Reactions of pholasin with peroxidases and hypochlorous acid

The ability of myeloperoxidase (MPO) and horseradish peroxidase (HRP) to induce chemiluminescence (CL) in Pholasin (Knight Scientific, Plymouth, UK), the photoprotein of the Common Piddock Pholas dactylus, was studied. The oxidation of Pholasin by compound I or II of HRP induced an intense light emission, whereas native HRP showed only a small effect. The luminescence observed upon incubation of Pholasin with native MPO was diminished by preincubation with catalase. Considering the high instability of diluted MPO, it is concluded that traces of hydrogen peroxide in water converted MPO to its active forms, compound I and/or II, which are able to oxidize Pholasin. Indeed, the addition of hydrogen peroxide to a mixture of MPO and Pholasin induced an intense burst of light. This emission was enhanced in degree and duration in the absence of chloride. Hypochlorous acid, the reaction product of Cl(-) and compound I of MPO, was itself able to elicit a luminescent response in Pholasin and this luminescence was strongly inhibited by methionine and taurine. However, both of these HOCl scavengers only slightly reduced the light emission induced by MPO/H(2)O(2) in both the presence or absence of chloride. Thus, hypochlorous acid produced by the MPO/H(2)O(2)/Cl(-) system, under the conditions described in this study, did not contribute to Pholasin luminescence. The Pholasin luminescence elicited by formyl-leucyl-methionyl-phenylalanine (fMLP)-stimulated neutrophils depends both on superoxide anion radicals and higher oxidation states of myeloperoxidase (but not on hypochlorous acid). This is shown by the inhibition of luminescence with superoxide dismutase and potassium cyanide, together with the lack of effect of both methionine and taurine. The luminescence response is about eight times greater in cells stimulated with fMLP/cytochalasin B than with fMLP alone.     

The photoprotein pholasin as a luminescence substrate for detection of superoxide anion radicals and myeloperoxidase activity in stimulated neutrophils

Pholasin, the photoprotein of the common piddock Pholas dactylus, emits an intense luminescence upon oxidation. The contribution of superoxide anion radicals and myeloperoxidase (MPO) to Pholasin luminescence in stimulated neutrophils was investigated. Data on Pholasin luminescence were compared with results of superoxide anion radical generation detected by the cytochrome c test as well as with the release of elastase and MPO. In N-formyl-methionyl-leucyl-phenylalanine (fMLP) stimulated neutrophils, most of the luminescence is caused by superoxide anion radicals, whereas MPO shows only a small effect as shown by coincubation with superoxide dismutase (SOD) as well as potassium cyanide (KCN), an inhibitor of MPO. However, both, O₂^- and MPO contribute to light emission in fMLP/cytochalasin B and phorbol myristoyl acetate (PMA) stimulated cells. Thus, the kinetics of O₂^- generation and MPO release can be very well detected by Pholasin luminescence in stimulated neutrophils.

Degranulation of azurophilic granules was assessed using an ELISA test kit for released MPO or detection of elastase activity with MeO-Suc-Ala-Ala-Pro-Val-p-nitroanilide in the supernatant of stimulated cells. Both approaches revealed concurrently similar results concerning the amount and kinetics of enzyme release with data of Pholasin luminescence. Both, cytochrome c measurements and Pholasin luminescence indicate that fMLP/cytochalasin B and PMA stimulated neutrophils produce more O₂^- than fMLP stimulated cells. Thus, Pholasin luminescence can be used to detect, sensitively and specifically, O₂^- production and MPO release from stimulated neutrophils.     

