination in Escherichia coli nitrate reductase A: investigation by site-directed mutagenesis of the conserved his-50 residue in the NarG subunit.

Most of the molybdoenzymes contain, in the amino-terminal region of their catalytic subunits, a conserved Cys group that in some cases binds an [Fe-S] cluster. In dissimilatory nitrate reductases, the first Cys residue of this motif is replaced by a conserved His residue. Site-directed mutagenesis of this residue (His-50) was performed on the NarG subunit from Escherichia coli nitrate reductase A. The results obtained by EPR spectroscopy enable us to exclude the implication of this residue in [Fe-S] binding. Additionally, we showed that the His-50 residue does not coordinate the molybdenum atom, but its substitution by Cys or Ser introduces a perturbation of the hydrogen bonding network around the molybdenum cofactor. From potentiometric studies, it is proposed that the high-pH and the low-pH forms of the Mo(V) are both involved during the redox turnover of the enzyme. Perturbation of the Mo(V) pKV value might be responsible for the low activity reported in the His-50-Cys mutant enzyme. A catalytic model is proposed in which the protonation/deprotonation of the Mo(V) species is an essential step. Thus, one of the two protons involved in the catalytic cycle could be the one coupled to the molybdenum atom in the dissimilatory nitrate reductase of E. coli.

Macrocyclic [CuI/II(bite)]+/2+ (bite = biphenyldiimino dithioether): An Example of Fully-Gated Electron Transfer and Its Biological Relevance1

Template condensation of 2,2‘-diaminobiphenyl, 1,4-bis(2-formylphenyl)-1,4-dithiabutane, and copper(II) tetrafluoroborate yields the new macrocyclic compound [CuI(bite)](BF4) (bite = biphenyldiimino dithioether). [CuI(bite)]BF4 crystallizes in the orthorhombic space group P212121 with a = 14.379(3) Å, b = 21.370(3) Å, c = 8.046(2) Å, V = 2534.7(7) Å3, Z = 4, R1 = 0.045, and R2 = 0.048. The X-ray structure of [CuI(bite)](BF4) reveals distorted tetrahedral N2S2 coordination about copper, with one unusually short Cu−S(thioether) bond of 2.194(2) Å. Oxidation of [CuI(bite)](BF4) with nitrosyl tetrafluoroborate gives [CuII(bite)](BF4)2. [CuII(bite)](BF4)2 crystallizes in the tetragonal space group I41/a with a = 11.640(2) Å, c = 39.527(3) Å, V = 5355.6(7) Å3, Z = 8, R1 = 0.061, and R2 = 0.063. X-ray crystallography of [CuII(bite)](BF4)2 reveals an approximately square planar CuN2S2 structure with two distant axial BF4- anions (Cu−F 2.546(4) Å) completing a “pseudo-octahedral” coordination sphere. Comparative EXAFS studies of solid samples and acetonitrile solutions of [CuI(bite)](BF4) and [CuII(bite)](BF4)2 demonstrate that the primary coordination environments of both species are the same in solution as in the solid. Copper(I/II) electron self-exchange kinetics measured by 1H NMR line broadening of [CuI(bite)]+ in the presence of [CuII(bite)]2+ reveal an overall first-order process with a rate constant of 21.7(1.9) s-1 at 295 K in acetone-d6. This result represents the first example of fully-gated electron transfer by small-molecule copper(I). The gating process likely involves inversion at sulfur and the tetrahedral → square planar structural change coincident with electron transfer. Variable-temperature 1H NMR coalescence temperatures for methylene ligand protons of [CuI(bite)](BF4) (287 K) demonstrate possible correlation of fast electron transfer with high ligand mobility for this and related small-molecule copper(I/II) couples. Comparison with other small-molecule copper systems also reveals that fast electron transfer is not always observed with coordination-number-invariance and conserved geometry during redox turnover, contrary to popular interpretations of the entatic state hypothesis for blue-copper protein active sites.

Glutathione metabolism of human vascular endothelial cells under peroxidative stress

Glutathione (GSH) plays an important role in the cellular defense against (per-)oxidative stress. The capacity of this cellular defense system may be related to the oxygen tension, cells are normally subjected to in vivo; therefore, we studied the de novo synthesis of glutathione, and the redox turnover under peroxidative stress, in human umbilical vein and artery endothelial cells (HUVEC, HUAEC) and human skin fibroblasts. De novo synthesis in these cell types was studied in vitro by measuring the time course of intracellular GSH recovery after depletion with diamide. For fibroblasts, the initial rate of de novo synthesis after GSH depletion was twice that of the endothelial cell strains. In the endothelial cells (HUVE, HUAEC) the original intracellular GSH level is reached within 40 min, while in the same time span, the GSH level in fibroblasts returned to 75% of control level. The activity of the hexose monophosphate shunt (HMS) was determined under oxidative stress as a measure for the coupled redox turnover of intracellular GSH. Under control conditions the HMS in endothelial cells was twice as high as in fibroblasts. Cumene hydroperoxide (40 μM) induced a three-fold increase in HMS in both HUVEC and HUAEC, while fibroblasts exhibited an increase of 83%, During the same peroxidative stress, the intracellular GSH concentration of HUVEC, HUAEC and fibroblasts stayed at control level. So with respect to GSH metabolism there were no differences between the two endothelial cell strains. In comparison with the endothelial cells, the fibroblasts were less susceptible toward oxidative stress. We could confirm that the plasma of patients with diabetes or hyperlipoproteinaemia have an increased level of thiobarbituric acid reacting substances (TBRS). Moreover, we found that the HMS activity of HUVEC during incubation with patient plasma had increased by at least 40%. The increased levels of TBRS together with an increased endothelial HMS activity using blood plasma of diabetics and hyperlipidemics are suggestive for a chronic peroxidative-like stress on the endothelial cells of the vessel wall in these patients.

