Chlorpromazine inhibition of electron transport in Azobacter vinelandii membranes

Chlorpromazine was a potent inhibitor of O₂-dependent malate oxidation, but not of H₂ oxidation in Azotobacter vinelandii membranes. However, chlorpromazine did not significantly affect the activity of malate reductase or the reduction of cytochromes c and d. In the presence of chlorpromazine, cytochrome o failed to form a complex with CO. The site of action of chlorpromazine seems to be in the cytochromes c to cytochrome o branch, the pathway utilized by malate, succinate and NADH, but not by H₂.     

Effect of water activity control on the catalytic performance of surfactant–Arthromyces ramosus peroxidase complex in toluene

Arthromyces ramosus peroxidase (ARP) was successfully modified with a synthetic surfactant for one-electron oxidation reaction of a hydrophobic substrate in toluene. Although UV–visible absorption spectrum of surfactant–ARP complex in toluene showed slight red shift of Soret band compared to that in water, the complex can catalyze oxidation reaction of o-phenylenediamine (o-PDA) with hydrogen peroxide. It appeared that thermodynamic water activity in the reaction system has dominant effect on either the catalytic activity or the stability in the catalytic cycle. Steady-state kinetics under the optimal condition revealed that the specific constant (k_cat/K_m) of ARP complex for o-PDA was 2 orders of magnitude lower than that in aqueous media, while only 13-fold lower for hydrogen peroxide. The reduction of catalytic activity caused by altering the reaction media from water to toluene was found to be mainly due to the low specific constant of ARP complex for o-PDA rather than hydrogen peroxide.     

Oxidative Half-reaction of Arabidopsis thaliana Sulfite Oxidase: GENERATION OF SUPEROXIDE BY A PEROXISOMAL ENZYME*

Vertebrate forms of the molybdenum-containing enzyme sulfite oxidase possess a b-type cytochrome prosthetic group that accepts reducing equivalents from the molybdenum center and passes them on to cytochrome c. The plant form of the enzyme, on the other hand, lacks a prosthetic group other than its molybdenum center and utilizes molecular oxygen as the physiological oxidant. Hydrogen peroxide is the ultimate product of the reaction. Here, we present data demonstrating that superoxide is produced essentially quantitatively both in the course of the reaction of reduced enzyme with O₂ and during steady-state turnover and only subsequently decays (presumably noncatalytically) to form hydrogen peroxide. Rapid-reaction kinetic studies directly following the reoxidation of reduced enzyme demonstrate a linear dependence of the rate constant for the reaction on [O₂] with a second-order rate constant of k_ox = 8.7 × 10⁴ ± 0.5 × 10⁴ m⁻¹s⁻¹. When the reaction is carried out in the presence of cytochrome c to follow superoxide generation, biphasic time courses are observed, indicating that a first equivalent of superoxide is generated in the oxidation of the fully reduced Mo(IV) state of the enzyme to Mo(V), followed by a slower oxidation of the Mo(V) state to Mo(VI). The physiological implications of plant sulfite oxidase as a copious generator of superoxide are discussed.     

Respiratory components and oxidase activities in Alcaligenes eutrophus

1. Cells of the hydrogen bacterium Alcaligenes eutrophus are broken by gentle lysis using lysozyme treatment in hypertonic sucrose followed by osmotic shock. By this method, 93% of the in vivo activity of the H₂ oxidase is recovered and the ATPase remains particle bound. In contrast, cell disruption in a French pressure cell diminishes the in vivo activity of the H₂ oxidase by 50% and solubilizes the bulk of the ATPase.2. The bacterium contains a periplasmic cytochrome c with bands at 418, 521 and 550 nm (difference spectrum). In addition to cytochrome aa₃, b-560, c-553 and o, low temperature difference spectra of membranes show the presence of two further cytochromes (shoulders at 551 and 553 nm).3. The unsupplemented membrane fraction catalyses the oxidation of hydrogen, NADH, NADPH, succinate, formate and endogenous substrate (NAD linked) at rates 2–3-fold higher than membranes obtained from cells disrupted in a French pressure cell. With the exception of the H₂ oxidase all oxidase activities in lysozyme membranes are sensitive to carbonylcyanide m-chlorophenylhydrazone (20–100% stimulation of oxygen uptake).4. The cytoplasmic fraction contains a B-type cytochrome with absorption maxima at 436 and 560 nm, capable of combining with CO; it contains non-covalently bound protohaem. In alkaline solutions a spectral transition to the haemochrome type with bands at 423, 526 and 556 nm occurs. The addition of NADH to an aerobic suspension of this cytochrome elicits new absorption maxima at 418, 545 and 577 nm (difference spectrum), which are believed to represent an oxygenated form of the reduced cytochrome.     