The photoprotein pholasin as a luminescence substrate for detection of superoxide anion radicals and myeloperoxidase activity in stimulated neutrophils

Pholasin, the photoprotein of the common piddock Pholas dactylus, emits an intense luminescence upon oxidation. The contribution of superoxide anion radicals and myeloperoxidase (MPO) to Pholasin luminescence in stimulated neutrophils was investigated. Data on Pholasin luminescence were compared with results of superoxide anion radical generation detected by the cytochrome c test as well as with the release of elastase and MPO. In N-formyl-methionyl-leucyl-phenylalanine (fMLP) stimulated neutrophils, most of the luminescence is caused by superoxide anion radicals, whereas MPO shows only a small effect as shown by coincubation with superoxide dismutase (SOD) as well as potassium cyanide (KCN), an inhibitor of MPO. However, both, O₂^- and MPO contribute to light emission in fMLP/cytochalasin B and phorbol myristoyl acetate (PMA) stimulated cells. Thus, the kinetics of O₂^- generation and MPO release can be very well detected by Pholasin luminescence in stimulated neutrophils.

Degranulation of azurophilic granules was assessed using an ELISA test kit for released MPO or detection of elastase activity with MeO-Suc-Ala-Ala-Pro-Val-p-nitroanilide in the supernatant of stimulated cells. Both approaches revealed concurrently similar results concerning the amount and kinetics of enzyme release with data of Pholasin luminescence. Both, cytochrome c measurements and Pholasin luminescence indicate that fMLP/cytochalasin B and PMA stimulated neutrophils produce more O₂^- than fMLP stimulated cells. Thus, Pholasin luminescence can be used to detect, sensitively and specifically, O₂^- production and MPO release from stimulated neutrophils.     

Effects of nucleophils on kinetic parameters of peroxidase-catalyzed oxidative reactions]

The effect of imidazole on the kinetic parameters of reactions of potassium ferrocyanide and o-dianizidine peroxidation by hydrogen peroxide within a wide range of pH was studied. It was shown that imidazole activates the reaction of o-dianizidine peroxidation at pH greater than or equal to 6.5, but does not affect the oxidation of potassium ferrocyanide. It was also found that imidazole causes a similar increase in the kkat and Km, i. e. non-competitive activation occurs. The data obtained suggest a possible mechanism of the activator effect. Differences in the mechanism of interaction of various substrates uith peroxidase are discussed.     

Pholasin--a bioluminescent indicator for detecting activation of single neutrophils

Pholasin is the protein-bound luciferin from the bivalve mollusc Pholas dactylus which reacts with its luciferase and molecular oxygen to produce light. Pholasin was purified 226-fold with a yield of 58% from P. dactylus to give a preparation free from luciferase contamination. The ratio (k) of endogenous pholasin chemiluminescence to that when maximally stimulated by luciferase was 8.12 X 10(-6) +/- 0.87 X 10(-6) (mean +/- SD, n = 6), equivalent to a t 1/2 of 23.7 h at pH 9. Pholasin could detect reactive oxygen metabolite production from neutrophils stimulated by the chemotactic peptide N-formyl-Met-Leu-Phe, in the presence and absence of 2-chloroadenosine or cytochalasin B, and by latex beads in the presence and absence of cytochalasin B. Pholasin was also able to detect a longer-lived oxidative activity distinct from myeloperoxidase, and released from neutrophils activated by latex beads or chemotactic peptide; luminol could not. Under optimal conditions pholasin produced a signal some 50-100 times that of luminol in the presence of activated neutrophils. This enabled activation of a single neutrophil by chemotactic peptide (1 microM) to be detected, giving a signal of 194 +/- 21 chemiluminescent counts per second, some six times that of the background signal (mean +/- SD, n = 2). Pholasin thus provides an indicator sufficiently sensitive to establish whether neutrophil activation occurs through thresholds in individual cells.     

