An e.p.r. study of the non-equivalence of the copper sites of caeruloplasmin.

The two Type 1 (blue) copper-binding sites of caeruloplasmin were spectroscopically differentiated by the kinetic analysis of the e.p.r. spectra during the redox cycle. One blue copper, with a hyperfine splitting constant (A parallel) of 6.8 mT, which was rapidly reduced, was not reoxidized by oxygen, whereas it was reoxidized by H2O2. The other blue copper (A parallel = 5.8 mT), which was reduced slowly, was rapidly reoxidized by either oxygen or H2O2. A conformational change of the Type 2 copper was concomitant with the fast reduction of Type 1 copper, whereas its reduction occurred during the slow phase. This sequence of events was reversed in the reoxidation step, that is, the Type 2 copper reappeared rapidly as the species with altered conformation and reverted to the symmetry typical of the native state in the slow phase. The specific reaction of a blue-copper site with the H2O2 can tentatively be related to the established ability of caeruloplasmin to prevent 'oxidative' attack of proteins and lipids.     

Titrations with ferrocyanide of japanese-lacquer-tree (Rhus vernicifera) laccase and of the type 2 copper-depleted enzyme. Interrelation of the copper sites.

1. Redox titrations are reported of the metal centres in Japanese-lacquer-tree (Rhus vernicifera) laccase with ferrocyanide. 2. The redox potential of Type 1 Cu was found to increase with ferrocyanide concentration up to a limiting value similar to that for the Type 1 Cu in Type 2 Cu-depleted enzyme (which is independent of ferrocyanide concentration). 3. The redox potential of the two-electron acceptor (Type 3 Cu) is also independent of ferrocyanide concentration in Type 2 Cu-depleted enzyme and lower than values reported for the native enzyme. 4. The two-electron acceptor is present in the oxidized state in the Type 2 Cu-depleted enzyme, though the latter lacks the 330 nm absorption band. 5. The redox potential of Type 2 Cu also depends on ferrocyanide concentration, at least in the presence of azide. 6. The redox potentials are affected by freezing the solutions and/or addition of azide, the latter binding to Type 2 Cu with affinity dependent on the redox state of the two-electron acceptor.     

Oxidative protein damage in plasma of type 2 diabetic patients.

In this study, we evaluated protein oxidation in 84 patients with Type 2 diabetes with no complications and in 61 healthy volunteers who formed the control group, whose ages matched those of the patients. We determined plasma carbonyl and plasma thiol levels as markers of oxidative protein damage and erythrocyte glutathione, plasma ceruloplasmin and transferrin as markers of free radical scavengers. The concentrations (mean +/- SD) of both of plasma carbonyl (1.24 +/- 0.46 vs. 0.72 +/- 0.17 nmole/mg protein; p < 0.0001) and lipid hydroperoxides (1.8 +/- 0.63 vs. 1.3 +/- 0.21 micromole/l; p < 0.0001) were increased, and the concentration of plasma transferrin (3.85 +/- 0.65 vs. 4.59 +/- 0.79 g/l; p < 0.05) was decreased, respectively, in Type 2 diabetic patients compared with those of the controls. There were no significant differences in the concentrations of plasma thiol (0.0064 +/- 0.001 vs. 0.0068 +/- 0.001 micromole/mg protein), erythrocyte glutathione (2.54 +/- 0.57 vs. 2.65 +/- 0.56 mg/g Hb), plasma ceruloplasmin (548 +/- 107.30 vs. 609 +/- 93.34 mg/l) between the patients and the controls. These changes observed in diabetic patients contribute to the imbalance in the redox status of the plasma. We attribute this imbalance to oxidative protein damage in Type 2 diabetic patients clinically free of complications.     

Purification and properties of bovine caeruloplasmin.