Physical and catalytic properties of a peroxidase derived from cytochrome c

Except for its redox properties, cytochrome c is an inert protein. However, dissociation of the bond between methionine-80 and the heme iron converts the cytochrome into a peroxidase. Dissociation is accomplished by subjecting the cytochrome to various conditions, including proteolysis and hydrogen peroxide (H₂O₂)-mediated oxidation. In affected cells of various neurological diseases, including Parkinson's disease, cytochrome c is released from the mitochondrial membrane and enters the cytosol. In the cytosol cytochrome c is exposed to cellular proteases and to H₂O₂ produced by dysfunctional mitochondria and activated microglial cells. These could promote the formation of the peroxidase form of cytochrome c. In this study we investigated the catalytic and cytolytic properties of the peroxidase form of cytochrome c. These properties are qualitatively similar to those of other heme-containing peroxidases. Dopamine as well as sulfhydryl group-containing metabolites, including reduced glutathione and coenzyme A, are readily oxidized in the presence of H₂O₂. This peroxidase also has cytolytic properties similar to myeloperoxidase, lactoperoxidase, and horseradish peroxidase. Cytolysis is inhibited by various reducing agents, including dopamine. Our data show that the peroxidase form of cytochrome c has catalytic and cytolytic properties that could account for at least some of the damage that leads to neuronal death in the parkinsonian brain.     

Hydrogen peroxide formation and stoichiometry of hydroxylation reactions catalyzed by highly purified liver microsomal cytochrome P-450.

The stoichiometry of hydroxylation reactions catalyzed by cytochrome P-450 was studied in a reconstituted enzyme system containing the highly purified cytochrome from phenobarbital-induced rabbit liver microsomes. Hydrogen peroxide was shown to be formed in the reconstituted system in the presence of NADPH and oxygen; the amount of peroxide produced varied with the substrated added. NADPH oxidation, oxygen consumption, and total product formation (sum of hydroxylated compound and hydrogen peroxide) were shown to be equimolar when cyclohexane, benzphetamine, or dimethylaniline served as the substrate. The stoichiometry observed represents the sum of two activities associated with cytochrome P-450. These are (1) hydroxylase activity: NADPH + H⁺ + O₂ + RH → NADP⁺ + H₂O + ROH; and (2) oxidase activity: NADPH + H⁺ + O₂ → NADP⁺ + H₂O₂. Benzylamphetamine (desmethylbenzphetamine) acts as a pseudosubstrate in that it stimulates peroxide formation to the same extent as the parent compound (benzphetamine), but does not undergo hydroxylation. Accordingly, when benzylamphetamine alone is added in control experiments to correct for the NADPH and O₂ consumption not associated with benzphetamine hydroxylation, the expected 1:1:1 stoichiometry for NADPH oxidation, O₂ consumption, and formaldehyde formation in the hydroxylation reaction is observed.     

Δ1-Pyrroline-5-carboxylate: The product of proline dehydrogenase from Cucurbita moschata cotyledons

The product of oxidation of proline by pumpkin proline dehydrogenase reacted with o-aminobenzaldehyde to give a yellow compound that had an absorption spectrum similar to that obtained from chemically synthesized Δ¹-pyrroline-5-carboxylate. The product of the proline dehydrogenase reaction and synthetic Δ¹-pyrroline-5-carboxylate had identical R_f values. Both authentic Δ¹-pyrroline-5-carboxylate and the product of the enzyme gave a pink colour with acid ninhydrin on paper chromatograms and both had identical elution patterns on Dowex 50(H⁺) columns. Neither synthetic Δ¹-pyrroline-5-carboxylate nor the product of proline-dehydrogenase produced γ-amino butyrate with hydrogen peroxide.     

