Two-electron Oxidation Product Research Articles

Redox Chemistry of Dinuclear Molybdenum Dithiolene Complexes. Interconversion of the Bridging Disulfide and Sulfides

Abstract The redox properties of a photochromic dinuclear molybdenum complex, [Mo2(μ-S2)(μ-S2C2Ph2)2(S2C2Ph2)2] (1), were examined. Conventional and thin-layer cyclic voltammetric measurements showed that 1 undergoes two-electron reduction followed by chemical reactions to give [Mo2(μ-S)2(S2C2Ph2)4]2- (2), which was structurally characterized by X-ray crystallography. The overall two-electron oxidation product of 2 was converted to 1 via formation of four bonds.

Mechanistic Study of CH3OH + O2 Photoredox Reaction in a FeAlPO4 Sieve by Time-Resolved FT-IR Spectroscopy

The mechanism of the ligand-to-metal charge transfer (LMCT) induced reaction of methanol and O2 at the gas−micropore interface of a FeAlPO4-5 sieve has been studied by time-resolved FT-IR spectroscopy. Measurements using the rapid-scan method revealed that the final products, HCO2-···Fe and H2O, emerge with a rise time of 4 s (250 K) by decomposition of the two-electron-transfer intermediate, HO2CH2OH. Intermediacy of hydroxymethylhydroperoxide was established by direct detection by step-scan FT-IR spectroscopy on the 500 microsecond time scale and by monitoring of its photodissociation products HCO2H + H2O on the millisecond and second time scale. Formaldehyde was observed as minor two-electron oxidation product. The fast rise of the HO2CH2OH intermediate strongly suggests that it is formed by direct coupling of HOO and CH2OH radicals. This constitutes the most conclusive evidence yet for the formation of a hydroperoxy radical and hydroxymethyl radical as the primary one-electron-transfer products of the...

One- and two-electron oxidation reactions of trolox by peroxynitrite.

One and two electron oxidation reactions of peroxynitrite with trolox, an analogue of vitamin E were studied. Kinetics of the reaction was studied using a single mixing stopped-flow spectrometer in the pH range from 6 to 11 and the products were analyzed by HPLC/HPIC. One-electron oxidation reaction produced the phenoxyl radical of trolox having a characteristic absorption spectrum in the 300-500 nm region with absorption maximum at 430 nm. The rate constant for the formation of the trolox radical at 430 nm was determined to be 1.8 x 10(5) M(-1) s(-1). At low concentration of peroxynitrite the radical did not show any noticeable decay in the time scale of 20-50 s. However, when the concentration of peroxynitrite was increased, there was an appreciable increase in the decay of the trolox radical due to its reaction with excess peroxynitrite yielding a bimolecular rate constant of 6.0 x 10(3) M(-1) s(-1) for the reaction of trolox radical with peroxynitrite. The released nitrite and nitrate anions, and the two-electron oxidation product quinone produced as a result of these reactions, were estimated using ion chromatography and HPLC analysis. By following the changes in the yields of nitrite and quinone as a function of peroxynitrite concentration and from the above determined rate parameters, the relative importance of the one and two electron processes has been discussed.

Semiempirical molecular orbital calculations of biliverdin: study of dynamics and energetics of the self-association of a two-electron oxidation product

As an extension of our previous theoretical studies on biliverdin, a model molecule; octaethyl biliverdin (OEBV)+ is a good candidate for the present work. OEBV and its cationic form—octaethyl biliverdin oxide (OEBVO)+ are geometrically optimized using the semiempirical method. Employing OEBVO+, dynamics as well as energetics of the production of a dimer model molecule OEBVOD2+ with helical conformation is investigated. X-ray crystallography; via formation of intramolecular hydrogen bonding such as N–H⋯O, N–H⋯N, and H⋯O between pyrrolone rings of the same symmetry of ions; proves the self-association of ions causing OEBVOD2+ to form. A comparison between experimental X-ray results and our theoretical data for the model compounds shows good agreement which lends support to our approach, to come up with the same proposed mechanism for self-association of natural biliverdin oxide (BVO)+, thereby generating biliverdin oxide dimer (BVOD)2+. Our calculations predict the dynamics and energetics of formation of dimers such as (BVOD)2+ and we propose the geometry as well as molecular parameters for this species.

