Rate of electron transfer between cytochrome b561 and extravesicular ascorbic acid

Cytochrome b561 transfers electrons across secretory vesicle membranes in order to regenerate intravesicular ascorbic acid. To show that cytosolic ascorbic acid is kinetically competent to function as the external electron donor for this process, electron transfer rates between cytochrome b561 in adrenal medullary chromaffin vesicle membranes and external ascorbate/semidehydroascorbate were measured. The reduction of cytochrome b561 by external ascorbate may be measured by a stopped-flow method. The rate constant is 450 (+/- 190) M-1 s-1 at pH 7.0 and increases slightly with pH. The rate of oxidation of cytochrome b561 by external semidehydroascorbate may be deduced from rates of steady-state electron flow. The rate constant is 1.2 (+/- 0.5) x 10(6) M-1 s-1 at pH 7.0 and decreases strongly with pH. The ratio of the rate constants is consistent with the relative midpoint reduction potentials of cytochrome b561 and ascorbate/semidehydroascorbate. These results suggest that cytosolic ascorbate will reduce cytochrome b561 rapidly enough to keep the cytochrome in a mostly reduced state and maintain the necessary electron flux into vesicles. This supports the concept that cytochrome b561 shuttles electrons from cytosolic ascorbate to intravesicular semidehydroascorbate, thereby ensuring a constant source of reducing equivalents for intravesicular monooxygenases.     

Nitric Oxide in Plants: The Roles of Ascorbate and Hemoglobin

Ascorbic acid and hemoglobins have been linked to nitric oxide metabolism in plants. It has been hypothesized that ascorbic acid directly reduces plant hemoglobin in support of NO scavenging, producing nitrate and monodehydroascorbate. In this scenario, monodehydroascorbate reductase uses NADH to reduce monodehydroascorbate back to ascorbate to sustain the cycle. To test this hypothesis, rates of rice nonsymbiotic hemoglobin reduction by ascorbate were measured directly, in the presence and absence of purified rice monodehydroascorbate reductase and NADH. Solution NO scavenging was also measured methodically in the presence and absence of rice nonsymbiotic hemoglobin and monodehydroascorbate reductase, under hypoxic and normoxic conditions, in an effort to gauge the likelihood of these proteins affecting NO metabolism in plant tissues. Our results indicate that ascorbic acid slowly reduces rice nonsymbiotic hemoglobin at a rate identical to myoglobin reduction. The product of the reaction is monodehydroascorbate, which can be efficiently reduced back to ascorbate in the presence of monodehydroascorbate reductase and NADH. However, our NO scavenging results suggest that the direct reduction of plant hemoglobin by ascorbic acid is unlikely to serve as a significant factor in NO metabolism, even in the presence of monodehydroascorbate reductase. Finally, the possibility that the direct reaction of nitrite/nitrous acid and ascorbic acid produces NO was measured at various pH values mimicking hypoxic plant cells. Our results suggest that this reaction is a likely source of NO as the plant cell pH drops below 7, and as nitrite concentrations rise to mM levels during hypoxia.     

Platinum(II) catalysis and radical intervention in reductions of platinum(IV) antitumor drugs by ascorbic acid

Reductions of four platinum(IV) amine complexes, cis-diamminetetrachloroplatinum(IV), tetraammine-cis-dichloroplatinum(IV), cis,cis,trans-diamminedichlorodihydroxoplatinum(IV), and cis,trans,cis-dichloro-dihydroxo-bis(isopropylamine)platinum(IV) by ascorbic acid were catalyzed by platinum(II) at pH 7.3 and 22 degrees C. Except for the first mentioned compound, initial slow uncatalyzed reductions yielded platinum(II) products which served as catalyst as revealed by the presence of induction periods and their disappearance by the addition of the platinum(II) products. The platinum(II) catalysis generated ascorbate bound platinum(IV) intermediates. An internal electron transfer process within these intermediates led to the formation of platinum(II) complexes. Although the rate constants for the uncatalyzed reductions vary greatly depending on the nature of the ligands and their spatial arrangements, the magnitudes of the platinum(II) catalyzed rate constants fall in the narrow range, 100 to 300 M(-2) s(-1). The values of the uncatalyzed reductions lie in the range 5 x 10(-2) to 15 M(-1) s(-1), the tetrachloroplatinum(IV) complex suffered the faster reduction. The reduction of iproplatin with two hydroxide ligands in trans configuration was the slowest. The internal electron transfer rate constants span two orders of magnitude, from 0.15 to 4 x 10(-3) s(-1). These reactions were accompanied by the formation of the ascorbate radical which persists throughout the entire reaction. Although the tetrachloro species exhibited simple second order reduction, first order in each of the reactants, the rate of reduction was also accelerated by the addition of cis-diamminedichoroplatinum(II) indicating the presence of catalysis in this reaction as well.     