Isolation and purification of the cytochrome oxidase of Azotobacter vinelandii

A membrane-bound cytochrome oxidase from Azobacter vinelandii was purified 20-fold using a detergent-solubilization procedure. Activity was monitored using an ascorbate-TMPD oxidation assay. The oxidase was ‘solubilized’ from a sonic-type electron-transport particle (R₃ fraction) using Triton X-100 and deoxycholate. Low detergent concentrations first solubilized the flavoprotein oxidoreductases, then higher concentrations of Triton X-100 and KCl solubilized the oxidase, which was precipitated at 27–70% (NH₄)₂SO₄. The highly purified cytochrome oxidase has a V of 60–78 μgatom O consumed/min per mg protein. TMPD oxidation by the purified enzyme was inhibited by CO, KCN, NaN₃ and NH₂OH; NaNO₂ (but not NaNO₃) also had a potent inhibitory effect. Spectral analyses revealed two major hemoproteins, the c-type cytochrome c₄ and cytochrome o; cytochromes a₁ and d were not detected. The Azotobacter cytochrome oxidase is an integrated cytochrome c₄?o complex, TMPD-dependent cytochrome oxidase activity being highest in preparations having a high c-type cytochrome content. This TMPD-dependent cytochrome oxidase serves as a major oxygen-activation site for the A. vinelandii respiratory chain. It appears functionally analogous to cytochrome a+a₃ oxidase of mammalian mitochondria.     

Lipoperoxide radical production during oxidation of cardiolipin in the complex with cytochrome c

The key stage of apoptosis is lipid peroxidation which causes cytochrome c efflux from mitochondria. Cardiolipin-bound cytochrome c on the surface of the inner mitochondrial membrane is supposed to be a main lipoperoxidation catalyst. In this work, lipoperoxide radical (LOO·) production in the complex of cytochrome c (Cyt C) with bovine heart cardiolipin (BCL) was investigated with the method of chemiluminescence (CL) in the presence of a physical activator, coumarin dye C-525. It was shown that a CL flash with a half quenching time of 1.12 min was observed after the addition of Cyt C to a BCL+C-525 solution in the absence of hydrogen peroxide. At H₂O₂ concentrations of 0.1–0.5 mM, quenching time reduced at constant CL flash amplitude and at H₂O₂ concentrations of 1–5 mM, the amplitude of CL increased with the growth of peroxide concentration. It testifies to different mechanisms of BCL oxidation: the lipoxygenase mechanism in the absence of H₂O₂ and at low H₂O₂ concentrations, and the peroxidase mechanism at higher H₂O₂ concentrations. When small H₂O₂ amounts were added, another CL flash was observed in the course of a lipoxygenase reaction whose light sum increased with time in parallel with the extent of the following inhibition of CL. Iron chelators EDTA and o-phenanthroline made no significant effect on the CL associated with cytochrome c lipoxygenase action, while desferal, a well-known peroxidase and lipoxygenase inhibitor, inhibited CL by half in a concentration of 18 μM. A scheme of reactions resulting in LOO· radical production on BCL oxidation by the Cyt C-cardiolipin complex in the absence and in the presence of H₂O₂ was suggested.     

Oxygen reduction in chloroplast thylakoids results in production of hydrogen peroxide inside the membrane

Hydrogen peroxide production in isolated pea thylakoids was studied in the presence of cytochrome c to prevent disproportionation of superoxide radicals outside of the thylakoid membranes. The comparison of cytochrome c reduction with accompanying oxygen uptake revealed that hydrogen peroxide was produced within the thylakoid. The proportion of electrons from water oxidation participating in this hydrogen peroxide production increased with increasing light intensity, and at a light intensity of 630 μmol quanta m^− 2 s^− 1 it reached 60% of all electrons entering the electron transport chain. Neither the presence of a superoxide dismutase inhibitor, potassium cyanide or sodium azide, in the thylakoid suspension, nor unstacking of the thylakoids appreciably affected the partitioning of electrons to hydrogen peroxide production. Also, osmolarity-induced changes in the thylakoid lumen volume, as well as variation of the lumen pH induced by the presence of Gramicidin D, had negligible effects on such partitioning. The flow of electrons participating in lumen hydrogen peroxide production was found to be near 10% of the total electron flow from water. It is concluded that a considerable amount of hydrogen peroxide is generated inside thylakoid membranes, and a possible mechanism, as well as the significance, of this process are discussed.     