Ascorbate-dependent recycling of the vitamin E homologue Trolox by dihydrolipoate and glutathione in murine skin homogenates

In the redox antioxidant network, dihydrolipoate can synergistically enhance the ascorbate-dependent recycling of vitamin E. Since the major endogenous thiol antioxidant in biological systems is glutathione (GSH) it was of interest to compare the effects of dihydrolipoate with GSH on ascorbate-dependent recycling of the water-soluble homologue of vitamin E, Trolox, by electron spin resonance (ESR). Trolox phenoxyl radicals were generated by a horseradish peroxidase (HRP)–hydrogen peroxide (H 2O 2) oxidation system. In the presence of dihydrolipoate, Trolox radicals were suppressed until both dihydrolipoate and endogenous levels of ascorbate in skin homogenates were consumed. Similar experiments made in the presence of GSH revealed that Trolox radicals reappeared immediately after ascorbate was depleted and that GSH was not able to drive the ascorbate-dependent Trolox recycling reaction. However, at higher concentrations GSH was able to increase ascorbate-mediated Trolox regeneration from the Trolox radical. ESR and spectrophotometric measurements demonstrated the ability of dihydrolipoate or GSH to react with dehydroascorbate, the two-electron oxidation product of ascorbate in this system. Dihydrolipoate regenerated greater amounts of ascorbate at a much faster rate than equivalent concentrations of GSH. Thus the marked difference between the rate and efficiency of ascorbate generation by dihydrolipoate as compared with GSH appears to account for the different kinetics by which these thiol antioxidants influence ascorbate-dependent Trolox recycling.

Two new bimetallic phosphido carbonyl complexes of nickel(0): [Ni2(CO)2(PPh3)2(μ-CO)(μ-PPh2)]− and [Ni2(CO)4(μ-PPh2)2]2−

Toluene solutions of Ni(CO)2(PPh3)2 react with excess potassium metal to give the [Ni2(CO)4(μ-PPh2)2]2− ion (2) which contains two tetrahedral Ni centers fused along a common edge. Each Ni achieves an 18 electron configuration without the formation of a Ni–Ni bond (dNi–Ni = 3.397(1) A) in contrast to the two-electron oxidation product, Ni2(CO)4(μ-PPh2)2 (1) (dNi–Ni = 2.510(2) A). The anion 2 shows a reversible oxidation at −1.5 V and a quasi-reversible oxidation at −1.1 V vs. FeCp2/FeCp2+. The negative ion electrospray mass spectrum of 2 shows the one-electron oxidation product of 2, [Ni2(CO)4(μ-PPh2)2]−, along with three decarbonylation products [Ni2(CO)3(μ-PPh2)2]−, [Ni2(CO)2(μ-PPh2)2]−, and [Ni2(CO)(μ-PPh2)2]− where [Ni2(CO)3(μ-PPh2)2]− is the base peak. Ethylenediamine solutions of Sn94− react with Ni(CO)2(PPh3)2 in the presence of 2,2,2-crypt (4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8.8.8]hexacosane) to reproducibly give [Ni2(CO)2(PPh3)2(μ-CO)(μ-PPh2)]− (3), as one of three products. The anion 3 also has an edge-shared bitetrahedral geometry but, unlike 2, possesses a Ni–Ni bond (dNi–Ni = 2.4930(9) A).

