DPNH Dehydrogenase Research Articles

Electron Paramagnetic Resonance-detectable Electron Acceptors in Beef Heart Mitochondria: REDUCED DIPHOSPHOPYRIDINE NUCLEOTIDE UBIQUINONE REDUCTASE SEGMENT OF THE ELECTRON TRANSFER SYSTEM

Abstract The EPR-detectable (≥4.2 K) electron acceptors of the DPNH-ubiquinone reductase segment of the electron transfer system of beef heart mitochondria were studied in preparations of varying complexity. Four distinct iron-sulfur centers associated with this segment of the electron transfer system were identified. They occur in approximately equal concentrations, and this concentration approximates that of the bound flavin of DPNH dehydrogenase. Quantitative reductive titrations with DPNH and dithionite showed that the order of the oxidation-reduction potentials of the ironsulfur centers is: Center 3 ≥ 2 >> 4 g 1, indicating that there are two centers of high and two of low potential. Complex I accepts approximately 20 electron neq per mg of protein from DPNH. In rapid reaction studies using the freeze-quench method the rates of reduction of the iron-sulfur centers by DPNH could not be resolved. With acetylpyridine DPNH, however, the rates of appearance of the signals were in this sequence: Center 2 g 3 + 4 g 1.

Catalytic activity and EPR signals of DPNH dehydrogenase in relation to the acquisition and loss of piericidin sensitivity and of coupling site 1

The events accompanying the development of piericidin sensitivity and of energy coupling site 1 during the transition of Candida utilis cells from the log phase to the late stationary phase of growth have been investigated. There is a large increase in DPNH dehydrogenase (DPNHD) activity, and major increases in the EPR signals of iron-sulfur centers 1, 2, and 3 of the enzyme, as measured at 13°K. The increase in DPNHD activity, however, does not reflect the increased development of the same enzyme as is present in the log phase, for the enzyme being synthesized in the stationary phase is different from that present in the log phase, as judged by juglone reductase and DPNH oxidase activities, which declined during the transition, and by stability, kinetics, and in the type of EPR signal present, which are different in the log and stationary phases. On catabolite repression of stationary phase cells the converse occurs: the specific activities of juglone reductase and DPNH oxidase rise, DPNHD activity and EPR signals corresponding to centers 1 and 2 disappear. This process of catabolite repression by ethanol is prevented by cycloheximide.

Purification, properties and reconstitutive activity of a DPNH dehydrogenase

A DPNH dehydrogenase has been solubilized from cardiac submitochondrial particles using Triton X-100 and purified by (NH 4) 2SO 4 fractionation, chromatography and density gradient centrifugation. It is an iron-flavoprotein free of lipid; the ratio of FMN:Fe:S was 1:28:28. The dehydrogenase can use CoQ as electron acceptor and the reaction is inhibited by Amytal and rotenone. The purified enzyme reacts with the cytochrome b - c 1 particle to reconstitute antimycin A sensitive DPNH cytochrome c reductase.

Studies of the Electron Transport Chain of Extremely Halophilic Bacteria: VII. SOLUBILIZATION PROPERTIES OF MENADIONE REDUCTASE

Abstract When NaCl concentration was lowered, membrane vesicle-bound menadione reductase of Halobacterium cutirubrum showed 6- to 7-fold increased activity. The enzyme with this increased activity was initially found in a transient, membrane-bound form, followed by release from the membranes. Several detergents caused solubilization and a similar increase in enzyme activity, but sodium dodecyl sulfate, which increased activity, gave only limited solubilization. Unlike mitochondrial DPNH dehydrogenase, the halophilic system showed only minor changes in substrate affinity upon these treatments. Conditions that resulted in highly active, membrane-bound enzyme also released trapped horseradish peroxidase, which had been added as a marker of the interior of these vesicles. These results suggest that menadione reductase is attached to the interior surface of the vesicles and becomes accessible to the substrates upon the breakage of the vesicles during the course of solubilization. Half of the menadione reductase content of the vesicles was released into the supernatant in 1 m NaCl but was still attached to large membrane fragments. As NaCl concentration was further lowered the enzyme in this fraction was detached and could be eluted from an Agarose column in a position corresponding to 36,000 molecular weight. In addition, at NaCl concentrations lower than 1 m, large membrane fragments containing the second portion of the enzyme were released. Tween 80 solubilized half of the total enzyme in the vesicles; the solubilized enzyme was eluted at 48,000 molecular weight, which probably corresponds to a protein-detergent complex. The sequential action of lowered NaCl concentration and Tween 80 resulted in the recovery of all the enzyme in this low molecular weight fraction.