Preparation of Labeled Casein fron Goat Milk after Lysine-14C Injection

Hydrogen peroxide in methylotrophic yeasts can be metabolized in at least two distinct ways. Addition of exogenous hydrogen peroxide removes the dependance of catalase on endogenously-produced hydrogen peroxide resulting enhanced rates of alcohol oxidation. Exogenous hydrogen peroxide is also efficiently degraded by cytochrome c peroxidase (CCP), a competitive reaction which does not result in enhanced alcohol oxidation. To overcome the influence of cytochrome c peroxidase, artificial peroxisomes were prepared by coimmobilization of alcohol oxidase and catalase. These artificial peroxisomes mimic the peroxide-induced rate enhancement observed with whole cells.     

Reaction center complex from an aerobic photosynthetic bacterium,Erythrobacter species OCh 114

A photosynthetic reaction center complex has been purified from an aerobic photosynthetic bacterium, Erythrobacter species OCh 114. The reaction center was solubilized with 0.45% lauryldimethylamine N-oxide and purified by DEAE-Sephacel column chromatography. Absorption spectra of both reduced and oxidized forms of the reaction center were very similar to those of the reaction center from Rhodopseudomonas sphaeroides R-26 except for the contributions due to cytochrome and carotenoid. 1 mol reaction center contained 4 mol bacteriochlorophyll a, 2 mol bacteriopheophytin a, 4 mol cytochrome c-554, 2 mol ubiquinone-10, and carotenoid. The reaction center consisted of four different polypeptides of 26, 30, 32 and 42 kDa. The last one retained heme c. Absorbance at 450 nm oscillated with the period of two on consecutive flashes. The light-minus-dark difference spectrum had two peaks at 450 nm and 420 nm, indicating that odd flashes generated a stable ubisemiquinone anion and even flashes generated quinol. o-Phenanthroline accelerated the re-reduction of flash-oxidized reaction centers, indicating that o-phenanthroline inhibited the electron transfer between Q_A and Q_B. The cytochrome (cytochrome c-554) in the reaction center was oxidized on flash activation. The midpoint potential of the primary electron acceptor (Q_A) was determined by measuring the extent of oxidation of cytochrome c-554 at various ambient potentials. The mid-point potential of Q_A was −44 mV, irrespective of pH between 5.5 and 5.9.     

Electrocatalytic four-electron reduction of oxygen at the cytochrome c3-adsorbed electrode

The electrocatalytic activity of cytochrome c₃ for the reduction of molecular oxygen was characterized from the studies of the adsorption of cytochrome c₃ and the co-adsorption of cytochrome c₃ with cytochrome c on the mercury electrode by the a.c. polarographic technique. The adsorption of cytochrome c₃ on the mercury electrode is irreversible and is diffusion-controlled. The maximum amount of cytochrome c₃ adsorbed was 0.92 · 10^?11 mol · cm^?2 at ?0.90 V. The amount of cytochrome c₃ in the mixed adsorbed layer with cytochrome c was determined from the differential capacitance measurement. It was shown that the fractional coverage of cytochrome c₃ can be estimated from its bulk concentration and the diffusion coefficient (1.05 · 10^?6 cm² · s^?1). Cytochrome c₃ catalyzes the electrochemical reduction of molecular oxygen from the two-electron pathways via hydrogen peroxide to the four-electron pathway at the mercury electrode in neutral phosphate buffer solution. The catalytic activity varies with the bulk concentration of cytochrome c₃. The highest catalytic activity for the oxygen reduction (no hydrogen peroxide formation) is attained when one-half of the mercury electrode surface is covered by cytochrome c₃. The addition of cytochrome c or bovine serum albumin to the cytochrome c₃ solution inhibits the catalytic activity of cytochrome c₃. The reversible polarographic behavior of cytochrome c₃ through the mixed adsorbed layer of cytochrome c₃ and cytochrome c was also investigated.     