Spectroscopic and Quantum Chemical Studies of Excited States of One- and Two-Electron Oxidation Products of a Lutetium Triple-Decker Phthalocyanine Complex

Electronic and MCD (magnetic circular dichroism) spectra of dilutetium(III) tris(phthalocyanate), Lu2(Pc)3 (Pc = phthalocyanine), its one-electron oxidation product [Lu2(Pc)3]+, and its two-electron oxidation product [Lu2(Pc)3]2+ are reported. Lu2(Pc)3 was prepared by a new synthetic route in which PcLu(CH3COO)(H2O)2 is used as the starting material. This new route enabled a purification of the triple-decker compound to a level necessary for spectroscopic measurements. Each of the two chemical oxidation steps occurred keeping isosbestic points. The first oxidation product [Lu2(Pc)3]+ shows a relatively intense band in the near-IR region. This is assigned to the LUMO-to-SOMO (singly occupied orbital) transition. The band has a transition moment perpendicular to the Pc planes. A single intense band was observed in the Q-band region in [Lu2(Pc)3]+ in contrast to the two bands in Lu2(Pc)3. Between the Q band and the near-IR band, several weak bands are observed. Among them, two bands show clear MCD A-term dispersions. In the region between the Q band and the B band, [Lu2(Pc)3]+ shows a characteristic band for Pc π radicals. Its MCD pattern suggests the presence of a negative A term. The second oxidation product [Lu2(Pc)3]2+ displays an intense band with two distinct vibronic bands in the near-IR region. None of the bands in the region shows an A-term contribution in MCD. The Q band remains single. There is only one band which shows an MCD A term between the near-IR band and the Q band. Semiempirical quantum chemical calculations were carried out for the ground and excited states of [Lu2(Pc)3]+ and [Lu2(Pc)3]2+. Band assignments are discussed using the theoretical excitation energies, oscillator strengths, and MCD A terms.

UV-B-Induced photooxidation of vitamin E in mouse skin.

Topically applied alpha-tocopherol (alpha-TH, vitamin E) inhibits UV-B (290-320 nm) photocarcinogenesis, yet alpha-TH undergoes rapid photooxidation by UV-B in vitro. To examine the effect of UV-B on alpha-TH in vivo, we studied the fate of alpha-TH in UV-B-irradiated mouse skin. [14C]-alpha-TH was applied to mouse skin at various times prior to UV-B irradiation. UV-B irradiation for 1 h at a dose rate of 2.6-2.9 J m-2 s-1 resulted in consumption of 40-60% of the applied dose and formation of oxidation products. The major product fraction formed in UV-B-irradiated mice treated topically with alpha-TH contained an alpha-TH dihydroxy dimer and its two-electron oxidation product, a spirodimer. Products previously identified as being derived from photochemical or peroxyl radical scavenging reactions of alpha-TH were also observed, including alpha-tocopherolquinone (alpha-TQ), alpha-tocopherolquinone 2, 3-epoxide (alpha-TQE 1), alpha-tocopherolquinone 5,6-epoxide (alpha-TQE 2), and 8a-(hydroperoxy)epoxytocopherones. These results indicate that topically applied alpha-TH is extensively oxidized in skin and suggest that alpha-TH photoproducts may be involved in the observed effects of topically applied vitamin E in UV-B-irradiated skin.

Regeneration of phenolic antioxidants from phenoxyl radicals: An ESR and electrochemical study of antioxidant hierarchy

Radicals from the flavonoids quercetin, (+)-catechin, (±)-taxifolin and luteolin, and from all-rac-α-tocopherol have been generated electrochemically by one-electron oxidation in deaerated dimethylformamide (DMF), and characterised by electron spin resonance spectroscopy (ESR) after spin-trapping by 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). Simulations of the ESR spectrum based on estimated coupling constants of the spin-trapped quercetin radical, confirmed that this antioxidant radical is oxygen-centered. The complex mixture of radicals, quinoid intermediates and stable two-electron oxidation products, were for each antioxidant allowed to react with each of the four other antioxidants, and the progression of reaction followed by ESR after addition of DMPO, and the product solution further analysed by HPLC. All-rac-α-tocopherol was found to be most efficient in regenerating each of the other antioxidants from their oxidation products with a regeneration index (defined as moles regenerated of the oxidised phenolic antioxidant divided with moles of all-rac-α-tocopherol consumed) of 0.90 ± 0.16 for quercetin, 0.48 ± 0.11 for (+)-catechin, 0.48 ± 0.06 for (±)-taxifolin and 0.50 ± 0.10 for luteolin in equimolar 1.00 mM solution. Quercetin was found to have the highest regeneration index among the flavonoids: 0.88 ± 0.13 for (+)-catechin, 0.41 ± 0.03 for (±)-taxifolin and 0.41 ± 0.02 for luteolin. The antioxidant hierarchy based on the reduction potentials determined by cyclic voltammetry under similar conditions (0.93 V for all-rac-α-tocopherol, 1.07 V for quercetin, 1.15 V for luteolin, 1.16 V for (+)-catechin and 1.20 V for (±)-taxifolin) is compared with the observed over-all regeneration (34% for quercetin, 34% for (+)-catechin, 52% for (±)-taxifolin and 43% for luteolin by all-rac-α-tocopherol).