Light emission from tryptophan oxidation by hypobromous acid

The emission of ultraweak light from cells is a phenomenon associated with the oxidation of biomolecules by reactive oxygen species. The indole moiety present in tryptophan, serotonin and melatonin is frequently associated with the emission of light during the oxidation of these metabolites. This study presents results for hypobromous acid (HOBr) oxidation of tryptophan as a putative endogenous source of ultraweak light emission. We found that chemiluminescence elicited by the oxidation of tryptophan by HOBr was significantly higher than by hypochlorous acid (HOCl). This difference was related to secondary oxidation reactions, which were more intense using HOBr. The products identified during oxidation by HOCl, but depleted by using HOBr, were N‐formylkynurenine, kynurenine, 1,2,3,3a,8,8a‐hexahydro‐3a‐hydroxypyrrolo[2,3‐b]‐indole‐2‐carboxylic acid, oxindolylalanine and dioxindolylalanine. The emission of light is dependent on the free α‐amino group of tryptophan, and hence, the indole of serotonin and melatonin, although efficiently oxidized, did not produce chemiluminescence. The emission of light was even greater using taurine monobromamine and dibromamine as the oxidant compared to HOBr. A mechanism based on bromine radical intermediates is suggested for the higher efficiency in light emission. Altogether, the experimental evidence described in the present study indicates that the oxidation of free tryptophan or tryptophan residues in proteins is an important source of ultraweak cellular emission of light. This light emission is increased in the presence of taurine, an amino acid present in large amounts in leukocytes, where this putative source of ultraweak light emission is even more relevant. Copyright © 2012 John Wiley & Sons, Ltd.     

Synthesis, mechanism and fluorescence properties of 8-(aryl)-3-beta-D-ribofuranosylimidazo[2,1-i]purine 5'-phosphate derivatives

The synthesis of new fluorescent nucleotides is described. This synthesis comprises two parallel reactions, the Kornblum oxidation and imidazole formation, which lead to 8-(aryl)-3-beta-D-ribofuranosylimidazo[2,1-i]purine 5'-phosphates 2 from AMP or ATP. A detailed mechanism is proposed based on monitoring the reaction by 1H- and 13C-NMR spectroscopy, MS, FAB, HPLC, and pH meter. The spectral and fluorescent properties of the new derivatives at various pH values are described. Excitation and emission maxima for 3 were observed at 290 and 420 nm, respectively, in both basic and neutral media. In acidic media, the emission maximum shifted to 410 nm, however, the fluorescence intensity increased 1.5-fold. ATP analogues 2b and 3b exhibited relative stability regarding hydrolysis by type II ATPDase. Compound 3b is relatively chemically stable at pH 10.4 and 7.4.     

Characteristics of chemiluminescence observed in the horseradish peroxidase-hydrogen peroxide-tyrosine system.

Electrolysis or horseradish peroxidase (HRP)-catalyzed oxidation of tyrosine and bityrosine in aqueous solution at pH 7.4 resulted in light emission in the visible region. Electrolysis of tyrosine emitted light which peaked at 490 nm and was almost completely quenched by superoxide dismutase (SOD), while emission by bityrosine peaked at 530 nm. In the HRP-H(2)O(2)-tyrosine system the oxidation-reduction of tyrosine emitted light with two prominent peaks, 490 and 530 nm, and was not quenched by SOD. The phenoxyl neutral radical of the tyrosine in HRP-H(2)O(2)-tyrosine system was detected by electron spin resonance (ESR) spectrometry using tert-nitrosobutane as a spin trap; the spin adduct was found to adhere to the HRP molecule during the enzymatic reaction. Further, bityrosine was detected in the HRP-H(2)O(2)-tyrosine reaction system. Changes in absorption spectra of HRP and chemiluminescence intensities during HRP-catalyzed oxidation of tyrosine suggest that for photon emission compound III is a candidate superoxide donor to the phenoxyl cation radical of tyrosine on the enzyme molecule. The luminescence observed in this study might be originated from at least two exciplexes involved with the tyrosine cation radical (Tyr(*+)) and the bityrosine cation radical (BT(*+))     

Generation of hydrogen peroxide, superoxide and hydroxyl radicals during the oxidation of dihydroxyfumaric acid by peroxidase.