Effect of Silicotungstate on Reduced Diphosphopyridine Nucleotide Oxidation in Submitochondrial Particles

Abstract 1. Silicotungstate inhibits DPNH oxidase, succinoxidase, and 32Pi-ATP exchange activity at relatively low concentrations (150 µg per mg of protein). The inhibition is reversed by bovine serum albumin. 2. When the concentrations of silicotungstate are increased (0.3 to 1.4 mg per mg of protein), DPNH oxidase activity appears which is insensitive to rotenone. Addition of serum albumin restores rotenone sensitivity. 3. Silicotungstate was shown to interact directly with DPNH dehydrogenase. The higher levels of silicotungstate convert both the particulate and soluble DPNH dehydrogenase but not succinate dehydrogenase into an oxidase enzyme.

EPR studies on the iron-sulfur centers of dpnh dehydrogenase during the redox cycle of the enzyme

Summary Preparations of the inner mitochondrial membrane, upon treatment with DPNH in the aerobic state, undergo a cycle of absorbance changes, which may be monitored at 470–500 mμ. There is rapid initial bleaching, followed by return of the color on exhaustion of DPNH. In the presence of rotenone or piericidin the cycle is prolonged, the return of color (reoxidation) is inhibited and the extent of reoxidation less than in uninhibited particles. The EPR signals of four different iron-sulfur centers associated with DPNH dehydrogenase have thus far been resolved at temperatures between 4–20°K. Of these the iron-sulfur center of lowest potential, center 1, is reoxidized by the respiratory chain when DPNH is exhausted, but the high potential center 2 remains reduced. ATP reoxidizes iron-sulfur center 2 and restores full color to the chromophore. It is suggested that the energy for the reoxidation of the chromophore and iron-sulfur center 2 is supplied by coupling site I. On the basis of these and of other observations it is tentatively proposed that coupling site I is directly associated with DPNH dehydrogenase and is located on the 0 2 side of iron-sulfur center 1 and the substrate side of both center 2 and the specific binding sites of rotenone and piericidin.

The DPNH dehydrogenase of the mitochondrial respiratory chain.

The DPNH dehydrogenase of the mitochondrial respiratory chain.

Effect of ATP on the redox cycle of respiratory chain-linked DPNH dehydrogenase and the localization of coupling site I

On adding DPNH to a phosphorylating membrane preparation in the presence of O 2 the reduction of a chromophore, which has been tentatively identified as nonheme iron of DPNH dehydrogenase, may be observed by dual wavelength spectrophotometry at 470–500 mμ. With rotenone or piericidin present a part of the chromophore remains permanently bleached even after exhaustion of the DPNH. ATP, however, causes rapid and complete reoxidation of the chromophore. This effect is abolished by oligomycin and dinitrophenol. Control experiments show that under these conditions the rotenone block between DPNH dehydrogenase and cytochrome b is substantially complete. Analysis of the data suggest that coupling site I is located between the specific binding site of rotenone and the nonheme irons responsible for the g=1.94 signal, both of which appear to be constituents of DPNH dehydrogenase.

Rhein, a selective inhibitor of the DPNH-flavin step in mitochondrial electron transport