Inhibition of copper-induced LDL oxidation by vitamin C is associated with decreased copper-binding to LDL and 2-oxo-histidine formation

Oxidatively modified low-density lipoprotein (LDL) has numerous atherogenic properties, and antioxidants that can prevent LDL oxidation may act as antiatherogens. We have previously shown that vitamin C (L-ascorbic acid, AA) and its two-electron oxidation product dehydro-L-ascorbic acid (DHA) strongly inhibit copper (Cu)-induced LDL oxidation. These findings are unusual, as AA is known to act not only as an antioxidant, but also a pro-oxidant in the presence of transition metal ions in vitro, and DHA has no known reducing capacity. Here we report that human LDL (0.4 mg protein/ml) incubated with 40 μM Cu 2+ binds 28.0 ± 3.3 Cu ions per LDL particle (mean ± SD, n = 10). Co-incubation of LDL with AA or DHA led to the time- and concentration-dependent release of up to 70% of bound Cu, which was associated with the inhibition of LDL oxidation. Incubation of LDL with Cu and AA or DHA also led to the time-dependent formation of 2-oxo-histidine, an oxidized derivative of histidine with a low affinity for Cu. Addition of free histidine prevented the formation of the LDL-Cu complexes and inhibited LDL oxidation, despite the fact that Cu remained redox-active. Interestingly, histidine was more effective than AA or DHA at limiting Cu binding to LDL, but at low concentrations AA and DHA were more effective than histidine at inhibiting LDL oxidation. These data suggest that there are at least two types of Cu binding sites on LDL: those that bind Cu in a redox-active form critical for initiation of LDL oxidation, and those that bind Cu in a redox-inactive form not contributing to LDL oxidation. The former sites may be primarily histidine residues of apolipoprotein B-100 that are oxidized to 2-oxo-histidine in the presence of Cu and AA or DHA, thus explaining, at least in part, the unusual inhibitory effect of vitamin C on Cu-induced LDL oxidation.

Cholesteryl Ester Hydroperoxide Formation in Myoglobin-Catalyzed Low Density Lipoprotein Oxidation: Concerted Antioxidant Activity of Caffeic and p-Coumaric Acids with Ascorbate

Two diet-derived phenolic acids, caffeic and p-coumaric acids, interplayed with ascorbate in the protection of low density lipoproteins (LDL) from oxidation promoted by ferrylmyoglobin. Ferrylmyoglobin, a two-electron oxidation product from the reaction of metmyoglobin and H 2O 2, was able to oxidize LDL, degrading free cholesterol and cholesteryl esters. Upon exposure to ferrylmyoglobin, LDL became rapidly depleted of cholesteryl arachidonate and linoleate, which turn into the corresponding hydroperoxides. Cholesteryl oleate and cholesterol were, comparatively, more resistant to oxidation. Caffeic (2 μM) and p-coumaric (12 μM) acids efficiently delayed oxidations, as reflected by an increase in the lag times required for linoleate hydroperoxide and 7-ketocholesterol formation as well as for cholesteryl linoleate consumption. At the same concentration, ascorbate, a standard water-soluble antioxidant, was less efficient than the phenolic acids. Additionally, phenolic acids afforded a protection to LDL that, conversely to ascorbate, extends along the time, as inferred from the high levels of cholesteryl linoleate and cholesteryl arachidonate left after 22 hr of oxidation challenging. Significantly, the coincubation of LDL with ascorbate and each of the phenolic acids resulted in a synergistic protection from oxidation. This was inferred from the lag phases of cholesteryl linoleate hydroperoxide (the major peroxide found in LDL) formation in the presence of mixtures of ascorbate with phenolic acids longer than the sum of individual lag phases of ascorbate and the phenolic acids. A similar description could be drawn for the accumulation of a late product of oxidation, 7-ketocholesterol. It is concluded that ferrylmyoglobin induces a typical pattern of LDL lipid peroxidation, the oxidation rate of cholesteryl esters being a function of unsaturation; furthermore, there is a synergistic antioxidant activity of diet-derived phenolic acids with ascorbate in the protection of LDL from oxidation, a finding of putative physiological relevance.