1. Dihydroxyfumarate slowly autoxidizes at pH6. This reaction is inhibited by superoxide dismutase but not by EDTA. Mn2+ catalyses dihydroxyfumarate oxidation by reacting with O2 leads to to form Mn3+, which seems to oxidize dihydrofumarate rapidly. Cu2+ also catalyses dihydroxyfumarate oxidation, but by a mechanism that does not involve O2 leads to. 2. Peroxidase catalyses oxidation of dihydroxyfumarate at pH6; addition of H2O2 does not increase the rate. Experiments with superoxide dismutase and catalase suggest that there are two types of oxidation taking place: an enzymic, H2O2-dependent oxidation of dihydroxyfumarate by peroxidase, and a non-enzymic reaction involving oxidation of dihydroxyfumarate by O2 leads to. The latter accounts for most of the observed oxidation of dihydroxyfumarate. 3. During dihydroxyfumarate oxidation, most peroxidase is present as compound III, and the enzymic oxidation may be limited by the low rate of breakdown of this compound. 4. Addition of p-coumaric acid to the peroxidase/dihydroxyfumarate system increases the rate of dihydroxyfumarate oxidation, which is now stimulated by addition of H2O2, and is more sensitive to inhibition by catalase but less sensitive to superoxide dismutase. Compound III is decomposed in the presence of p-coumaric acid. p-Hydroxybenzoate has similar, but much smaller, effects on dihydroxyfumarate oxidation. However, salicylate affects neither the rate nor the mechanism of dihydroxyfumarate oxidation. 5. p-Hydroxybenzoate, salicylate and p-coumarate are hydroxylated by the peroxidase/dihydroxyfumarate system. Experiments using scavengers of hydroxyl radicals shown that OH is required. Ability to increase dihydroxyfumarate oxidation is not necessary for hydroxylation to occur.     

The chromophore of pholasin: A highly luminescent protein

Pholasin is the photoprotein extracted from the marine bivalve Pholas dactylus. It undergoes an oxidative chemiluminescent reaction to oxypholasin with superoxide anion, hypochlorite, peroxidases and other oxidants. Since the observed absorbance and chemiluminescent emission spectra of pholasin solutions cannot be brought about solely by the amino acids composing the protein, there has to be a chemiluminescent chromophore. However, little is known about the chemical nature of this molecule. This work seeks to identify the chemical structure of the luminescent prosthetic group of pholasin. Pholasin could not be reactivated using chromophores from the hydroid Obelia geniculata (coelenterazine) and from the ostracod shrimp Vargula (formerly Cypridina) hilgendorfi. Furthermore, the reaction product of the Vargula chromophore could not be detected in solutions containing oxypholasin. Fluorescence analysis of such a solution revealed a compound with an emission spectrum (γ_max 480 nm; excitation at 320 nm), resembling the emission spectrum of the chemiluminescent reaction. This fluorescent substance was separated by gel filtration. It exhibited an apparent molecular mass of < 2000. Fluorescence masurements of extracts of partially purified pholasin suggested that a flavin moiety may be involved in pholasin luminescence.     

Importance of hydroxyl radical in the vanadium-stimulated oxidation of NADH

Vanadium compounds are known to stimulate the oxidation of NAD(P)H, but the mechanism remains unclear. This reaction was studied spectrophotometrically and by electron spin resonance spectroscopy (ESR) using vanadium in the reduced state (+4, vanadyl) and the oxidized state (+5, vanadate). In 25 mM sodium phosphate buffer at pH 7.4, vanadyl was slightly more effective in stimulating NADH oxidation than was vanadate. Addition of a superoxide generating system, xanthine/xanthine oxidase, resulted in a marked increase in NADH oxidation by vanadyl, and to a lesser extent, by vanadate. Decreasing the pH with superoxide present increased NADH oxidation for both vanadate and vanadyl. Addition of hydrogen peroxide to the reaction mixture did not change the NADH oxidation by vanadate, regardless of concentration or pH. With vanadyl however, addition of hydrogen peroxide greatly enhanced NADH oxidation which further increased with lower pH. Use of the spin trap DMPO in reaction mixtures containing vanadyl and hydrogen peroxide or a superoxide generating system resulted in the detection by ESR of hydroxyl. In each case, the hydroxyl radical signal intensity increased with vanadium concentration. Catalase was able to inhibit the formation of the DMPO--OH adduct formed by vanadate plus superoxide. These results show that the ability of vanadium to act in a Fenton-type reaction is an important process in the vanadium-stimulated oxidation of NADH.     

