Redox cycling of polycyclic aromatic hydrocarbon o-quinones: metal ion-catalyzed oxidation of catechols bypasses inhibition by superoxide dismutase.

Several two-electron quinone reductases catalyze the redox cycling of polycyclic aromatic hydrocarbon (PAH) o-quinones. When the carbonyl reductase of human placenta catalyzes the cycling of 9,10-phenanthrenequinone in aqueous phosphate buffer, reactive oxygen species are produced. Superoxide dismutase (SOD) inhibits the cycling by more than 90%, but the addition of 1 microM Cu2+ or 15 microM ferricytochrome c (cyt c3+) completely restores the cycling rate to that of the control. Similar results are obtained for 5,6-chrysenequinone, 5,6-benz[a]anthracenequinone, 4,5-benzo[a]pyrenequinone, and 7,8-benzo[a]pyrenequinone in assay mixtures which contain dimethyl sulfoxide. The 17beta-hydroxysteroid dehydrogenase (17beta-HSD) of human placenta also catalyzes the redox cycling of these quinones, and cycling is inhibited by SOD. Although free metal ions (Cu2+ and Fe3+) inhibit the 17beta-HSD, cyt c3+ does not inhibit the enzyme. If cyt c3+ is added to assay mixtures containing SOD, cycling rates are equal to those of the corresponding controls. These experiments suggest that SOD may not protect cells from the toxic effects of PAH o-quinone cycling if certain metal ions or metal chelates are also present.

Redox cycling of polycyclic aromatic hydrocarbon o-quinones: reversal of superoxide dismutase inhibition by ascorbate.

When redox cycling of four polycyclic aromatic hydrocarbon o-quinones is catalyzed by the 17 beta-hydroxysteroid dehydrogenase, autooxidation of the hydroquinone is a free radical chain reaction in which superoxide anion is the propagating species. Superoxide dismutase inhibits the redox cycling of these quinones, and ascorbate reverses this inhibition. Studies of the mechanism, using 9,10-phenanthrenequinone, show that ascorbate competes with superoxide dismutase for the superoxide anion; the ascorbyl radical formed then oxidizes the hydroquinone. In this mechanism, ascorbyl radical participates in chain propagation. The reversal of superoxide dismutase inhibition by ascorbate is observed when other two-electron reductases catalyze the cycling, and it occurs in the absence of metal ions. Although ascorbate is generally thought to be an antioxidant, it behaves as a prooxidant in the experiments reported here.

Polycyclic aromatic hydrocarbon quinone-mediated oxidation reduction cycling catalysed by a human placental 17beta-hydroxysteroid dehydrogenase.

The human placental 17beta-hydroxysteroid dehydrogenase reduces a number of polycyclic aromatic hydrocarbon (PAH) o-quinones; some of the quinones undergo redox cycling at rates that approach or exceed the rate of reduction of estrone by the enzyme. The non-K-region o-quinone, 7,8-benzo[a]pyrenequinone, is the best o-quinone substrate tested. Cycling of all the quinone substrates is inhibited by superoxide dismutase; cycling is also inhibited by 17beta-estradiol and other estrogens. Since 19 alpha-estradiol is a competitive inhibitor of 9,10-phenanthrenequinone by the 17beta-hydroxysteroid dehydrogenase, it is likely that both reactions occur at the same active site on the enzyme. In the presence of the 17beta-hydroxysteroid dehydrogenase, the equilibrium between 17beta-estradiol, estrone, NADP, and NADPH is shifted by 7,8-benzo[a]pyrenequinone because the rapid redox cycling of this quinone results in the oxidation of NADPH. Unlike a number of hydroxysteroid dehydrogenases, the placental 17beta-hydroxysteroid dehydrogenase does not oxidize any of the six PAH trans-dihydrodiols tested.

Effect of ascorbate on the DT-diaphorase-mediated redox cycling of 2-methyl-1,4-naphthoquinone.

Following the two-electron reduction of 2-methyl-1,4-naphthoquinone by rat liver DT-diaphorase (also called NAD(P)H: (quinone acceptor) oxidoreductase, EC 1.6.99.2), the hydroquinone product is slowly autoxidized to the quinone in buffered solutions at pH 7.0. The autoxidation, which generates the superoxide radical (O2-.) and other reactive oxygen species, is the rate-limiting step in the oxidation-reduction (redox) cycling of the quinone. The addition of ascorbate to these reaction mixtures increases the rate of redox cycling. Two mechanisms are proposed to explain this increase: (1) ascorbate reduces the quinone in a one-electron reduction and (2) if Fe(3+)-EDTA is present, ascorbate reduces the metal chelate in a one-electron reduction. Both mechanisms produce O2-. which initiates the free radical chain reaction that results in autoxidation of the hydroquinone. Although ascorbate may be a physiologically important antioxidant under some conditions, the studies reported here show that ascorbate is a prooxidant in the redox cycling of 2-methyl-1,4-naphthoquinone and, as such, could increase the potential toxicity of this quinone.

A chaperone-mimetic effect of serum albumin on rhodanese.

Reactivation of denatured rhodanese (thiosulfate:cyanide sulfurtransferase, EC 2.8.1.1) was found to be aided by the presence of serum albumin. Both the rate and the extent of reactivation of the urea-denatured enzyme were optimal at low rhodanese and moderate serum albumin concentrations. Similarly, stabilization of the sulfurtransferase activity of rhodanese that had been partially unfolded at 40 degrees C was aided by the presence of serum albumin. All the observations are in accord with a model in which enzyme that has been partially refolded from the urea-denatured state or partially unfolded thermally interacts directly with serum albumin in a way that prevents rhodanese self-association. Serum albumin thus acts as a molecular chaperone in these systems.

