Effects of ligands on reduction of oxygen by vanadium(IV) and vanadium(III).

V(IV) and V(III) reduce molecular oxygen with increasing rates as the pH is raised from 6.0 to 7.4. Under all conditions tested, V(IV) is the more efficient reductant. EDTA and ATP generally inhibit the reduction of oxygen by V(III) and V(IV). In contrast, desferrioxamine accelerates the reduction of oxygen by V(IV) but with decreasing effectiveness at pH 7.4 compared to pH 6.0, while desferrioxamine accelerates the reduction of oxygen by V(III) only at pH 6.0. Histidine enhances the reduction of oxygen by V(IV) at pH 7.0 and 7.4. The observed rates of oxygen reduction by V(III) and V(IV) imply that the intracellular distribution of vanadium among its redox states reflects not an equilibrium but a steady state.     

Reduction of vanadium(V) to vanadium(IV) by NADPH, and vanadium(IV) to vanadium(III) by cysteine methyl ester in the presence of biologically relevant ligands

To better understand the mechanism of vanadium reduction in ascidians, we examined the reduction of vanadium(V) to vanadium(IV) by NADPH and the reduction of vanadium(IV) to vanadium(III) by L-cysteine methyl ester (CysME). UV-vis and electron paramagnetic resonance spectroscopic studies indicated that in the presence of several biologically relevant ligands vanadium(V) and vanadium(IV) were reduced by NADPH and CysME, respectively. Specifically, NADPH directly reduced vanadium(V) to vanadium(IV) with the assistance of ligands that have a formation constant with vanadium(IV) of greater than 7. Also, glycylhistidine and glycylaspartic acid were found to assist the reduction of vanadium(IV) to vanadium(III) by CysME.     

Reduction of vanadium(V) by <Emphasis Type="Italic">Enterobacter cloacae</Emphasis> EV-SA01 isolated from a South African deep gold mine

Dissimilatory reduction of vanadium(V) by Enterobacter cloacae EV-SA01, isolated from a gold mine at 1.6 km below surface, is shown to occur anaerobically as well as aerobically. Growth rates were unaffected by up to 2 mM V₂O₅. Reduction of vanadium(V) was growth phase-dependent and resulted in cell deformities and precipitation of the vanadium in its lower oxidation states. The vanadate reductase activity was membrane-associated and coupled the oxidation of NADH to the reduction of vanadate.     

Kinetic studies on oxidized and partially reduced cytochrome c oxidase.

1. Kinetic studies have been performed with beef-heart cytochrome c oxidase, with the enzyme either in its oxidized, resting state or pretreated anaerobically with different amounts of reduced cytochrome c. The techniques used for the study have been stopped-flow spectrophotometry and electron paramagnetic resonance (EPR) spectroscopy. 2. The results show that the one-electron equivalent-reduced enzyme rapidly oxidizes one further equivalent of aerobically or anaerobically added ferrocytochrome c, with a rate constant of 5 . 10(6) M-1 . s-1. 3. When an excess of ferrocytochrome c in the presence of oxygen is added to the one-electron-reduced enzyme, the same turnover rate is obtained as in experiments with the resting enzyme. 4. The one-electron equivalent-enzyme reacts with CO with a rate constant of 4 . 10(4) M-1 . s-1 to yield approx. 35% of the CO compound as compared with the reaction between the fully reduced enzyme and CO. 5. It is shown that on reduction the enzyme is converted into an active form, but it is concluded that the enzyme does not have to be fully reduced before it is catalytically active.     

The permeability and cytotoxicity of insulin-mimetic vanadium (III,IV,V)-dipicolinate complexes