Studies on Auxin Protectors: XI. Inhibition of Peroxidase-Catalyzed Oxidation of Glutathione by Auxin Protectors and o-Dihydroxyphenols

Commercial horseradish peroxidase, when supplemented with dichlorophenol and either manganese or hydrogen peroxide, will rapidly oxidize glutathione. This peroxidase-catalyzed oxidation of glutathione is completely inhibited by the presence of auxin protectors. Three auxin protectors and three o-dihydroxyphenols were tested; all inhibited the oxidation. Glutathione oxidation by horseradish peroxidase in the presence of dichlorophenol and Mn is also completely inhibited by catalase, implying that the presence of Mn allows the horseradish peroxidase to reduce oxygen to H₂O₂, then to use the H₂O₂ as an electron acceptor in the oxidation of glutathione. Catalase, added 2 minutes after the glutathione oxidation had begun, completely inhibited further oxidation but did not restore any gluthathione oxidation intermediates. In contrast, the addition of auxin protectors, or o-dihydroxyphenols, not only inhibited further oxidation of gluthathione by horseradish peroxidase (+ dichlorophenol + Mn), but also caused a reappearance of glutathione as if these antioxidants reduced a glutathione oxidation intermediate. However, when gluthathione was oxidized by horseradish peroxidase in the presence of dichlorophenol and H₂O₂ (rather than Mn), then the inhibition of further oxidation by auxin protectors or o-dihydroxyphenols was preceded by a brief period of greatly accelerated oxidation. The data provide further evidence that auxin protectors are cellular redox regulators. It is proposed that the monophenol-diphenol-peroxidase system is intimately associated with the metabolic switches that determine whether a cell divides or differentiates.     

A covalent complex between horse heart cytochrome c and yeast cytochrome c peroxidase: kinetic properties

The kinetic properties of a 1:1 covalent complex between horse-heart cytochrome c and yeast cytochrome c peroxidase (ferrocytochrome-c:hydrogen-peroxide oxidoreductase, EC 1.11.1.5) have been investigated by transient-state and steady-state kinetic techniques. Evidence for heterogeneity in the complex is presented. About 50% of the complex reacts with hydrogen peroxide with a rate 20–40% faster than that of native enzyme; 20% of the complex exists in a conformation which does not react with hydrogen peroxide but converts to the reactive form at a rate of 20 ± 5 s⁻¹; 30% of the complex does not react with hydrogen peroxide to form the oxidized enzyme intermediate, cytochrome c peroxidase Compound I. Intramolecular electron transfer between covalently bound ferrocytochrome c and an oxidized site in cytochrome c peroxidase Compound I is too fast to measure, but a lower limit of 600 s⁻¹ can be estimated at 5°C in a 10 mM potassium phosphate buffer at pH 7.5. Free ferrocytochrome c reduces cytochrome c peroxidase Compound I covalently bound to ferricytochrome c at a rate 10⁻⁴ to 10⁻⁵-times slower than for free Compound I. The transient-state ferrocytochrome c reduction rates of Compound I covalently linked to ferricytochrome c are about 70-times too slow to account for the steady-state catalytic properties of the 1:! covalent complex. This indicates that hydrogen peroxide can interact with the 1:1 complex at sites other than the heme of cytochrome c peroxidase, generating additional species capable of oxidizing free ferrocytochrome c.     

Tropolone as a substrate for horseradish peroxidase

Tropolone (2,4,6-cycloheptatrien-1-one), in the presence of hydrogen peroxide but not in its absence, can serve as a donor for the horseradish peroxidase catalysed reaction. The product formed is yellow and is characterized by a new peak at 418 nm. The relationship between the rate of oxidation of tropolone (ΔA at 418 nm/min) and various concentrations of horseradish peroxidase, tropolone and hydrogen peroxide is described. The yellow product obtained by the oxidation of tropolone by horseradish peroxidase in the presence of hydrogen peroxide was purified by chromatography on Sephadex G-10 and its spectral properties at different pHs are presented. The M, of the yellow product was estimated to be ca 500, suggesting that tropolone, in the presence of horseradish peroxidase and hydrogen peroxide is converted to a tetratropolone.     