Kinetics of Reduction of Hypervalent Iron in Myoglobin by Crocin in Aqueous Solution

Crocin in aqueous solution is oxidized by ferrylmyoglobin, MbFe(IV)=O, in a second order reaction with k = 183 1 · mol-1 · s-1, AH‡298 = 55.0 kJ · mol-1, and ΔLS‡298 = -17 J · mol-1 K-1 (pH = 6.8, ionic strength 0.16 (NaCl), 25°C), as studied by stopped-flow spectroscopy. The reaction has 1:1 stoichiometry to yield metmyoglobin, MbFe(III), and has AGo = -11 kJ · mol-1, as calculated from the literature value E0 = +0.85 V (pH = 7.4) vs. NHE for MbFe(IV)=O/MbFe(III) and from the half-peak potential +0.74 V (vs. NHE in aqueous 0.16 NaCl, pH = 7.4) determined by cyclic voltammetry for the one-electron oxidation product of crocin, for which a cation radical structure is proposed and which has a half-peak potential of +0.89 V for its formation from the two-electron oxidation product of crocin. The fer-rylmyoglobin protein-radical, MbFe(IV)=O, reacts with crocin with 2:l stoichiometq to yield MbFe(IV)= 0, as determined by ESR spectroscopy, in a reaction faster than the second order protein-radical generating reaction between H2O2 and MbFe(III), for which latter reaction k = 137 L · mol-1 · s-1, ΔH‡298 = 51.5 kJ · mol-1, and ΔH‡298 = -31 J · mol-1 · K-1 (pH = 6.8, ionic strength = 0.16 (NaCI), 25°C) was determined. Based on the difference between the stoichiometry for the reaction between crocin and each of the two hypervalent forms of myoglobin, it is concluded in agreement with the determined half peak reduction potentials, that the crocin cation radical is less reducing compared to crocin, as the cation radical can reduce the protein radical but not the iron(IV) centre in hypervalent myoglobin.

Mechanism of Simultaneous Iodination and Coupling Catalyzed by Thyroid Peroxidase

Thyroid peroxidase (TPO) simultaneously catalyzes two very different types of reaction in the thyroid gland—iodination and coupling. The present study addresses the mechanism of this simultaneous dual activity. Compound I, the two-electron oxidation product of TPO, exists in two different forms—an oxoferryl porphyrin π-cation radical and an oxoferryl protein radical. It has been proposed that iodination is mediated by the porphyrin π-cation radical form of TPO compound I, while coupling is mediated by the protein radical form. However, results obtained in the present study favor the view that both iodination and coupling are mediated by the porphyrin π-cation radical form of compound I. In the first part of the study, we compared coupling and iodination activities of two peroxidases with very similar crystal structures—cytochromecperoxidase (CcP) and lignin peroxidase (LiP). Although these two peroxidases have very similar three-dimensional structures, CcP forms a compound I only of the protein radical type, whereas compound I of LiP exists only as a porphyrin π-cation radical. Comparison of the catalytic activities of the two enzymes showed that diiodotyrosine (DIT)-stimulated coupling activity of LiP was significantly greater than that of CcP. Moreover, lignin peroxidase displayed very significant iodinating activity at acid pHs, whereas iodination with CcP was negligible at all pHs tested. Our findings with these two structurally similar peroxidases suggested that TPO-catalyzed iodination and coupling could both be mediated by the porphyrin π-cation radical form of compound I. More direct evidence in support of this view was obtained in the second part of this study, employing TPO and lactoperoxidase (LPO) model systems in which iodination and coupling occurred simultaneously. Heme spectral analysis was used to correlate formation of the protein radical form of compound I with the kinetics of the iodination and coupling reactions. Formation of the compound I protein radical was not observed until the iodination and coupling reactions had almost been completed. In separate experiments it was shown that the spontaneous conversion of the porphyrin π-cation radical form of TPO or LPO compound I to the protein radical form was markedly inhibited by a low concentration of iodide, especially in the presence of an iodide acceptor. These studies provide compelling evidence that both iodination and coupling are mediated by the porphyrin π-cation radical form of compound I. This was further substantiated by the finding that coupling was inhibited in the presence of excess iodide, an observation readily explained by competition between iodide and DIT residues in thyroglobulin for oxidation by the porphyrin π-cation radical.