Studies on the interaction between hydroxylamine and hydrazine as substrate analogues and the water-oxidizing enzyme system in isolated spinach chloroplasts

The functional interaction between the photosynthetic water-oxidizing enzyme system and the substrate analogues hydroxylamine and hydrazine has been analyzed in isolated class II chloroplasts by measuring the effect of these species on the characteristic oscillation pattern of oxygen yield induced by a flash train. The following was found. (1) At concentrations where both substances cause the pronounced two-flash phase shift (Bouges, B. (1971) Biochim. Biophys. Acta 234, 103–112) the dark equilibration is rather slow with half-times of approx. 1 min. (2) The numerical evaluation of the oscillation patterns reveals quantitative differences between hydroxylamine and hydrazine. The interaction with hydroxylamine is complex. It involves one- and two-electron processes as well as fast reaction steps during the flash sequence. The fast reactions take place only with redox states S 2 and S 3 of the water-oxidizing enzyme. Furthermore, the redox turnover in the presence of hydroxylamine leads to an S 1-state that differs markedly in its susceptibility to hydroxylamine from that of S 1 in control chloroplasts. (3) Below a threshold concentration which varies for different preparations the hydrazine effect can be quantitatively described by the assumption that after dark equilibration the agent becomes consumed irreversibly via a reaction with two oxidizing redox equivalents produced by PS II. This process is accomplished during the first two flashes. No further interaction occurs during the flash sequence, so that besides the two-flash phase shift the water-oxidizing enzyme system reveals the normal oxygen-evolution pattern. (4) Based on the analysis of the concentration dependence hydrazine is inferred to interact with the catalytic center of the water-oxidizing enzyme system via a cooperative mechanism including two binding sites. The data are discussed in terms of the kinetics of the dark interaction and its possible rate limitation. Mechanistic aspects (ligand-ligand exchange at the functional manganese cluster and transport step) are considered. Furthermore, possible mechanisms for the redox reaction of hydrazine at the catalytic site are briefly discussed.

On the distribution of iron and manganese at the sediment/water interface: thermodynamic versus kinetic control

Iron and manganese solubility at the sediment/water interface has been studied at a water depth of 20 m in Kiel Bight, Western Baltic. By means of an in situ bell jar system enclosing 3.14 m 2 sediment surface and 2094 l water a complete redox turn-over in the bottom water was simulated in an experiment lasting 99 days. The concentration of dissolved Fe in the bell jar water never exceeded 0.041 μmol · dm −3during the first 50 days of the experiment and then rose abruptly as the Eh fell from +600 to −200 mV. The concentration of dissolved Fe under oxic and anoxic conditions seems to be limited by equilibria with solid Fe-phases (hydroxides and amorphous sulphide, respectively). In contrast to Fe, manganese was released continuously from the bottom during the first 50 days of the experiment leading to exponentially increasing manganese concentrations in the bell jar water. During this time dissolved O 2 had become ready depleted and pH had dropped from 8.3 to 7.5. Contrary to iron, manganese being solubilized in reduced sediment layers can penetrate oxic strata in metastable form due to slow oxidation kinetics; when the redoxcline moves upwards Mn 2+ is enriched in bottom waters. The maximum concentration of dissolved Mn under anoxic conditions is controlled by a solid phase with solubility properties similar to MnCO 3 (rhodochrosite). Bottom water enrichment in dissolved Mn 2+ could be traced to originate from excess solid manganese within the top 3 cm of the sediment.

Bacterial cycling of sulfur in a Baltic sediment: An in situ study in closed systems

Bacterial cycling of sulfur at the sediment‐water interface was studied in situ at 10‐m water depth in both transparent and opaque closed‐box systems over a period of 245 days. Reducing processes dominated at Eh values below +200 mV. In the transparent systems, oxygen was produced because of photosynthesis and the redox turnover was delayed 30 days compared to the opaque systems in which reducing conditions were established in 10 days. The initial rates of bacterial sulfate reduction were 0.22 and 0.35 g SO4‐S m‐2 day‐1 for the opaque and transparent systems, respectively. The bacteria generated sulfide at rates corresponding to the depletion of sulfate. Photosynthetic sulfur bacteria developed in the transparent systems, and sulfide was oxidized at twice the rate of its formation, equal to 0.7 g sulfide‐S m‐2day‐1. A stepwise oxidation of sulfide, via elemental sulfur to sulfate, was demonstrated. Sulfate reduction dominated the opaque systems and also the transparent ones with insufficient light.