Cytochrome c catalyzes the in vitro synthesis of arachidonoyl glycine

Long chain fatty acyl glycines are an emerging class of biologically active molecules that occur naturally and produce a wide array of physiological effects. Their biosynthetic pathway, however, remains unknown. Here we report that cytochrome c catalyzes the synthesis of N-arachidonoyl glycine (NAGly) from arachidonoyl coenzyme A and glycine in the presence of hydrogen peroxide. The identity of the NAGly product was verified by isotope labeling and mass analysis. Other heme-containing proteins, hemoglobin and myoglobin, were considerably less effective in generating arachidonoyl glycine as compared to cytochrome c. The reaction catalyzed by cytochrome c in vitro points to its potential role in the formation of NAGly and other long chain fatty acyl glycines in vivo.     

Functional flexibility of electron flow between quinol oxidation Qo site of cytochrome bc1 and cytochrome c revealed by combinatory effects of mutations in cytochrome b,iron-sulfur protein and cytochrome c1

Transfer of electron from quinol to cytochrome c is an integral part of catalytic cycle of cytochrome bc₁. It is a multi-step reaction involving: i) electron transfer from quinol bound at the catalytic Q_o site to the Rieske iron-sulfur ([2Fe-2S]) cluster, ii) large-scale movement of a domain containing [2Fe-2S] cluster (ISP-HD) towards cytochrome c₁, iii) reduction of cytochrome c₁ by reduced [2Fe-2S] cluster, iv) reduction of cytochrome c by cytochrome c₁.In this work, to examine this multi-step reaction we introduced various types of barriers for electron transfer within the chain of [2Fe-2S] cluster, cytochrome c₁ and cytochrome c. The barriers included: impediment in the motion of ISP-HD, uphill electron transfer from [2Fe-2S] cluster to heme c₁ of cytochrome c₁, and impediment in the catalytic quinol oxidation. The barriers were introduced separately or in various combinations and their effects on enzymatic activity of cytochrome bc₁ were compared. This analysis revealed significant degree of functional flexibility allowing the cofactor chains to accommodate certain structural and/or redox potential changes without losing overall electron and proton transfers capabilities. In some cases inhibitory effects compensated one another to improve/restore the function. The results support an equilibrium model in which a random oscillation of ISP-HD between the Q_o site and cytochrome c₁ helps maintaining redox equilibrium between all cofactors of the chain. We propose a new concept in which independence of the dynamics of the Q_o site substrate and the motion of ISP-HD is one of the elements supporting this equilibrium and also is a potential factor limiting the overall catalytic rate.     

Cytochrome c-linked reactions in Rhodopseudomonas palustris grown photosynthetically on thiosulfate

The nonsulfur purple bacterium Rps. palustris was adapted to grow photoautotrophically with thiosulfate as substrate. An isolated cell-free fraction catalyzed the enzymatic transfer of electrons from thiosulfate to endogenous and/or added mammalian cytochrome c. Antimycin A, NOQNO, rotenone, amytal and atebrin did not inhibit the thiosulfate-cytochrome c reductase. The products of thiosulfate oxidation were primarily tetrathionate, trithionate, and sulfate, suggesting oxidation via the polythionate pathway. Succinate, formate and NADH were also effective electron donors in this system showing Michaelis constants of 40, 30 and 0.025 mm, respectively for cytochrome c reduction. The NADH-cytochrome c reductase was not inhibited by flavoprotein inhibitors and by Antimycin A or NOQNO. The cell-free extracts also contained an active cytochrome c-O₂ oxidoreductase which was inhibited by cyanide, azide and EDTA, and these inhibitions were overcome by the addition of Cu²⁺. The oxidase activity was stimulated by the addition of uncoupling agents such as CCCP and DNP, as well as by Antimycin A and NOQNO. Reduced + CO minus reduced difference absorption spectra revealed the presence of cytochrome components of the a and o types which may function as the terminal oxidase(s).