Previous findings in the literature that rhein inhibits DPNH-linked mitochondrial oxidations by acting in the DPNH dehydrogenase region of the respiratory chain have been confirmed and extended. In the micromolar range rhein inhibits DPNH oxidase and DPNH-ferricyanide activities and the energy-linked reduction of DPN by succinate in membrane preparations from heart, as well as the DPNH dehydrogenase and transhydrogenase activities of the soluble, purified enzyme. The inhibition of the activities of the soluble enzyme are purely competitive with respect to substrate. These facts localize the primary inhibition site of rhein between substrate and FMN. In. heart ETP a second noncompetitive inhibition is also present but is detectable only at very low (<10 μM) rhein concentrations. Rhein also inhibits DPNH dehydrogenase in Candida utilis mitochondria and the purified enzyme from liver. On conversion of the heart enzyme to the low molecular weight DPNH-cytochrome reductase the typical effect of rhein disappears and is replaced by a slight stimulation or inhibition, depending on the electron acceptor used, showing that the substrate-binding site is modified in this form of the enzyme. In beef liver mitochondria DPNH oxidation may appear insensitive to rhein, probably because of the strong binding of rhein to other proteins. To a lesser extent unspecific binding of rhein and resultant interference with the inhibition of DPNH dehydrogenase is also shown by BSA and by proteins in heart ETP. Rhein also inhibits transhydrogenations in mitochondria and at higher concentrations lactate and malate dehydrogenases but has no effect on succinate, alcohol (liver and yeast), and glucose-6-P dehydrogenases or on Neurospora DPN-ase, glucose-6-phosphatase, and amine oxidase.

Ultrastructural and histochemical correlations of experimental muscle regeneration

A correlative ultrastructural and light microscopic histochemical investigation was performed in cold-injured gastrocnemius muscle in normal adult mice. Three zones were in evidence after the injury: undamaged muscle, a necrotic area, and an intermediate region. In the necrotic area, degenerating myofibres contain mainly fragments of myofibrils; they are stained by the myofibrillar ATP-ase and the menadione-mediated α-glycerophosphate dehydrogenase reactions. Necrotic fibres are not stained by the reactions for 4 oxidative enzymes (succinate, lactate, malate, and DPNH dehydrogenase) or the phosphorylase reaction. In the intermediate region, many persistent myofibres possess collections of enlarged mitochondria; they are darkly-stained with the 4 oxidative enzyme reactions mentioned above. Early regenerating sarcoblasts are rich in ribosomes, have central nuclei with adjacent accumulations of mitochondria, and myofibrils developing at the periphery of the cell; their cytoplasm is strongly basophilic and the myofibrils are well-stained with the myofibrillar ATP-ase. Sarcoblasts present dark irregular dots between myofibrils and centrally, adjacent to nuclei, with the oxidative enzyme reactions used. Later, regenerated myofibres are recognized by central nuclei, large mitochondria and an undeveloped sarcotubular system; they display an intense but irregular staining with the oxidative enzyme reactions and are darkly-stained by the ATP-ase reaction. Large regenerated myofibres acquire a positive phosphorylase reaction.

Studies of the Electron Transport Chain of Extremely Halophilic Bacteria: II. Salt dependence of reduced diphosphopyridine nucleotide oxidase

Abstract A scheme of electron transport in Halobacterium cutirubrum is proposed based on the following findings. (a) The slow rate of reduction of b-type cytochromes by DPNH is not consistent with the participation of these components in the main pathway of the oxidation of DPNH. (b) DPNH oxidation proceeds through a fast, 2-heptyl-4-hydroxyquinoline N-oxide (QO)-sensitive and a slow, QO-insensitive path. Succinate oxidation, which is slow, is not QOsensitive. These results suggest a main branch of electron transport including a flavoprotein, reduced by DPNH, and a second branch consisting of flavoproteins and cytochrome b(559), reduced by DPNH and succinate. The branches join at the c-type cytochromes. Cytochrome b(563), although reduced by the dehydrogenase of the main branch, lies outside the main DPNH oxidase pathway. The relation of DPNH oxidase activity to NaCl concentration and the irreversible inactivation of the enzyme in the absence of salt reflect the behavior of a single component of the system, DPNH dehydrogenase. The place of the b-type cytochromes in the proposed electron transport scheme is confirmed through the determination of salt dependence curves.