Localization of the sulfur-cyanolysis site of serum albumin to subdomain 3-AB.

The results of kinetic experiments measuring the effects of a variety of ligands on the sulfur-cyanolysis reaction catalyzed by serum albumin point to the conclusion that the active site for cyanolysis is on subdomain 3-AB. Relationships among the inhibition by short-chain fatty acids, the activation by p-nitrophenyl acetate, and the influence of bilirubin and L-tryptophan on these effects indicate that the cyanolysis active site and the known primary binding site for indoles are both near, but on opposite sides of, tyrosine-409 of bovine albumin (tyrosine-411 of human albumin).

Competitive partial inhibitors of serum albumin-catalyzed sulfur cyanolysis.

Efforts to locate the active site for sulfur cyanolysis catalyzed by bovine serum albumin have led to systematic tests of several compounds that inhibit the catalyzed reaction. Hexanoate and 5-dimethylaminonaphthalene-1-sulfonate bind at the same site and are partial inhibitors competitive with cyanide, uncompetitive with respect to sulfur. Various dansyl amino acids and 1-anilino-8-naphthalene sulfonate display the same inhibitory behavior but bis (1-anilino-8-naphthalene sulfonate) is a total inhibitor competitive with cyanide. These findings are interpreted to indicate that the cyanolysis active site is near, but not at, one of the short-chain fatty acid binding sites on albumin subdomain 2-AB or 3-AB. Both ionic repulsion and steric considerations are implicated in the mechanisms of inhibition.

Appendix to the sulfur-cyanolysis sites of serum albumin: metabolite competition studies. Tight-binding inhibitions that yield atypical Henderson plots.

Ordinary tight-binding inhibition in steady-state enzyme systems is conveniently evaluated by means of the Henderson plot. This is a linear plotting form that has an ordinate intercept equal to the total enzyme concentration. However, there are two experimental situations that yield deviations from the common Henderson plot form. These are inhibitor binding in a separate, noninhibitory mode that depletes the concentration of free inhibitor, and partial inhibition, i.e., the retention of partial activity by the enzyme-inhibitor complex. Noninhibitory depletion results in Henderson plots with elevated ordinate intercepts. Competitive partial inhibition yields a characteristic pattern of parabolic Henderson plots.

The sulfur-cyanolysis sites of serum albumin: metabolite competition studies.

In the presence of a source of sulfane sulfur, a cyanolysis reaction catalyzed by serum albumin may contribute to cyanide detoxication. The active site for this catalysis by serum albumin has been investigated in competition studies with ligands that have known albumin binding sites. Despite complications caused by the occurrence of multiple primary and secondary sites for many ligands, the results show that the primary sites for bilirubin, steroids, indoles, aspirin, and palmitate are distinct from that for sulfur. Laurate is a tight-binding partial inhibitor of the cyanolysis reaction, competitive with cyanide rather than with sulfur. In view of the formal mechanism previously established for the catalyzed reaction, this result indicates that the sulfur-cyanolysis site is probably near the site occupied by laurate.

Serum albumin and cyanide detoxication. Kinetic characterization of a reactive albumin-sulfur complex.

Kinetic studies of the serum albumin-catalyzed reaction of cyanide with colloidal elemental sulfur have been carried out to elucidate the formal mechanism of this reaction. The results indicate that one of the several sulfur-binding sites on the protein molecule is involved in a single displacement catalytic cycle in the presence of cyanide. At high velocities the rate of thiocyanate production is limited by an albumin-independent second order process which produces the form of sulfur that serves as substrate. The catalyzed reaction is also subject to substrate inhibition at high colloidal sulfur concentrations.

Evidence for dynamically determined conformational states in rhodanese catalysis.

Physical and kinetic studies have been used to explore hysteretic effects that are observed in rhodanese catalysis at pH 5 and also at neutral pH when the ionic strength of the medium is high. Experiments that involve observation of changes in intrinsic protein fluorescence of the enzyme and kinetic investigation of its interactions with product thiocyanate anion at pH 5 have implicated enzyme isomerization as the cause of hysteresis. Taken all together, the data indicate that the conformations of enzyme forms in the catalytic cycle are dynamically determined, depending on the relative rates of conformational relaxation and catalysis as influenced by the concentrations of substrates and products.

3-Mercaptopyruvate sulfurtransferase: rapid equilibrium-ordered mechanism with cyanide as the acceptor substrate.

A steady-state kinetic analysis of 3-mercaptopyruvate sulfurtransferase (EC 2.8.1.2) using cyanide as the sulfur-acceptor substrate was performed. Measurement of pyruvate production gave initial velocity patterns and secondary plots characteristic of a rapid equilibrium-ordered sequential mechanism. Initial velocity data obtained by measuring the formation of thiocyanate, which is the other reaction product, revealed a discrepancy between the rates of pyruvate and thiocyanate production; the yield of thiocyanate per unit time was smaller than that of pyruvate for each reaction mixture. This velocity discrepancy, which diminished with approach to cyanide saturation, suggests that sulfur is not discharged from the enzyme as thiocyanate, but as elemental sulfur, and that thiocyanate is formed in a subsequent nonenzymic step. A formal mechanism which has a rapid equilibrium-ordered catalytic cycle and elemental sulfur as one of the initial reaction products is proposed. Computer simulation is used to show that this model is in agreement with all of the kinetic data.