A novel method is reported for isolation of bovine caeruloplasmin from plasma; it involves a rapid and mild procedure, namely two column chromatographies with stepwise elution and one (NH4)2SO4 precipitation, and results in a proteolytically undegraded homogeneous protein. The general structure of the protein, as evaluated by molecular-weight determination and amino acid composition, is very similar to that established for human and rat caeruloplasmin. Copper determination and e.p.r. spectral analysis on the native and NO-treated protein gave a metal-to-protein stoichiometry of six atoms of copper per molecule. Three copper atoms were detectable by e.p.r., with Type 2/Type 1 ratio = 1 : 3 in most samples. The protein is very sensitive to storage and/or handling. A component was isolated from aged samples, which was found to contain approximately four copper atoms per 125000 daltons, two of which were detectable by e.p.r. with the characters of Type 2 copper. However, the same component was found to be present, although to a lesser extent, in the fresh preparation and does not seem to be related to proteolytic degradation. This component has no oxidase activity. On the basis of these results it is suggested that caeruloplasmin molecules are intrinsically heterogeneous with respect to both copper content and copper type, and this can explain the intriguing stoichiometry regarding the different types of copper centres.     

Effect of glycine on insulin secretion and action in healthy first-degree relatives of type 2 diabetes mellitus patients.

The aim of this study was to identify the effect of glycine on insulin secretion and action in healthy first-degree relatives of Type 2 diabetes mellitus patients. A randomized, double-blind, placebo-controlled clinical trial was performed in 12 healthy, non-obese volunteers who were first-degree relatives of Type 2 diabetes mellitus patients. Six volunteers received a morning dose of glycine 5 g orally and the other six received placebo. At baseline without drugs and after pharmacological intervention, a metabolic profile and, to assess insulin secretion and action, a hyperglycemic-hyperinsulinemic clamp study were performed. There were no significant differences in baseline metabolic profile, insulin secretion or action between groups. Changes from baseline of early (p < 0.05), late (p < 0.05), and total insulin (p < 0.02) responses were higher in the glycine group than in controls. There were no significant differences in the changes from baseline of insulin action between groups. In conclusion, a morning dose of glycine 5 g orally increased early, late and total insulin responses without changes in insulin action in healthy first-degree relatives of Type 2 diabetes mellitus patients.     

Electron-paramagnetic-resonance measurements of the electron-transfer components of the reaction centre of Rhodopseudomonas viridis. Oxidation–reduction potentials and interactions of the electron acceptors

Oxidation-reduction potentiometry was carried out on Rhodopseudomonas viridis chromatophores. Measurements of e.p.r. signals of the semiquinone-iron type at g=1.82 have revealed a more complex situation than previously reported. The presence of three different components is indicated. The midpoint potential (E(m)) of the primary acceptor quinone/semiquinone couple was found to be approx. -165mV at pH10, with a pK being reached at around pH7.5. The primary acceptor also accepts a second electron with an E(m) of -525mV, but this redox transition exhibits a hysteresis effect. Interaction effects indicate the presence of another component with E(m) values at pH10 of approx. -165mV (pK reached at around pH7.5) for single reduction and -350mV (pK at pH10 or greater) for double reduction. It is suggested that this component is the secondary acceptor. Another semiquinone-iron-type component which gives a g=1.82 signal is also present. This component is distinguishable from the primary acceptor by its e.p.r. spectrum, which shows a double peak at g=1.82 and a g(x) line at g=1.76. This component has E(m) values at pH10 for single and double reduction of -15mV and approx. -150mV respectively. Both of these E(m) values are pH-dependent. The presence of an interaction between this component and the photoreduced primary acceptor indicates the close proximity of these components. However, the midpoint potential of this component indicates a function as a secondary electron-transport component rather than an electron acceptor in the reaction centre. The dependence of the bacteriopheophytin intermediate (I) doublet e.p.r. signal on the presence of the semiquinone-iron form of the primary acceptor is demonstrated. The midpoint potential of the I/I(-) couple is estimated to be lower than -600mV.     