Studies on the reactions of ferric iron with ascorbic acid. A study of solution chemistry using Mössbauer spectroscopy and stopped-flow techniques

We report studies on the interaction of iron(III) and ascorbic acid as a function of pH in pure water, pure methanol and mixtures of these solvents.Mössbauer data indicates the iron(III) is reduced in water at low pH to iron(II). Rapid mixing studies and pH jump investigations using stopped flow spectrophotometry have been used to follow the reactions and show evidence for blue intermediates in the reduction pathway of iron at low pH values. A scheme is proposed to account for the complex reaction between iron and ascorbate in aqueous solvent. Binding constants between iron(III) and ascorbate are given.     

Effect of organic phosphates on methemoglobin reduction by ascorbic acid.

The rate of methemoglobin reduction by ascorbic acid was accelerated in the presence of ATP,2,3-diphosphoglycerate (2,3-DPG), and inositol hexaphosphate (IHP). The acceleration was as much as three times, four times, and ten times in the presence of ATP, 2.3-DPG, and IHP at pH 7.0, respectively. The changes of the concentrations of methemoglobin and ascorbic acid during the methemoglobin reduction were determined, and the reaction was found to proceed stoichiometrically in the presence of IHP. The reduction rate of methemoglobin by ascorbic acid was compared at different concentrations of organic phosphates (ATP,2,3-DPG, and IHP) at various pH values (6.3, 7.0, 7.7). From the changes in the reduction rate under different concentrations of organic phosphates, the dissociation constants of ATP, 2,3-DPG, and IHP to methemoglobin could be determined and were estimated to be 3.3 X 10(-4) M, 2 X 10(-3) M, and 8 X 10(-6) M at pH 7.0, respectively. On the basis of these results, the acceleration mechanism of methemoglobin reduction by ascorbic acid due to the presence of organic phosphates was described. The physiological role of 2,3-DPG in human red cells was discussed in relation to the reduction of methemoglobin by ascorbic acid.     

Reaction of ascorbic acid with cytochrome b561. Concerted electron and proton transfer.

Rate constants for reduction of cytochrome b561 by internal ascorbate (k0A) and oxidation by external ferricyanide (k1F) were determined as a function of pH from rates of steady-state electron transfer across chromaffin-vesicle membranes. The pH dependence of electron transfer from cytochrome b561 to ferricyanide (k1F) may be attributed to the pH dependence of the membrane surface potential. The rate constant for reduction by internal ascorbate (k0A), like the previously measured rate constant for reduction by external ascorbate (k-1A), is not very pH-dependent and is not consistent with reduction of cytochrome b561 by the ascorbate dianion. The rate at which ascorbate reduces cytochrome b561 is orders of magnitude faster than the rate at which it reduces cytochrome c, despite the fact that midpoint reduction potentials favor reduction of cytochrome c. Moreover, the rate constant for oxidation of cytochrome b561 by ferricyanide (k1F) is smaller than the previously measured rate constant for oxidation by semidehydroascorbate, despite the fact that ferricyanide has a higher midpoint reduction potential. These results may be reconciled by a mechanism in which electron transfer between cytochrome b561 and ascorbate/semidehydroascorbate is accelerated by concerted transfer of a proton. This may be a general property of biologically significant electron transfer reactions of ascorbic acid.     