Vanadium (III,IV,V)-dipicolinate complexes with different redox properties were selected to investigate the structure-property relationship of insulin-mimetic vanadium complexes for membrane permeability and gastrointestinal (GI) stress-related toxicity using the Caco-2 cell monolayer model. The cytotoxicity of the vanadium complexes was assayed with 3-(4,5-dimethylthiazoyl-2-yl) 2,5-diphenyltetrazolium bromide (MTT) assays and the effect on monolayer integrity was measured by the trans-epithelial electric resistance (TEER). The three vanadium complexes exhibited intermediate membrane permeability (P(app) = 1.4-3.6x10(-6) cm/s) with low cellular accumulation level (<1%). The permeability of all compounds was independent of the concentration of vanadium complexes and excess picolinate ligands. Both V(III) and V(V)-dipicolinate complexes induced 3-4-fold greater reactive oxygen and nitrogen species (RONS) production than the V(IV)-dipicolinate complex; while the vanadium (III)-dipicolinate was 3-fold less damaging to tight junction of the Caco-2 cell monolayer. Despite the differences in apparent permeability, cellular accumulation, and capacity to induce reactive oxygen and nitrogen species (RONS) levels, the three vanadium complexes exhibited similar cytotoxicity (IC50 = 1.7-1.9 mM). An ion pair reagent, tetrabutylammonium, increased the membrane apparent permeability by 4-fold for vanadium (III and IV)-dipicolinate complexes and 16-fold for vanadium (V)-dipicolinate as measured by decrease in TEER values. In addition, the ion pair reagent prevented damage to monolayer integrity. The three vanadium (III,IV,V)-dipicolinate complexes may pass through caco-2 monolayer via a passive diffusion mechanism. Our results suggest that formation of ion pairs may influence compound permeation and significantly reduce the required dose, and hence the GI toxicity of vanadium-dipicolinate complexes.     

Flavoenzymes reduce vanadium(V) and molecular oxygen and generate hydroxyl radical.

ESR spectroscopic evidence is presented for the formation of vanadium(IV) in the reduction of vanadium(V) by three typical, NADPH-dependent, flavoenzymes: glutathione reductase, lipoyl dehydrogenase, and ferredoxin-NADP+ oxidoreductase. The vanadium(V)-reduction mechanism appears to be an enzymatic one-electron reduction process. Addition of superoxide dismutase (SOD) showed that the generation of vanadium(IV) does not involve the superoxide (O2-) radical significantly. Measurements under anaerobic atmosphere showed, however, that the enzymes-vanadium-NADPH mixture can cause the reduction of molecular oxygen to generate H2O2. The H2O2 and vanadium(IV) thus formed react to generate hydroxyl (.OH) radical. The .OH formation is inhibited strongly by catalase and to a lesser degree by SOD, but it is enhanced by exogenous H2O2, suggesting the occurrence of a Fenton-like reaction. The inhibition of vanadium(IV) formation by N-ethylmaleimide indicates that the SH group on the flavoenzyme's cystine residue plays an important role in the enzyme's vanadium(V) reductase function. These results thus reveal a new property of the above-mentioned, NADPH-dependent flavoenzymes--their function as vanadium(V) reductases, as well as that as generators of .OH radical in the vanadium(V) reduction mechanism.     

The microbial metabolism of C1 compounds. The electron-transport chain of Pseudomonas AM1

Pseudomonas AM1, Hyphomicrobium X and Pseudomonas MS all contain cytochrome a/a(3) and a b-type cytochrome able to react with CO. Pseudomonas AM1 and Hyphomicrobium X also have a CO-binding cytochrome c. The purified cytochrome c (redox potential 0.26V) of Pseudomonas AM1 was not susceptible to oxidation by molecular oxygen. CO reacted slowly with the reduced form giving a CO difference spectrum with a peak at 412nm and troughs at 420nm and 550nm. Similar results were obtained with the cytochrome c of Hyphomicrobium (aerobically grown or anaerobically grown with nitrate) and with that of Pseudomonas extorquens. The results given in the present paper are incompatible with an oxygenase or oxidase function for the soluble cytochrome c of methylotrophs. Studies with whole cells of Pseudomonas AM1 and a cytochrome c-deficient mutant have demonstrated that cytochrome b (redox potential 0.009V) is the first cytochrome in the electron-transport chain for oxidation of all substrates except methanol (and ethanol) whose oxidation does not involve this cytochrome. All substrates are usually oxidized by way of cytochrome c and cytochrome oxidase (cytochrome a/a(3)), but there is an alternative route for the reduction of cytochrome a/a(3) in the mutant lacking cytochrome c. Results of experiments on cyanide inhibition of respiration and cytochrome oxidation support the suggestion that the susceptibility of cytochrome b to oxidation by molecular oxygen (reflected in its ability to react with CO) is probably irrelevant to the normal physiology of Pseudomonas AM1.     

Reaction of vanadium(V) with thiols generates vanadium (IV) and thiyl radicals

The in vivo toxicity of vanadium(V) has been found to correlate with the depletion of cellular glutathione and related non-protein thiols. With a view to understanding the mechanism for this observation, we have investigated the oxidation of glutathione, cysteine N-acetylcysteine and penicillamine by vanadium(V), using electron spin resonance (ESR) and ESR spin trapping methodology. The spin trap used was 5,5-dimethyl-1-pyrroline 1-oxide (DMPO). It is found that the oxidation of these thiols by vanadium(V) generates the corresponding thiyl radicals and vanadium- (IV) complexes. The results suggest that free radical reactions play a significant role in the depletion of cellular thiols by vanadium(V) and hence in vanadium(V) toxicity.     