Chlorogenic Acid as a Natural Scavenger for Hypochlorous Acid

The effect of chlorogenic acid (CGA) on the killing of Escherichia coli by hypochlorous acid (HOCl) was examined. CGA prevented E. coli from bactericidal action of HOCl in a concentration-dependent manner. HOCl reacted rapidly with CGA. By comparison with the one- and two-electron oxidation products produced by peroxidase and phenolase reactions, respectively, the main product was identified as o-quinone of CGA via the formation of o-semiquinone of CGA as an intermediate The quinone form of CGA reacted further with HOCl to yield unstable product(s).

Vitamin C prevents metal ion-dependent initiation and propagation of lipid peroxidation in human low-density lipoprotein

Lipid peroxidation and oxidative modification of low-density lipoprotein (LDL) have been implicated as causal factors in the pathogenesis of atherosclerosis, and prevention of LDL oxidation by antioxidants may be an effective strategy to inhibit the progression of the disease. We investigated the effects of the reduced form of vitamin C ( l-ascorbic acid, AA) and its two-electron oxidation product (dehydro- l-ascorbic acid, DHA) upon metal ion-dependent oxidative modification of human LDL. We found that low micromolar concentrations of both AA and DHA protect LDL against oxidation induced by Cu 2+ or by hemin and hydrogen peroxide. In a dose-dependent manner, AA and DHA prevented the initiation of lipid peroxidation in LDL, as determined by a sensitive and selective assay for lipid hydroperoxides utilizing HPLC with chemiluminescence detection. AA and DHA also preserved the LDL-associated antioxidants α-tocopherol, β-carotene, and lycopene, but not ubiquinol-10. Furthermore, AA was able to stop propagation of lipid peroxidation in LDL, whereas DHA lacked this ability. The addition of 60 μM AA to LDL containing up to 38 nmol/mg protein of pre-formed lipid hydroperoxides led to their rapid disappearance; this activity of AA was dependent on the presence of redox-active copper, but did not lead to the formation of lipid hydroxides, the reduced form of lipid hydroperoxides. Our data show that in Cu 2+-exposed LDL (i) vitamin C primarily spares, rather than regenerates, α-tocopherol and other endogenous antioxidants, except for ubiquinol-10; (ii) AA and DHA, unlike the LDL-associated antioxidants, act as preventive rather than chain-breaking antioxidants, i.e., AA and DHA prevent initiation of lipid peroxidation in LDL; and (iii) AA can terminate lipid peroxidation, thereby protecting partially oxidized LDL against further oxidative modification.

Solid-state self-association of the two-electron oxidation product of a biliverdin analogue

Oxidation of octaethylbilindione with one molar equivalent of iodine produces an open-chain tetraaza cation [2+] which self-associates in the solid state through a set of hydrogen bonds to form a dimeric entity that consists of two interpenetrating C-shaped units.