Ascorbate- and dehydroascorbic acid-mediated reduction of free radicals in the human erythrocyte

Nitroxides were used as models of persistent free radicals to study the antioxidant function of ascorbic acid in the human erythrocyte. It was concluded that: 1) ascorbate and other reductant(s) derived from dehydroascorbic acid (DHA) in the presence of thiols are the only significant reducing agents for nitroxides, 2) glutathione and DHA reduce nitroxides by a process that cannot be inhibited by ascorbic acid oxidase, 3) erythrocytes can be depleted of ascorbic acid by exhaustive washing in the presence of membrane-permeable cationic nitroxides such as N,N-dimethylamino-Tempo, 4) ascorbate-depleted cells do not reduce nitroxides; however, nitroxide reduction is restored when the cells are incubated with DHA, 5) reduction of nitroxides in ascorbate-depleted, DHA-treated cells is significantly faster than in buffered solutions of DHA and glutathione, 6) several equivalents of nitroxide are reduced relative to the intracellular ascorbate pool, 7) sustained nitroxide reduction is observed even when most of the intracellular ascorbate is oxidized, 8) spin trapping of oxyradicals in tert-butyl hydroperoxide-treated cells is accelerated with ascorbate depletion and inhibited with ascorbate loading, 9) ascorbate can be quantified within intact cells by analyzing the initial reduction rates of membrane-permeable cationic nitroxides, and 10) DHA-stimulated reduction of cationic nitroxides is slower and less extensive in erythrocytes deficient in glucose-6-phosphate dehydrogenase than in normal erythrocytes.     

Reversible reduction of nitroxides to hydroxylamines: roles for ascorbate and glutathione

Biological applications of stable nitroxyl radicals, NR, include their use as contrast agents for magnetic resonance imaging, spin labels, superoxide dismutase mimics, and antioxidants. The rapid reduction of NR in biological samples into hydroxylamines (HA) significantly limits their application. In turn, reoxidation of HA back to the NR has been used for detection of reactive oxygen species (ROS). In this work comparative studies of the reduction of pyrrolidine, imidazoline, and imidazolidine NR by ascorbate were performed taking advantage of recently synthesized tetraethyl-substituted NR with much higher stability toward reduction both in vitro and in vivo. Surprisingly, these NR kept 10-50% of initial intensity of electron paramagnetic resonance signal for about 1 h in the presence of 100-fold excess of ascorbate. To explain these data, reoxidation of the corresponding HA by ascorbate radical and dehydroascorbic acid back to the NR was proposed. This hypothesis was supported by direct measurement of the NR appearance from the HA on ascorbate radical generation by ascorbate oxidase, or in the presence of the dehydroascorbic acid. The reversible reaction between NR and ascorbate was observed for the various types of NR, and the rate constants for direct and reverse reactions were determined. The equilibrium constants for one-electron reduction of the tetraethyl-substituted NR by ascorbate were found to be in the range from 2.65x10(-6) to 10(-5) which is significantly lower than corresponding values for the tetramethyl-substituted NR (more or about 10(-4)). This explains the establishment of an EPR-detectable quasi-equilibrium level of tetraethyl-substituted NR in the presence of an excess of ascorbate. The redox reactions of the NR-HA couple in ascorbate-containing media were found to be significantly affected by glutathione (GSH). This effect was attributed to the reduction of ascorbate radicals by GSH, and the rate constant of this reaction was found to be equal to 10 M-1 s-1. In summary, the data provide new insight into the redox chemistry of NR and HA, and significantly affect interpretation and strategy of their use as redox- and ROS-sensitive probes, or as antioxidants.     

6-Bromo-6-deoxy-L-ascorbic acid: an ascorbate analog specific for Na+-dependent vitamin C transporter but not glucose transporter pathways