Chemiluminescence probe with Cypridina luciferin analog, 2-methyl-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-3-one, for estimating the ability of human granulocytes to generate O2-

The Cypridina luciferin analog, 2-methyl-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-3-one (CLA), in Hanks' balanced salt solution, emitted a weak luminescence which was not affected by superoxide dismutase or catalase and was not augmented by resting human granulocytes. In contrast, activated granulocytes caused a dramatic increase in the luminescence of CLA. The light emission by CLA in the presence of activated granulocytes was inhibited by superoxide dismutase, but not by catalase or benzoate. Azide at 0.5 mM did not inhibit light emission significantly. These results indicate that O2-, rather than H2O2, HO., singlet oxygen, or HOCl, was the agent responsible for eliciting the chemiluminescence of CLA. Moreover, the intensity of light emission by CLA correlated with the rate of production of O2- either by activated neutrophils or by the xanthine oxidase reaction.     

pH dependent competition between Y(Z) and Y(D) in photosystem II probed by illumination at 5 K

The photosystem II (PSII) reaction center contains two redox active tyrosines, YZ and YD, situated on the D1 and D2 proteins, respectively. By illumination at 5 K, oxidation of YZ in oxygen-evolving PSII can be observed as induction of the Split S1 EPR signal from YZ* in magnetic interaction with the CaMn4 cluster, whereas oxidation of YD can be observed as the formation of the free radical EPR signal from YD*. We have followed the light induced induction at 5 K of the Split S1 signal between pH 4-8.5. The formation of the signal, that is, the oxidation of YZ, is pH independent and efficient between pH 5.5 and 8.5. At low pH, the split signal formation decreases with pKa approximately 4.7-4.9. In samples with chemically pre-reduced YD, the pH dependent competition between YZ and YD was studied. Only YZ was oxidized below pH 7.2, but at pH above 7.2, the oxidation of YD became possible, and the formation of the Split S1 signal diminished. The onset of YD oxidation occurred with pKa approximately 8.0, while the Split S1 signal decreased with pKa approximately 7.9 demonstrating that the two tyrosines compete in this pH interval. The results reflect the formation and breaking of hydrogen bonds between YZ and D1-His190 (HisZ) and YD and D2-His190 (HisD), respectively. The oxidation of respective tyrosine at 5 K demands that the hydrogen bond is well-defined; otherwise, the low-temperature oxidation is not possible. The results are discussed in the framework of recent literature data and with respect to the different oxidation kinetics of YZ and YD.     

Histochemical observations on endogenous lactoperoxidase activity of the bovine submandibular salivary gland

Synopsis Seromucous demilunar cells of glutaraldehyde-fixed bovine submandibular salivary glands are intensely stained when sections are incubated in a benzidine-or a 3,3-diaminobenzidine-hydrogen peroxide medium in the pH range 6.0–9.0 whereas mucous acinar cells are completely unreactive. The histochemical reaction is completely inhibited by 3-amino-1,2,4-triazole. In contrast 2,4-dichlorophenol or potassium cyanide has little or no effect on the staining of demilunar cells. Striated duct cells also display a positive reaction with the diaminobenzidine method; this staining reaction, however, is most intense at pH 6.0. Furthermore, this reaction is markedly affected by potassium cyanide. The positive histochemical benzidine and diaminobenzidine reactions of demilunar cells probably corresponds to endogenous lactoperoxidase activity. On the other hand, the positive reaction shown by striated ducts, with optimal staining at pH 6.0 and which is completely inhibited by potassium cyanide, seems to be due to cytochromal oxidation of diaminobenzidine.     