Mesoferriheme regeneration from its hemoprotein enzyme compound I analog: absorption and kinetic properties of one- and two-electron oxidation products

Spectral and kinetic data obtained in studies of the biphasic in situ regeneration of mesoferriheme (mfh) from oxidized species formed through the NaOCl oxidation of heme are utilized to calculate extinction coefficients of dinuclear iron species which have been proposed as models of peroxidase Compounds I and II (A and B below). For the proposed regeneration mechanism, involving consecutive one-electron redox processes, the calculated values of ϵ A and ϵ B are 4x10 4 and 5x10 4 M −1 cm −1, respectively. ▪ These values are somewhat greater than those previously calculated for analogous derivatives of deuteroferriheme. Both k 1 and k 2 are independent of pH over the range 6.9–9.1 and of total phosphate buffer concentration from 8 to 50 mM at pH 6.9 suggesting that, under these conditions, there is an absence of significant dissociation of the proposed dinuclear iron species.

30] Hydrogen peroxide-mediated ferrylhemoglobin generation in Vitro and in red blood cells

The oxidation of methemoglobin by hydrogen peroxide is explained by a heterolytic cleavage of the O—O bond of the coordinated peroxide, yielding a two-electron oxidation product of the hemoprotein, known as “ferrylhemoglobin.” The first oxidation equivalent provided by this reaction is in the form of the oxo-ferryl complex, FeIV=O, similar to compound II reported for peroxidases, and the second oxidation equivalent is detected as a transient apoprotein radical as reported for myoglobin. Therefore, the formation of the radical form of the ferryl species bears the important implication that it bridges the discrepancy between the retention of one oxidizing equivalent in the ferrylheme complex and the observed catalysis of two-electron oxidations. The reason that ferrylhemoglobin is regarded as an oxidizing agent capable of promoting oxidation of molecules of biological relevance, it is of some value (1) to obtain ferrylhemoglobin as an oxidizing experimental tool in various systems and (2) to characterize ferrylhemoglobin in systems in which it may be produced to distinguish it from other species with similar spectral profiles. This chapter describes procedures for the preparation and spectral characterization of ferrylhemoglobin.

Facile aerial oxidation of a porphyrin. Part 13. : Generation of π-cation radicals via acid addition to mosotetrakis (3,5-di-t-bu-4-hydroxyphenyl)porphyrin and its two-electron oxidation product.

Addition of acidified 1,1,1-trichloroethane to the oxidised porphyrin 2 produces the porphyrin π-cation radical 3: evidence is presented to show that a long-lived radical produced via the aerial oxidation of porphyrin 1 in TFA, is due to a porphyrin π-cation radical dimer.

Laser flash photolysis study of the kinetics of electron transfer reactions of flavocytochrome b2 from Hansenula anomala: further evidence for intramolecular electron transfer mediated by ligand binding.

Intramolecular electron transfer between the heme and flavin cofactors of flavocytochrome b2 is an obligatory step during the enzymatic oxidation of L-lactate and subsequent reduction of cytochrome c. Previous kinetic studies using both steady-state and transient methods have suggested that such intramolecular electron transfer is inhibited when pyruvate, the two-electron oxidation product of L-lactate, is bound at the active site of Hansenula anomala flavocytochrome b2. In contrast to this, we have recently demonstrated using laser flash photolysis that intramolecular electron transfer could be observed in the flavocytochrome b2 from Saccharomyces cerevisiae only when pyruvate was present [Walker, M., & Tollin, G. (1991) Biochemistry 30, 5546-5555], despite a large thermodynamic driving force of 100 mV and apparently favorable cofactor geometry as indicated by crystallographic studies. In the present study, we have utilized laser flash photolysis to investigate intramolecular electron transfer in the flavocytochrome b2 from H. anomala in an effort to address these apparently conflicting interpretations with respect to the influence of pyruvate on enzyme properties. The results obtained are closely comparable to those we reported using the protein from Saccharomyces. Thus, in the absence of pyruvate, bimolecular reduction of both the heme and FMN cofactors by deazaflavin semiquinone occurs (k approximately 10(9) M-1 s-1), followed by a protein concentration dependent intermolecular electron transfer from the semiquinone form of the FMN cofactor to the heme (k approximately 10(7) M-1 s-1).(ABSTRACT TRUNCATED AT 250 WORDS)