Vitamin C intracellular accumulation is mediated by Na(+)-dependent vitamin C transporters SVCT1 and -2 and dehydroascorbic acid transporters GLUT1 and -3. It is unclear which pathways dominate in vivo. As a new step to resolve this issue, we identified and tested 6-bromo-6-deoxy-L-ascorbic acid as a specific candidate for SVCTs. In high performance liquid chromatography and electron paramagnetic resonance analyses, the reduced compounds ascorbic acid and 6-bromo-6-deoxy-L-ascorbic acid were similar. The oxidized products 6-bromo-6-deoxy dehydroascorbic acid (BrDHA) and dehydroascorbic acid (DHA) had comparable stabilities, based on reduction recoveries. Upon expression of GLUT1 or GLUT3 in Xenopus oocytes, BrDHA was neither transported nor bound, in contrast to robust transport of DHA. The findings were not explained by differences in the oocyte reduction of DHA and BrDHA because lysed oocytes reduced both compounds equally. Further, there was no transport of the reduced compound, 6-bromo-6-deoxy-L-ascorbic acid, by GLUT1 or GLUT3. As a prerequisite for investigating 6-bromo-6-deoxy-L-ascorbic acid transported by SVCTs, SVCT2 transport activity in oocytes was enhanced 14-fold by construction and use of a vector that added a fixed poly(A) tail to the 3' end of cRNA. For SVCT1 and SVCT2 expressed in oocytes, similar K(m) and V(max) values were observed for ascorbic acid and 6-bromo-6-deoxy-L-ascorbic acid. In human fibroblasts, predicted to have SVCT-mediated ascorbate accumulation, K(m) and V(max) values were again comparable for ascorbic acid and 6-bromo-6-deoxy-L-ascorbic acid. Using activated human neutrophils, predicted to have ascorbate accumulation mediated predominantly by DHA and GLUT transporters, 6-bromo-6-deoxy-L-ascorbic acid accumulation was <1% of accumulation when compared with ascorbic acid. We conclude that 6-bromo-6-deoxy-L-ascorbic acid is the first transport substrate identified as completely specific for SVCTs, but not GLUTs, and provide a new strategy to determine the contribution of each pathway to ascorbate accumulation.     

Kinetic studies of the copper nitrite reductase from Achromobacter cycloclastes and its interaction with a blue copper protein

Transient state, burst and steady state kinetics of reactions of the blue copper nitrite reductase (NIR) and blue copper protein from Achromobacter cycloclastes are investigated. The two copper-containing species are reacted with each other and where possible with dithionite, ascorbate and nitrite. Both copper proteins are fully reduced by dithionite with both S2O4(2-) and SO2-. species active. NIR is only partially reduced by ascorbate in an unusual biphasic reaction consistent with complete reduction of type-one copper followed by partial reduction of type-two copper. The rate of reduction of the type-one copper is accelerated using phenazine methosulfate as mediator. Nitrite can oxidize dithionite-reduced NIR but cannot reduce oxidized NIR. Rate constants were determined for all observed reactions.     

Ascorbic acid, metal ions and the superoxide radical.

1. No evidence could be found for production of the superoxide radical, O2-, during autoxidation of ascorbic acid at alkaline pH values. Indeed, ascorbate may be important in protection against O2- genat-d in vivo. 2. Oxidation of ascorbate at pH 10.2 was stimulated by metal ions. Stimulation by Fe2+ was abolished by superoxide dismutase, probably because of generation of O2-- during reduction of O2 by Fe2+, followed by reaction of O2-- with ascorbate. EDTA changed the mechanism of Fe2+-stimulated ascorbate oxidation. 3. Stimulation of ascorbate oxidation by Cu2+ was also decreased by superoxide dismutase, but this appears to be an artifact, since apoenzyme or bovine serum albumin showed similar effects.     

Involvement of ascorbic acid and ab-type cytochrome in plant plasma membrane redox reactions

Summary Higher plant plasma membranes contain ab-type cytochrome that is rapidly reduced by ascorbic acid. The affinity towards ascorbate is 0.37 mM and is very similar to that of the chromaffin granule cytochromeb ₅₆₁. High levels of cytochromeb reduction are reached when ascorbic acid is added either on the cytoplasmic or cell wall side of purified plasma membrane vesicles. This result points to a transmembrane organisation of the heme protein or alternatively indicates the presence of an effective ascorbate transport system. Plasma membrane vesicles loaded by ascorbic acid are capable of reducing extravesicular ferricyanide. Addition of ascorbate oxidase or washing of the vesicles does not eliminate this reaction, indicating the involvement of the intravesicular electron donor. Absorbance changes of the cytochromeb -band suggest the electron transfer is mediated by this redox component. Electron transport to ferricyanide also results in the generation of a membrane potential gradient as was demonstrated by using the charge-sensitive optical probe oxonol VI. Addition of ascorbate oxidase and ascorbate to the vesicles loaded with ascorbate results in the oxidation and subsequent re-reduction of the cytochromeb. It is therefore suggested that ascorbate free radical (AFR) could potentially act as an electron acceptor to the cytochrome-mediated electron transport reaction. A working model on the action of the cytochrome as an electron carrier between cytoplasmic and apoplastic ascorbate is discussed.Abbreviations AFR ascorbate free radical - AO ascorbate oxidase - DTT dithiothreitol - FCCP carbonylcyanidep-trifluorome-thoxyphenylhydrazon - Hepes N-(2-hydroxyethyl)-piperazine-N-(2-ethanesulfonic acid) - Oxonol VI bis(3-propyl-5-oxoisoxazol-4-yl) penthamethine oxonol - PMSF phenylmethylsulfluoride     