Electrochemical reactions of horse heart cytochrome c at graphite electrodes

The interaction of cytochrome c with a paraffin-wax-impregnated spectroscopic graphite electrode (WISGE) was studied in a medium consisting of 0.1 M potassium phosphate, pH 7.0, by means of differential pulse and cyclic voltammetry. Ferricytochrome c yields on voltammograms a single cathodic peak C around a potential of -0.3 V (vs. Ag/AgCl) and two anodic peaks AI and AII around the potentials of 0.66 and 0.89 V, respectively. Cathodic peak C corresponds to a catalytic reaction during which ferricytochrome c is reduced to ferrocytochrome c: ferricytochrome c is then regenerated by chemical oxidation of ferrocytochrome c by oxygen adsorbed at the WISGE surface. The first, more negative anodic peak AI corresponds to anodic electrochemical oxidation of tyrosine residues, whereas the second, more positive anodic peak (peak AII) corresponds to an anodic reaction of haemin. Voltammetry at a WISGE may provide a valuable technique for obtaining data about cytochrome c properties on electrically charged surface.     

Oxidation of D-galacturonic acid by vanadium(V)

The kinetics of the oxidation of D-galacturonic acid by vanadium(V) in acid solution have been studied. The reaction is of the first order with respect to both vanadium(V) and the organic substrate. Formic acid and oxovanadium(IV) are the final reaction products. The reaction rate is increased with increasing acidity, suggesting that variously protonated vanadium(V) species are active in the substrate oxidation.     

Superoxide-independent reduction of vanadate by rat liver microsomes/NAD(P)H: vanadate reductase activity.

It has been reported that vanadate-stimulated oxidation of NAD(P)H by microsomal systems can proceed anaerobically, in contrast to the general notion that the oxidation proceeds exclusively by an O(2-)-dependent free radical chain mechanism. The current study indicates that microsomal systems are endowed with a vanadate-reductase property, involving a NAD(P)H-dependent electron transport cytochrome P450 system. Our ESR measurements demonstrated the formation of a vanadium(IV) species in a mixture containing vanadate, rat liver microsomes, and NAD(P)H. This vanadium(IV) species was identified as the vanadyl ion (VO2+) by comparison with the ESR spectrum of VOSO4. The initial rate of vanadium(IV) formation depends linearly on the concentration of microsomes. The Michaelis-Menten constants were found to be: km = 1.25 mM and Vmax = 0.066 mumol (min)-1 (mg microsomes)-1, respectively. Pretreatment of the microsomes with carbon monoxide or K3Fe(CN)6 reduced vanadium(IV) generation, suggesting that the NAD(P)H-dependent electron transport cytochrome P450 system plays a significant role in the microsomal reduction of vanadate. Measurements under argon or in the presence of superoxide dismutase caused only minor (less than 10%) reductions in vanadium(IV) generation. The VO2+ species was also detected in NAD(P)H oxidation by fructose plus vanadate, a reaction known to proceed via an O(2-)-mediated chain mechanism. However, the amount of vanadium(IV) generated by this reaction was an order of magnitude smaller than that by the microsomal system and was inhibitable by superoxide dismutase, affirming the conclusion that the microsomal/NAD(P)H system is endowed with the (O(2-)-independent) vanadium(V) reductase property.     

Spectrophotometric and ESR evidence for vanadium(IV) deferoxamine complexes.

Complexes of vanadium(IV), vanadyl, are reported to be formed with the trihydroxamic acid deferoxamine (H3DF+). One complex exhibits a reddish-violet color, with a major absorbance peak at 386 nm and a smaller peak at 520 nm. This complex is potentially useful for the microdetermination of vanadyl. The apparent molar absorptivity is 3.91 mM-1 cm-1, and the complex obeys Beer's law in the concentration range of 0.6-63 ppm. Electron spin resonance studies indicate the formation of two vanadyl complexes that are 1:1 in vanadyl and deferoxamine, but have two or three bound hydroxamate groups. ESR and spectrophotometric evidence indicate that the red, low pH form, involves an octahedral vanadium (4+) ion coordinated by three hydroxamate ligands. One of these hydroxamates is displaced by an oxygen at pH greater than 2.8 according to the following equilibria: VO2+ + H3DF+ in equilibrium with VIV(DF)2+ + H3O+, VIV(DF)2+ + H2O in equilibrium with VO(HDF)+ + H+, where pk2 = 2.8.     