Participation of histidine-51 in catalysis by horse liver alcohol dehydrogenase

Histidine-51 in horse liver alcohol dehydrogenase (ADH) is part of a hydrogen-bonded system that appears to facilitate deprotonation of the hydroxyl group of water or alcohol ligated to the catalytic zinc. The contribution of His-51 to catalysis was studied by characterizing ADH with His-51 substituted with Gln (H51Q). The steady-state kinetic constants for ethanol oxidation and acetaldehyde reduction at pH 8 are similar for wild-type and H51Q enzymes. In contrast, the H51Q substitution significantly shifts the pH dependencies for steady-state and transient reactions and decreases by 11-fold the rate constant for the transient oxidation of ethanol at pH 8. Modest substrate deuterium isotope effects indicate that hydride transfer only partially limits the transient oxidation and turnover. Transient data show that the H51Q substitution significantly decreases the rate of isomerization of the enzyme-NAD(+) complex and becomes a limiting step for ethanol oxidation. Isomerization of the enzyme-NAD(+) complex is rate limiting for acetaldehyde reduction catalyzed by the wild-type enzyme, but release of alcohol is limiting for the H51Q enzyme. X-ray crystallography of doubly substituted His51Gln:Lys228Arg ADH complexed with NAD(+) and 2,3- or 2,4-difluorobenzyl alcohol shows that Gln-51 isosterically replaces histidine in interactions with the nicotinamide ribose of the coenzyme and that Arg-228 interacts with the adenosine monophosphate of the coenzyme without affecting the protein conformation. The difluorobenzyl alcohols bind in one conformation. His-51 participates in, but is not essential for, proton transfers in the mechanism.     

Glutathione status in constituted physiological fluids containing albumin

1. Glutathione (GSH) and cysteine, added to the constituted incubation medium, rapidly disappeared from the medium in the presence of bovine serum albumin (BSA). The major portions of added GSH and cysteine were oxidized. Only a fraction was recovered as cysteine-GSH mixed disulfide in case of GSH. About 15-30% cysteine or GSH were not recovered in the media. 2. The rate of GSH oxidation was linear with time, however, GSH disappearance was not linear with GSH concentrations. 3. Oxidation of GSH to GSSG in the albumin supplemented media was greater under O2 atmosphere, but was significantly decreased under N2 atmosphere. 4. Catalase, a peroxy radical scavenger, but not dimethyl pyroline N-oxide (DMPO), N-tertbutyl-2(-2 sulfophenyl)-nitrone (NTBSPN), mannitol or superoxide dismutase (SOD), decreased BSA mediated GSH oxidation. 5. GSH oxidation was abolished when mono- or divalent metal ions were absent in the BSA supplemented media. 6. Alkaline pH favored and acidic pH inhibited GSH oxidation. GSH oxidation was maximum above pH 7.4. GSH oxidation was minimal in the media containing boiled BSA. 7. A reaction mechanism involving the mixed GSH-BSA disulfide formation, followed by the reduction of these disulfides by GSH and subsequent release of GSSG is proposed.     

ESR studies on the oxidation of N,N-dimethyl-p-anisidine and its analogues catalyzed by myeloperoxidase