Stopped-flow investigation of the reaction of vitamin C with tocopheroxyl radical in aqueous triton X-100 micellar solutions. The structure-activity relationship of the regeneration reaction of tocopherol by vitamin C

A kinetic study of the reaction between vitamin C (L-ascorbic acid, AsH2) and a tocopheroxyl radical (7-tert-butyl-5-isopropyltocopheroxyl) in Triton X-100 micellar solution has been performed using stopped-flow spectrophotometry. The second-order rate constants (k2) obtained showed notable pH dependence with a broad maximum around pH 8. For instance, the k2 values obtained were 26 M-1 S-1 at pH 3, 322 M-1 S-1 at pH 7, and 273 M-1 S-1 at pH 10. A good correlation between the rate constants and the mole fraction of ascorbate monoanion (AsH-) was observed, showing that ascorbate (AsH-) can regenerate the tocopherol from tocopheroxyl in biological systems. Furthermore, the results indicate that reduced ascorbic acid (AsH2) does not have the ability to regenerate the tocopherol in aqueous solution. On the other hand, it was found that AsH2 can reduce the tocopheroxyl to tocopherol in benzene/ethanol (2:1) mixtures, although the rate of reaction is only approximately 15% of that observed in micellar solution at pH 7.     

Kinetics of the reactions of hypochlorous acid and amino acid chloramines with thiols, methionine, and ascorbate

Thiol oxidation by hypochlorous acid and chloramines is a favorable reaction and may be responsible for alterations in regulatory or signaling pathways in cells exposed to neutrophil oxidants. In order to establish the mechanism for such changes, it is necessary to appreciate whether these oxidants are selective for different thiols as compared with other scavengers. We have measured rate constants for reactions of amino acid chloramines with a range of thiols, methionine, and ascorbate, using a combination of stopped-flow and competitive kinetics. For HOCl, rate constants are too fast to measure directly by our system and values relative to reduced glutathione were determined by competition with methionine. For taurine chloramine, the rate constants for reaction with 5-thio-2-nitrobenzoic acid, GSH, methionine, and ascorbate at pH 7.4 were 970, 115, 39, and 13 M(-1) s(-1), respectively. Values for 10 thiols varied by a factor of 20 and showed an inverse relationship to the pK(a) of the thiol group. Rate constants for chloramines of glycine and N-alpha-acetyl-lysine also showed these relationships. Rates increased with decreasing pH, suggesting a mechanism involving acid catalysis. For hypochlorous acid, rates of reaction with 5-thio-2-nitrobenzoic acid, GSH, cysteine, and most of the other thiols were very similar. Relative reactivities varied by less than 5 and there was no dependence on thiol pK(a). Chloramines have the potential to be selective for different cellular thiols depending on their pK(a). For HOCl to be selective, other factors must be important, or its reactions could be secondary to chloramine formation.     

Reduction of cytochrome C by ascorbic free radical.

The rate of "in vivo" reduction of cytochrome c by ascorbic acid (AA) increases from 69 nmoles of cytochrome c for minute, to 202 nanomoles when ascorbate oxidase is added. Since the AA oxidation by AA oxidase is a system to generate ascorbic free radical (AFR), data suggest that AFR is a better reducing compound than ascorbate in cytochrome c reduction. Since the addition of oxidized glutathione and human immunoglobulins (-S-S- bridge containing compounds) in the medium produces a remarkable decrease in cytochrome c reduction, it is suggested that AFR could also reduce -S-S- groups.     