Activation of Pseudomonas cytochrome oxidase by limited proteolysis with subtilisin

Oxidized Pseudomonas cytochrome oxidase (ferrocytochrome c2: oxygen oxidoreductase; E.C.1.9.3.2) can be digested with subtilisin under controlled conditions that convert the original parent polypeptide chain (Mr on SDS gels approximately equal to 60,000) to a slightly smaller species (Mr on SDS gels approximately equal to 58,000). Under the conditions used (0.33% subtilisin, w/w, pH 7.4), the product formed from the oxidase was relatively stable to further digestion. Cytochrome oxidase activity was assayed at intervals during proteolysis by following the rate of oxidation of Pseudomonas ferrocytochrome c-551 by the enzyme in the presence of oxygen. The activity increased to a plateau that was more than two times the value for an untreated control. These observations suggest that clipping a small peptide from Pseudomonas cytochrome oxidase either facilitates the rate-limiting electron transfer between the intraprotein heme c and heme d1, enhances the interaction of the enzyme with ferrocytochrome c-551, or both.     

Synthesis, spectroscopy, and biological properties of vanadium(IV)-hydrazide complexes

The synthesis, spectroscopic, enzyme-inhibition, and free-radical-scavenging properties of a series of vanadium(IV) complexes, compounds 1-10, were investigated. These complexes exhibit a dimeric structure with hydrazide ligands coordinated in a bidentate fashion. All complexes are stable in the solid state, but exhibit varying degrees of stability in solution. In coordinating solvent such as DMSO, stepwise binding of two solvent molecules at the 6th positions trans to the V double bond O bond of the dimeric unit is observed. The dimeric compounds are converted to monomeric species in which both solvent molecules and the hydrazide ligands are coordinated to the V(IV) center. The free hydrazide ligands 11-20 were inactive against alpha-glucosidase, but the V(IV) complexes showed varying degrees of inhibition, depending on the type of ligand. The DPPH-radical-scavenging activities of 1-20 were determined, which indicated that steric and/or electronic effects responsible for changes in geometry play important roles in terms of antioxidant potential.     

Reduction of cytochrome c peroxidase compounds I and II by ferrocytochrome c. A stopped-flow kinetic investigation

The oxidation of yeast cytochrome c peroxidase by hydrogen peroxide produces a unique enzyme intermediate, cytochrome c peroxidase Compound I, in which the ferric heme iron has been oxidized to an oxyferryl state, Fe(IV), and an amino acid residue has been oxidized to a radical state. The reduction of cytochrome c peroxidase Compound I by horse heart ferrocytochrome c is biphasic in the presence of excess ferrocytochrome c as cytochrome c peroxidase Compound I is reduced to the native enzyme via a second enzyme intermediate, cytochrome c peroxidase Compound II. In the first phase of the reaction, the oxyferryl heme iron in Compound I is reduced to the ferric state producing Compound II which retains the amino acid free radical. The pseudo-first order rate constant for reduction of Compound I to Compound II increases with increasing cytochrome c concentration in a hyperbolic fashion. The limiting value at infinite cytochrome c concentration, which is attributed to the intracomplex electron transfer rate from ferrocytochrome c to the heme site in Compound I, is 450 +/- 20 s-1 at pH 7.5 and 25 degrees C. Ferricytochrome c inhibits the reaction in a competitive manner. The reduction of the free radical in Compound II is complex. At low cytochrome c peroxidase concentrations, the reduction rate is 5 +/- 3 s-1, independent of the ferrocytochrome c concentration. At higher peroxidase concentrations, a term proportional to the square of the Compound II concentration is involved in the reduction of the free radical. Reduction of Compound II is not inhibited by ferricytochrome c. The rates and equilibrium constant for the interconversion of the free radical and oxyferryl forms of Compound II have also been determined.     

Reduction of vanadium(V) with Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans

Biotechnological leaching has been proposed as a suitable method for extraction of vanadium from spent catalysts and oil ash. In the biological leaching process, the vanadium(V) can be reduced to vanadium(IV), which is a less toxic and more soluble form of the vanadium. The present investigation showed that Acidithiobacillus ferrooxidans efficiently reduced vanadium(V) in the form of vanadium pentaoxide, to vanadyl(IV) ions, and tolerated high concentrations of vanadium(IV) and vanadium(V). A. ferrooxidans was compared with Acidithiobacillus thiooxidans, which has previously been utilized for vanadium leaching and reduction. Vanadium pentaoxide and sodium vanadate were used as model compounds. The results of this study indicate possibilities to develop an economical and technically feasible process for biotechnological vanadium recovery.     