The molybdenum and iron-sulphur centres of Escherichia coli nitrate reductase are non-randomly oriented in the membrane

Oriented membrane multilayers, prepared from Escherichiacoli grown anaerobically with nitrate, allowed the spatial organisation of electron paramagnetic resonance (e.p.r.)-detectable signals from the respiratory nitrate reductase to be examined. At low temperatures (7 K), two signals (g = 2.02, g = 1.98) have been assigned to an iron-sulphur cluster and their magnitudes shown to be dependent on the angle that the multilayer makes with the magnetic field of the e.p.r. spectrometer. Signals seen at 45 K (g = 1.985, g = 1.96) have been attributed to an anisotropic molybdenum centre. These redox components of the nitrate reductase are thus non-randomly oriented in the cytoplasmic membrane.     

Characterization of the partially reduced cyanide-inhibited derivative of cytochrome c oxidase by optical, electron-paramagnetic-resonance and magnetic-circular-dichroism spectroscopy.

Optical. e.p.r. and near-infrared low-temperature m.c.d. (magnetic-circular-dichroism) spectroscopy were used to characterize the partially reduced cyanide-inhibited derivative of cytochrome c oxidase produced by anaerobic reductive titration with dithionite. The reductions of cytochrome a3+ and Cu2+a were followed by observation of the e.p.r. signals at g = 3.03, 2.21 and 1.5 and at g = 2.18, 2.03 and 1.99. As reduction proceeds new e.p.r. signals (g = 3.58 and 1.56) appear that quantify to give one haem per enzyme unit when a small excess of dithionite has been titrated in. The e.p.r. signal of the Cu2+a titrates in parallel with the disappearance of the band and 820nm in the optical absorption spectrum. The near-infrared m.c.d. spectrum shows the presence of the low-spin ferric haem, a3+, in the oxidized state of the enzyme, as a well-resolved positive peak at 1650nm. As reduction proceeds this band is replaced by one at 1550nm due to haem a3+(3)--CN in the partially reduced state. Hence as haem a3+(3)--CN becomes e.p.r.-detectable it also shows a near-infrared m.c.d. spectrum characteristic of a low-spin ferric haem. It is concluded that the partially reduced state of cyanide-inhibited cytochrome c oxidase contains a2+ . Cu+a . a3+(3)--CN . Cu+a3.     

Changes in the proportion of type I and type III collagen in the developing and retained bovine placentome

The proportions of Type I and Type III collagen were evaluated from gestational, postpartum-retained, and released bovine placental membranes. Placentomes were excised at 90, 150, 210, and 270 days of gestation (n = 32) and from postpartum-retained (2 and 12 h, n = 8) and released (2 h, n = 4) membranes. Placentome components were processed for collagen, hydroxyproline, protein, and dry weight determination. Collagen extracts were separated by SDS-PAGE. Densitometry was used to establish the proportions of collagen alpha chains (Type I = 2 alpha 1 + 1 alpha 2; Type III = 3 alpha 1). No difference in the proportion of maternal caruncular Type I and Type III collagen was found. The proportion of Type I fetal cotyledonary collagen was lowest (p less than 0.05) at Day 90 of gestation but did not differ between Days 150, 210, 270, or between retained and released fetal membranes. The proportion of Type III fetal cotyledonary collagen was greatest (p less than 0.05) at Day 90. Retained fetal cotyledons had a greater (p less than 0.05) proportion of Type III collagen than did released fetal cotyledons. Therefore, although hydroxyproline content was not different between retained and released fetal membranes, the retained bovine fetal cotyledon was characterized by disproportionate amounts of Type III collagen as compared to the fetal cotyledon that was not retained.     

Lysophosphatidylcholine molecular species in low density lipoprotein of type 2 diabetes.