N,N-Dimethyl-p-anisidine (DMA) was used as a substrate to differentiate between the direct, or chloride-independent, and the indirect, or chloride-dependent, pathways characteristic of myeloperoxidase (donor: hydrogen-peroxide oxidoreductase, EC 1.11.1.7). The chemical oxidation by sodium hypochlorite and the horseradish peroxidase-catalyzed oxidation by H2O2 were also investigated for a comparison. The chemical oxidation of DMA by NaOCl (DMA/NaOCl = 1) gave the p-N,N-dimethylaminophenoxy radical at pH 5 and 7. p-Benzoquinone and formaldehyde were determined as stable end-products. On the other hand, the cation radical of DMA was detected and p-benzoquinone was not obtained in the horseradish peroxidase-H2O2-Cl- system. In the presence of Cl- the myeloperoxidase-catalyzed oxidation at pH 5 gave nearly the same result as did the oxidation by NaOCl, whereas in the absence of Cl- the result of the oxidation was similar to that of the horseradish peroxidase-catalyzed oxidation, except for a low yield of formaldehyde formation, which was ascribed to the decomposition of H2O2 by the catalase activity of myeloperoxidase. Although the myeloperoxidase-catalyzed oxidation of DMA at pH 7 in the presence of Cl- gave only the cation radical of DMA, a fairly large amount of p-benzoquinone was obtained as a product. This result indicates that the indirect chloride-dependent oxidation is also operating at pH 7. The reaction mechanism for the myeloperoxidase-catalyzed oxidation of DMA is proposed.     

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.     

32P]ATP synthesis in steady state from [32P]Pi and ADP by Na+/K(+)-ATPase from ox brain and pig kidney. Activation by K+

The ouabain-sensitive synthesis of [32P]ATP from [32P]Pi and ADP (vsyn) was measured in parallel with the ouabain-sensitive hydrolysis of [32P]ATP (vhy) at steady state, at varying concentrations of sodium, potassium, magnesium, inorganic phosphate, ADP, ATP and oligomycin, and at varying pH. Na+ was necessary for ATP synthesis, but vsyn was decreased by high sodium concentrations. Oligomycin, depending on the Na+ concentration, either decreased or did not affect vsyn. Potassium, at low concentrations (1-5 mM) increased vsyn at all magnesium and sodium concentrations tested, lower potassium concentrations being needed to activate vsyn at lower sodium concentrations. vsyn was optimal below pH 6.7, decreasing abruptly at higher values of pH. At pH 6.7, vsyn was a hyperbolic function of the concentration of inorganic phosphate. In the presence of potassium, half-maximal rate was obtained at [Pi] congruent to 40 mM, whereas a higher concentration was needed to obtain half-maximal rate in the absence of K+. In contrast, increasing the concentration of ADP caused a nonhyperbolic activation of vsyn, the pattern obtained in the presence of potassium being different from that obtained in its absence. Increasing the ATP concentration above 0.5 mM decreased vsyn. The data are used to elucidate (1) which reaction steps are involved in the ATP-synthesis catalysed by the Na+/K(+)-ATPase at steady state in the absence of ionic gradients and (2) the mechanism by which K+ ions stimulate the reaction.     

Redox control of aryl sulfotransferase specificity

Aryl sulfotransferase IV from rat liver has the very broad substrate range that is characteristic of the enzymes of detoxication. With the conventional assay substrates, 4-nitrophenol and PAPS, sulfation was considered optimal at pH 5.5 whereas the enzyme in the physiological pH range was curiously ineffective. These properties would seem to preclude a physiological function for this cytosolic enzyme. Partial oxidation of the enzyme, however, results not only in a substantial increase in the rate of sulfation of 4-nitrophenol at physiological pH but also in a shift of the pH optimum to this range and radically altered overall substrate specificity. The mechanism for this dependence on redox environment involves oxidation at Cys66, a process previously shown to occur by formation of a mixed disulfide with glutathione or by the formation of an internal disulfide with Cys232. Oxidation at Cys66 acts only as a molecular redox switch and is not directly part of the catalytic mechanism. Underlying the activation process is a change in the nature of the ternary complex formed between enzyme, phenol, and the reaction product, adenosine 3',5'-bisphosphate. The reduced enzyme gives rise to an inhibitory, dead-end ternary complex, the stability of which is dictated by the ionization of the specific phenol substrate. Ternary complex formation impedes the binding of PAPS that is necessary to initiate a further round of the reaction and is manifest as profound, substrate-dependent inhibition. In contrast, the ternary complex formed when the enzyme is in the partially oxidized state allows binding of PAPS and the unhindered completion of the reaction cycle.