Model investigations for vanadium-protein interactions: vanadium(III) compounds with dipeptides and their oxovanadium(IV) analogues

The reaction of VCl(3) with 1,10-phenanthroline and a series of dipeptides (H(2)dip), having aliphatic as well as aromatic side chains, in methyl alcohol and in the presence of triethylamine affords vanadium(III) compounds of the general formula [V(III)(dip)(MeOH)(phen)]Cl. Aerial oxidation/hydrolysis of the vanadium(III) species gives their oxovanadium(IV) analogues of the general formula [V(IV)O(dip)(phen)]. X-ray crystallographic characterization of the [V(IV)O(dip)(phen)] compounds (where dip(2-)=Gly- L-Ala, Gly- L-Val and Gly- L-Phe) revealed that the vanadium atom possesses a severely distorted octahedral coordination and is ligated to a tridentate dip(2-) ligand at the N(amine) atom, the deprotonated N(peptide) atom and one of the O(carboxylate) atoms, as well as an oxo group and two phenanthroline nitrogen atoms. Circular dichroism characterization of the V(III)/V(IV)O(2+)-dipeptide compounds revealed a strong signal for the V(IV)O(2+) species in the visible range of the spectrum, with a characteristic pattern which may be exploited to identify the N(am), N(pep) and O(car) ligation of a peptide or a protein to V(IV)O(2+) center, and a weak Cotton effect of opposite sign to their vanadium(III) analogues. The visible spectra of the V(III)-dipeptide compounds revealed two d-d bands with high intensity, thus indicating that the covalency of the metal-donor atoms is significant, i.e. the vanadium d orbitals are significantly mixed with the ligand orbitals, and this is confirmed by the low values of their Racah B parameters. The high-intensity band of the V(IV)O(2+)-dipeptide compounds at approximately 460 nm implies also a strong covalency of the metal with the equatorial donor atoms and this was supported by the EPR spectra of these compounds. Moreover, the V(III)/V(IV)O(2+)-dipeptide complexes were characterized by EPR and IR spectroscopies as well as conductivity and magnetic susceptibility measurements.     

Cytochrome c oxidase from bakers' yeast. IV. Immunological evidence for the participation of a mitochondrially synthesized subunit in enzymatic activity.

In order to study the role of the individual subunits of yeast cytochrome c oxidase, rabbit antisera were prepared against Subunit II (a mitochondrially made polypeptide) and Subunit VI (a cytoplasmically made polypeptide). Antisera were also obtained against a mixture of the two mitochondrially made subunits (I PLUS II) and against mixtures of the following cytoplasmically made subunits: (IV PLUS VI); (V PLUS VII); and (IV PLUS V PLUS VI PLUS VII). Neither anti-II serum nor anti-VI serum cross-reacted with any of the other six subunits of cytochrome c oxidase as judged by a sensitive ring test or by double diffusion in agarose gels. Anti-II serum inhibited the oxidation of ferrocytochrome c by purified yeast cytochrome c oxidase or by freshly isolated as well as sonically fragmented yeast mitochondria. Anti-(V, VII) serum and anti-(IV, V, VI, VII) serum were also strongly inhibitory. Anti-VI serum and anti-(IV, VI) serum inhibited only weakly. If purified cytochrome c oxidase was inhibited with a saturating amount of anti-VI serum, anti-II serum elicited a further increment of inhibition, as would be expected if the inhibitory effects of these two antisera involved different antigenic sites on the holoenzyme. Each of the antisera precipitated all seven cytochrome c oxidase subunits from crude mitochondrial extracts. However, anti-VI and, particularly, anti-II were much less effective precipitants than antisera against Subunits IV to VII or antisera against the holoenzyme. These data suggest that the oxidation of ferrocytochrome c by cytochrome c oxidase required both mitochondrially as well as cytoplasmically made subunits.     

Microbial reduction and precipitation of vanadium by Shewanella oneidensis

Shewanella oneidensis couples anaerobic oxidation of lactate, formate, and pyruvate to the reduction of vanadium pentoxide (V(V)). The bacterium reduces V(V) (vanadate ion) to V(IV) (vanadyl ion) in an anaerobic atmosphere. The resulting vanadyl ion precipitates as a V(IV)-containing solid.