To reveal the importance of lysoposphatidylcholine (LPC) in patients with Type 2 diabetes (DM), LPC in low density lipoprotein (LDL) was determined by high performance liquid chromatography in 38 patients with Type 2 DM and 31 age and sex-matched non-diabetic controls. Stearoyl LPC (SLPC) and palmitoyl LPC (PLPC) were detected in LDL. The contents of both LPCs per gram protein in LDL were increased in diabetic patients compared with the non-diabetics (1.99+/-0.94mg SLPC and 3.02+/-1.81 mg PLPC vs 1.47+/-0.57 mg SLPC and 2.30+/-0.83 mg PLPC, mean +/- SD, p < 0.01 and p < 0.05, respectively). PLPC showed a weak correlation with the levels of fasting plasma glucose (FPG) and HbA1c (r=0.27 and r=0.33, p < 0.05 and p < 0.01, respectively). The diabetic patients with macroangiopathy showed higher levels of PLPC per gram protein compared to those without macroangiopathy (4.60+/-2.61 mg vs 2.53+/1.15 mg, respectively, p < 0.05). The LPC molecular species may participate in the atherogenicity of LDL in patients with Type 2 diabetes.     

An investigation of the iron-sulphur proteins of benzene dioxygenase from Pseudomonas putida by electron-spin-resonance spectroscopy.

Benzene dioxygenase from Pseudomonas putida comprises three components, namely a flavoprotein (NADH:ferredoxin oxidoreductase; Mr 81000), an intermediate electron-transfer protein, or ferredoxin (Mr 12000) with a [2Fe-2S] cluster, and a terminal dioxygenase containing two [2Fe-2S] iron-sulphur clusters (Mr 215000), which requires two additional Fe2+ atoms/molecule for oxygenase activity. The ferredoxin and the dioxygenase give e.s.r. signals in the reduced state with rhombic symmetry and average g values of 1.92 and 1.896 respectively. The mid-point redox potentials were determined by e.s.r. titration at pH 7.0 to be -155 mV and -112 mV respectively. The signal from the dioxygenase shows pronounced g anisotropy and most closely resembles those of 4-methoxybenzoate mono-oxygenase from Pseudomonas putida and the [2Fe-2S] 'Rieske' proteins of the quinone-cytochrome c region of electron-transport chains of respiration and photosynthesis.     

Peroxide and redox titrations of type 2 copper depleted laccase

The chemistry of Type 2 copper depleted T2D Rhus laccase has been investigated with regard to the binding of peroxide, and the ability of the enzyme to undergo reduction and reoxidation. Although the peroxide affinity is diminished in the T2D enzyme (10⁴ M^?1) relative to the holo-enzyme ((? 10⁸ M^?1) the actual mode of binding as a Type 3 μ-peroxo complex remains, as indicated by absorption and CD spectral measurements. Anaerobic reductive and reoxidative titrations with hydroquinone and hydrogen peroxide respectively revealed that the Type 3 copper pairwise interaction is disrupted during reduction but can be restored on reoxidation. The concept of separate Type 2 and Type 3 copper redox centers is suggested to be inadequate in view of the loss of functional integrity by the Type 3 site on removal of Type 2 copper.     

Fiber type-specific distribution of parvalbumin in rabbit skeletal muscle. A quantitative microbiochemical and immunohistochemical study.

A highly sensitive sandwich ELISA for parvalbumin (PA), based on a fluorometric detection system, was developed. This assay detected PA concentrations as low as 20 pg/ml (2 pg per assay) and was used for measuring PA contents in fragments of single muscle fibers isolated from freeze-dried 100-150 microns thick cross sections. The fibers were typed according to their histochemically assessed mATPase in parallel cross sections. Type I fibers from rabbit tibialis anterior (TA) and vastus lateralis (VL) muscles contained extremely low PA concentrations (2-5 micrograms/g w.wt.). Type IIA fibers displayed slightly higher values with mean values of 17 and 29 micrograms/g w.wt. (range 5-65) in TA and VL, respectively. Much higher PA concentrations were found in type IIB fibers with wide ranges from 75-1150 micrograms/g w.wt. in TA and 440-1370 micrograms/g w.wt. in VL. Whereas the IIB fibers of the TA displayed a continuum, two subgroups were distinguishable according to their PA contents (means of 590 and 1230 micrograms/g w.wt.) in VL. Possibly, the population with the lower PA content which was histochemically defined as type IIB in the present study, corresponds to fiber type IID. The finding that PA is predominantly present in type IIB fibers was also confirmed by the parallel decay of PA and type IIB fibers during chronic low-frequency stimulation. The use of freeze substitution, or alternatively, of freeze-drying, made it possible to demonstrate PA immunohistochemically without artifacts and to evaluate the staining intensity by microphotometry.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation of mannose-binding proteins from human and rat liver