Impairment of ascorbic acid's anti-oxidant properties in confined media: inter and intramolecular reactions with air and vanadate at acidic pH

The anti-oxidant properties of L-ascorbic acid were investigated in the confined medium produced by a sodium bis(2-ethylhexyl)sulfosuccinate (aerosol-OT, AOT) self-assembled reverse micelle. Using 1H-1H NOESY (proton-proton 2D nuclear overhauser enhancement correlation spectroscopy) NMR spectroscopy, the location of ascorbic acid was investigated and found to be at the AOT-interface in contrast to earlier studies where the ascorbate was assumed to be in the water pool in these microemulsions. The reaction of ascorbic acid with oxygen was investigated using EPR spectroscopy. A delocalized monoanionic ascorbate radical was observed in microemulsions prepared from pH 5.6 stock solutions. This is in contrast to studies carried out in aqueous media where no radical formation was observed. The oxidation of ascorbic acid by aqueous V(V) was investigated in reverse micelles. Modest changes in the kinetic parameters were observed for this system compared to that in water. Details of these reactions were examined and can be summarized as the microemulsion solvating and stabilizing reactive intermediates via rate inhibition or enhancement. The inhibition of the oxidation is due to solvation stabilization of ascorbic acid in microemulsion media. Since ascorbate is a valuable marker of oxidative stress, our results suggest that compartmentization can modify the stabilization of the ascorbate radical and the changes in properties could be important in biological systems.     

Rapid freeze- and chemical-quench studies of dopamine beta-monooxygenase: comparison of pre-steady-state and steady-state parameters

The copper-containing enzyme dopamine beta-monooxygenase has been studied with regard to pre-steady-state kinetics of tyramine hydroxylation and reduction of enzyme-bound Cu2+ by chemical- and freeze-quench EPR techniques. The bulk of the enzyme-bound copper (approximately 70%) is reduced in a single-exponential process with a limiting rate constant of 250 s-1, Km = 0.9 mM, consistent with participation of both copper ions in the redox events of catalysis. The remaining copper is reduced much more slowly (k approximately 2 s-1) or not at all, attributed to a distribution of copper into inhibitory binding sites and the presence of some inactive enzyme. Knowledge of the Cu2+ reduction rate, together with rate constants calculated from steady-state isotope effects [Miller, S. M., & Klinman, J. P. (1985) Biochemistry 24, 2114-2127], has allowed prediction of pre-steady-state product formation transients. Measurement of these transients under conditions of excess ascorbate shows close agreement with prediction, supporting the validity of individual rate constants obtained from steady-state data. Kinetic modeling shows further that the predominant steady-state enzyme form is the enzyme-product complex (E-P), which is expected to show a correspondingly large (approximately 70% of total copper) EPR signal for bound Cu2+. Surprisingly, the steady state is characterized by a low (19% of total copper) EPR signal. This lack of correlation between the anticipated and observed steady-state EPR signal suggests either antiferromagnetic coupling in binuclear copper centers or reduction of Cu2+ in this enzyme form by ascorbic acid.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reduction of horse heart ferricytochrome c by bovine liver ferrocytochrome b5. Experimental and theoretical analysis.

The reduction of horse heart ferricytochrome c by the tryptic fragment of bovine liver cytochrome b5 and its dimethyl ester heme (DME)-substituted derivative has been studied as a function of ionic strength, pH, and temperature under solution conditions where the reaction is bimolecular. The rate constant for ferricytochrome c reduction by native ferrocytochrome b5 is 1.8 (+/- 0.2) x 10(7) M-1 s-1 (25 degrees C) with delta H++ = 7.5 (+/- 0.2) kcal/mol and delta S++ = -0.3 (+/- 0.6) eu (pH 7.0, I = 0.348 M). Under the same solution conditions, the reduction of ferricytochrome c by DME-ferrocytochrome b5 proceeds with a rate constant of 1.7 (+/- 0.1) x 10(7) M-1 s-1 with delta H++ = 7.9 (+/- 0.4) kcal/mol and delta S++ = 1 (+/- 1) eu. The rate constants for both reactions are strongly dependent on ionic strength. A detailed electrostatic analysis of the proteins has been performed. Two relatively simple Brownian dynamics simulation models predict rate constants for the reaction between the two native proteins that demonstrate a dependence on ionic strength similar to that observed experimentally. In one of these models, the proteins are treated as spheres with reactive surface patches that are defined by a 5 degrees cone generated about the dipole vector calculated for each protein and aligned with the presumed electron-transfer site near the partially exposed heme edge. The second model replaces the reactive patch assumption with an exponential distance dependence for the probability of reaction that permits estimation of a value for the distance-dependence factor alpha. Calculations with this latter model in combination with the aligned dipole assumption provide a reasonable approximation to the observed ionic strength dependence for the reaction and are consistent with a value of alpha = 1.2 A-1.     

Mechanism of ascorbic acid oxidation by cytochrome b(561)

The 1 equiv reaction between ascorbic acid and cytochrome b(561) is a good model for redox reactions between metalloproteins (electron carriers) and specific organic substrates (hydrogen-atom carriers). Diethyl pyrocarbonate inhibits the reaction of cytochrome b(561) with ascorbate by modifying a histidine residue in the ascorbate-binding site. Ferri/ferrocyanide can mediate reduction of DEPC-treated cytochrome b(561) by ascorbic acid, indicating that DEPC-inhibited cytochrome b(561) cannot accept electrons from a hydrogen-atom donor like ascorbate but can still accept electrons from an electron donor like ferrocyanide. Ascorbic acid reduces cytochrome b(561) with a K(m) of 1.0 +/- 0.2 mM and a V(max) of 4.1 +/- 0.8 s(-1) at pH 7.0. V(max)/K(m) decreases at low pH but is approximately constant at pH >7. The rate constant for oxidation of cytochrome b(561) by semidehydroascorbate decreases at high pH but is approximately constant at pH <7. This suggests that the active site must be unprotonated to react with ascorbate and protonated to react with semidehydroascorbate. Molecular modeling calculations show that hydrogen bonding between the 2-hydroxyl of ascorbate and imidazole stabilizes the ascorbate radical relative to the monoanion. These results are consistent with the following mechanism for ascorbate oxidation. (1) The ascorbate monoanion binds to an unprotonated site (histidine) on cytochrome b(561). (2) This complex donates an electron to reduce the heme. (3) The semidehydroascorbate anion dissociates from the cytochrome, leaving a proton associated with the binding site. (4) The binding site is deprotonated to complete the cycle. In this mechanism, an essential role of the cytochrome is to bind the ascorbate monoanion, which does not react by outer-sphere electron transfer in solution, and complex it in such a way that the complex acts as an electron donor. Thermodynamic considerations show that no steps in this process involve large changes in free energy, so the mechanism is reversible and capable of fulfilling the cytochrome's function of equilibrating ascorbate and semidehydroascorbate.     

Kinetic studies on the reaction of compound II of myeloperoxidase with ascorbic acid. Role of ascorbic acid in myeloperoxidase function

Ascorbic acid is known to stimulate leukocyte functions. In a recent publication it was suggested that the role of ascorbic acid is to reduce compound II of myeloperoxidase back to the native enzyme (Bolscher, B. G. J. M., Zoutberg, G. R., Cuperus, R. A., and Wever, R. (1984) Biochim. Biophys. Acta 784, 189-191). In this paper we report rapid spectral scan and transient state kinetic results on the reaction of three myeloperoxidase compounds II, namely, human neutrophil myeloperoxidase, canine myeloperoxidase, and bovine spleen heme protein with ascorbate. We show by rapid scan spectra that compound II does not pass through any other intermediate when ascorbic acid reduces it back to native form. We also show that the reactions of all three compounds II involve a simple binding interaction before enzyme reduction with an apparent dissociation constant of 6.3 +/- 0.9 x 10(-4) to 2.0 +/- 0.3 x 10(-3)M and a first-order rate constant for reduction of 12.6 +/- 0.6 to 18.8 +/- 1.3 s-1. The optimum pH is 4.5, and at this pH the activation energy for the reaction is 13.2 kJ mol-1. Results of this work lend further evidence that the spleen green heme protein is very similar if not identical to leukocyte myeloperoxidase based on a comparison of spectral scans, pH-rate profiles, and kinetic parameters. We demonstrate that chloride cannot reduce compound II whereas iodide reduces compound II to native enzyme at a rate comparable to that of ascorbate. This explains why ascorbate accelerates chlorination but inhibits iodination. Formation of compound II is a dead end for the generation of hypochlorous acid; ascorbate regenerates more native enzyme to enhance the chlorination reaction namely: myeloperoxidase + peroxide----compound I followed by compound I + chloride----HOCl. On the other hand, ascorbate is a competitor with iodide for both compounds I and II and so inhibits iodination.