The interaction of e-aq., CO2-. and one-electron reduced nitroaromatics (RNO2-.) with ascorbate oxidase (AAO) was studied in aqueous solution at pH 6.0 and 7.5 by using the technique of pulse radiolysis. From observations at 330, 410 and 610 nm, interaction of e-aq. and CO2-. with AAO results in non-specific reduction of the protein followed by reduction of Type 1 Cu in a rate-determining intramolecular step. Only a few per cent of the reducing equivalents ultimately results in reduction of Type 1 Cu. With large excesses of reducing equivalents (e-aq. and CO2-.) with respect to the copper concentration, the amount of Type 1 copper reduced never exceeds 50% of the total amount of Type 1 copper after a single radiation pulse. With less-powerful reducing agents, e.g. RNO2-. reduction of Type 1 Cu occurs via a bimolecular step, and there is no evidence for formation of radicals on protein residues. From observations at 330 nm it is evident that Type 2 and/or Type 3 Cu may also be reduced along with Type 1 Cu. Almost stoichiometric reduction of AAO by RNO2-. was observed, e.g. the protein accepts 6-7 reducing equivalents. It is inferred that the various types of redox couples Cu2+/Cu+ are in equilibrium and that intramolecular electron transfer between the different types of Cu is not rate-determining when using RNO2-. as reducing agent.     

Spin–spin interaction between molybdenum and one of the iron–sulphur systems of xanthine oxidase and its relevance to the enzymic mechanism

1. Electron-paramagnetic-resonance (e.p.r.) studies at 9 and 35GHz at helium temperatures have given new information relating to the structure and mechanism of action of xanthine oxidase. 2. As reported by others, the enzyme gives two types of e.p.r. signal attributed to iron-sulphur systems. The first has g(av.)=1.95. Parameters of the second are determined as g(1) 2.12, g(2) 2.007 and g(3) 1.91, with g(av.)=2.01. This species seems to have a slightly higher redox potential than the former one. 3. Temperature-dependent changes in the form of Mo(v) e.p.r. signals from the enzyme, observed under certain conditions, are shown to be due to weak spin-spin interaction between Mo(v) and g(av.)=1.95 Fe/S. The phenomenon has been studied most fully for the Slow Mo(v) signal. Here, the spectral change takes the form of an additional approximately isotropic 11G splitting, detected below about 45 degrees K only. Samples without Fe/S reduced showed no such changes of spectrum. 4. Similar spectral changes were observed in the Rapid Mo(v) signals, obtained in rapid-freezing experiments, but only in samples corresponding to relatively long reaction times with the substrate. It is suggested therefore that the phenomenon may provide a means of distinguishing enzyme centres with Mo only reduced from those in which both Mo and Fe/S are reduced. 5. Additional rapid-freezing data tending to support a two- rather than a one-electron transfer of reducing equivalents from substrates to xanthine oxidase are reported.     

Enhanced efficiency of ATP hydrolysis during nitrogenase catalysis utilizing reductants that form the all-ferrous redox state of the Fe protein

The amount of MgATP hydrolyzed per pair of electrons transferred (ATP/2e) during nitrogenase catalysis (1.0 atm N(2), 30 degrees C) using titanium(III) citrate (Ti(III)) as reductant was measured and compared to the same reaction using dithionite (DT). ATP/2e values near 2.0 for Ti(III) and 5.0 for DT indicate that nitrogenase has a much lower ATP requirement using Ti(III) as reductant. Using reduced Azotobacter vinelandii flavoprotein (AvFlpH(2)), a possible in vivo nitrogenase reductant, ATP/2e values near 2.0 were also observed. When the reaction was conducted using Ti(III) under N(2), 5% CO in N(2), Ar, 5% CO in Ar, or acetylene, ATP/2e values near 2.0 were also observed. With Ti(III) as reductant, ATP/2e values near 2.0 were measured as a function of temperature, Fe:MoFe protein ratio, and MoFe:Fe protein ratio, in contrast to measured values of 4.0-25 when DT is used under the same conditions. Both Ti(III) and AvFlpH(2) are capable of forming the [Fe(4)S(4)](0) cluster state of the Fe protein whereas DT is not, suggesting that ATP/2e values near 2.0 arise from operation of the [Fe(4)S(4)](2+)/[Fe(4)S(4)](0) redox couple with hydrolysis of only 2 ATPs per pair of electrons transferred. Additional experiments showed that ATP/2e values near 2. 0 correlated with slower rates of product formation and that faster rates of product formation produced ATP/2e values near 5.0. ATP/2e values of 5.0 are consistent with the operation of the [Fe(4)S(4)](2+)/[Fe(4)S(4)](1+) redox couple while ATP/2e values of 2.0 could arise from operation of the [Fe(4)S(4)](2+)/[Fe(4)S(4)](0) redox couple. These results suggest that two distinct Fe protein redox couples may be functional during nitrogenase catalysis and that the efficiency of ATP utilization depends on which of these redox couples is dominant.     

Ca2+-dependent binding of cytosolic components to insulin-secretory granules results in Ca2+-dependent protein phosphorylation

Interaction of Cu(II) and Gly-His-Lys, a growth-modulating tripeptide from plasma, was investigated by 13C- and 1H-n.m.r. and e.p.r. spectroscopy. The n.m.r. line-broadening was interpreted in terms of major and minor species formed as a function of pH. The results indicate that the n.m.r. line-broadening is due to the presence of minor species in rapid exchange and not due to the major species in solution, which has a large tau M. It is concluded that the technique of 13C- and 1H-n.m.r. line broadening, caused by paramagnetic Cu(II) ion, should be undertaken with caution, since the method may not be useful for obtaining structural information on the major species. The e.p.r. spectra over a wide pH range are almost entirely due to similarly co-ordinating species. Starting at pH 5.5, the narrowest absorption near 340 mT shows superhyperfine structure, which comes out sharply in the pH region 6.0-9.6. The spectra in this pH range showed the seven lines of nitrogen superhyperfine splitting, indicating clearly the co-ordination of three nitrogen atoms to Cu(II). The e.p.r. parameters in the medium pH range, A parallel = 19.5 mT and g parallel = 2.21, fit well with the contention that Cu(II) is ligated to Gly-His-Lys through one oxygen atom and three nitrogen atoms in a square-planar configuration.     

Fiber type-specific distribution of parvalbumin in rabbit skeletal muscle

Summary A highly sensitive sandwich ELISA for parvalbumin (PA), based on a fluorometric detection system, was developed. This assay detected PA concentrations as low as 20 pg/ml (2 pg per assay) and was used for measuring PA contents in fragments of single muscle fibers isolated from freeze-dried 100–150 m thick cross sections. The fibers were typed according to their histo-chemically assessed mATPase in parallel cross sections. Type I fibers from rabbit tibialis anterior (TA) and vastus lateralis (VL) muscles contained extremely low PA concentrations (2–5 g/g w.wt.). Type IIA fibers displayed slightly higher values with mean values of 17 and 29 g/g w.wt. (range 5–65) in TA and VL, respectively. Much higher PA concentrations were found in type IIB fibers with wide ranges from 75–1150 g/g w.wt. in TA and 440–1370 g/g w.wt. in VL. Whereas the IIB fibers of the TA displayed a continuum, two subgroups were distinguishable according to their PA contents (means of 590 and 1230 g/g w.wt.) in VL. Possibly, the population with the lower PA content which was histochemically defined as type IIB in the present study, corresponds to fiber type IID. The finding that PA is predominantly present in type IIB fibers was also confirmed by the parallel decay of PA and type IIB fibers during chronic low-frequency stimulation. The use of freeze substitution, or alternatively, of freeze-drying, made it possible to demonstrate PA immunohistochemically without artifacts and to evaluate the staining intensity by microphotometry. Performing measurements on the same fibers with the two methods, it was possible to establish a relationship between immunohistochemical staining intensity and PA concentration. This correlation can be used to assess PA contents by evaluating immunohistochemical staining intensities in comparative measurements within the same section.     

Purification and properties of formate dehydrogenase from Pseudomonas aeruginosa. Electron-paramagnetic-resonance studies on the molybdenum centre.

Formate dehydrogenase from Pseudomonas aeruginosa contains molybdenum, a [4Fe-4S] cluster and cytochrome b. This paper reports the detection of molybdenum as Mo(V) by e.p.r. spectroscopy. In order to generate Mo(V) signals, addition of amounts of excess formate varying between 10- and 50-fold over enzyme, followed by 200-fold excess of sodium dithionite, were used. Two Mo(V) species were observed. One, the major component, has g1 = 2.012, g2 = 1.985 and g3 = 1.968, appeared at low concentrations of formate and increased linearly in intensity with increasing concentrations of formate up to 25-fold excess over the enzyme. At higher formate concentration this signal disappeared. The appearance and disappearance of this Mo(V) signal seems to parallel the state of reduction of the [4Fe-4S] clusters. A second, minor, Mo(V) species with g-values g1 = 1.996, g2 = 1.981 and g3 = 1.941 appears at a constant level during the formate-dithionite titration. No evidence has been obtained for nuclear hyperfine coupling to protons. The major Mo(V) species has unusual e.p.r. signals compared with other molybdenum-containing enzymes, except for that observed in the formate dehydrogenase from Methanobacterium formicicum [Barber, Siegel, Schauer, May & Ferry (1983) J. Biol. Chem. 258, 10839-10845]. The present work suggests that the enzyme is acting as a CO2 reductase, with dithionite as an electron donor to a [4Fe-4S] cluster, which in turn donates electrons to molybdenum, producing a Mo(V) species with CO2 bound to the metal.     

Comparative quantitative structure toxicity relationships for flavonoids evaluated in isolated rat hepatocytes and HeLa tumor cells

Quantitative structure activity relationship (QSAR) equations were obtained to describe the cytotoxicity of 22 polyphenols using toxicity (logLD50) representing the concentration for 50% cell survival in 2 h for isolated rat hepatocytes, log P representing octanol/water partitioning, and/or E(p/2) representing redox potential. One- and two-parameter equations were derived for the quantitative structure toxicity relationships (QSTR) for polyphenol induced hepatocyte cytotoxicity: e.g. log C(hepatocyte) (microM)=-0.65(-0.08)log P+4.12(-0.15) (n=19, r(2)=0.80, s=0.33, P<1 x 10(-6)). One- and two-parameter QSAR equations were also derived to describe the inhibitory effects of 13 polyphenols on tumor cell growth when incubated with HeLa cells for 3 days: e.g. log C(tumor) (microM)=-0.34(+/-0.04)log P+2.40(+/-0.07) (n=11, r(2)=0.90, s=0.13, P<1 x 10(-5)). These findings point to lipophilicity as a major characteristic determining polyphenol cytotoxicity. The E(p/2) also played a significant role in polyphenol cytotoxicity towards both cell types: e.g. log C(hepatocyte) (microM)=-0.60(+/-0.06)log P+2.01(+/-0.43)E(p/2) (V)+3.86(+/-0.12) (n=9, r(2)=0.96, s=0.15, P<0.005). The involvement of log P and E(p/2) could be explained if polyphenol cytotoxicity involved the formation of radicals, which interacted with the mitochondrial inner membrane resulting in a disruption of the membrane potential.