Inhibitors and Activators of the Mitochondrial Reduced Diphosphopyridine Nucleotide Dehydrogenase

Abstract The inhibitor response properties of the mitochondrial DPNH dehydrogenase have been studied with both the particle-bound and the soluble forms of the enzyme. Electron transfer from the particle-bound DPNH dehydrogenase to the respiratory chain is inhibited by barbiturates, Demerol, rotenone, and piericidin A, whereas the activities of the soluble enzyme are essentially unaffected by these inhibitors. In contrast, the ferricyanide reductase, as well as other diaphorase activities, of the soluble DPNH dehydrogenase is inhibited by mercurials, whereas the bound dehydrogenase appears to be protected against such mercurial attack. o-Phenanthroline interacts with the soluble dehydrogenase, inhibits its activities, and causes the release of labile sulfide from the enzyme. However, other bidentate iron chelators such as bathophenanthroline sulfonate and Tiron do not inhibit the enzyme but interact with it and protect it against inhibition by o-phenanthroline. These studies have indicated that in DPNH dehydrogenase the complex among iron, labile sulfide groups, and cysteine sulfur residues must have a tetrahedral structure. Adenosine phosphates inhibit DPNH dehydrogenase in the same manner as does DPN, but adenosine, nicotinamide mononucleotide, and NMNH are not inhibitory. Guanidinium compounds activate the soluble DPNH dehydrogenase, but inhibit the coenzyme Q reductase activity of the particulate DPNH-coenzyme Q reductase (Complex I). The activating effect of guanidinium compounds on the soluble dehydrogenase is associated not only with an increase in the value of Vmax, but also with a substantial decrease in the value of Kmdpnh (3-fold at 50 mm guanidine).

Isolation and Enzymatic Properties of the Mitochondrial Reduced Diphosphopyridine Nucleotide Dehydrogenase

Abstract The DPNH dehydrogenase of the mitochondrial electron transport system has been obtained in a highly purified, soluble form after resolution of DPNH-coenzyme Q reductase (Complex I) particles by chaotropic agents. The enzyme contains 1 mole of flavin mononucleotide, 4 g atoms of iron, and 4 moles of acid-labile sulfide per 70,000 g. The soluble, but not the particle-bound, enzyme behaves as a diaphorase and catalyzes the unspecific reduction of quinones and ferric compounds such as menadione, 2,6-dichloroindophenol, coenzymes Q, ferricyanide, and cytochrome c. Mercurials inhibit all the reductase activities of the soluble dehydrogenase, and DPNH (g10-4 m) inhibits its menadione and coenzyme Q reductase activities. The structure and function of DPNH dehydrogenase in both the soluble and the particle-bound form are discussed.

Inactivation of lyophilized DPNH dehydrogenase from Escherichia coli by oxygen

Lyophilized Escherichia coli membranes lose 50% of their DPNH oxidase activity when exposed to O 2. The oxidation of succinate or lactate is not affected by this treatment. The enzyme damaged by O 2 is the membrane DPNH dehydrogenase (DPNH-ferricyanide oxidoreductase, EC 1.6.99.3). The K m values for DPNH and the electron acceptor K 3Fe(CN) 6 are the same in the native and the inactivated enzyme. The v max of the latter is decreased by 50% for both substrates. The accessibility of non-heme iron to o-phenanthroline is partly increased. It is suggested that the non-heme iron of DPNH dehydrogenase participates in intramolecular electron conductance between the oxidizing and the reducing sites of the enzyme.

Optical Rotatory Dispersion of Mitochondrial Reduced Diphosphopyridine Nucleotide Dehydrogenase from Cardiac Tissue

Abstract Optical rotatory dispersion spectra of oxidized and reduced FMN and FAD are presented from 600 to 210 mµ and of riboflavin from 600 to 340 mµ. Optical rotatory dispersion behavior of soluble DPNH dehydrogenase was studied between 600 and 185 mµ with respect to the oxidation state of the enzyme. The optical rotatory dispersion spectra were analyzed and the extrinsic Cotton effects were resolved from the protein backbone rotations. Shechter-Blout's A' parameters were computed: A'193 = 147 and A'225 = -278° for the oxidized enzyme, and A'193 = 32 and A'225 = -210° for the reduced. The centers of the resolved extrinsic Cotton effects due to nonprotein chromophores were 437 mµ and 350 mµ for the oxidized and 436 mµ and 346 mµ for the reduced enzyme. The rotational strength, amplitude, and band width of these Cotton effects were dependent upon the oxidation state of the enzyme. In addition, anomalous rotations in the region of 550 mµ were also observed but were too small for accurate analysis. These observations indicate that the interactions between the chromophores and the protein are dependent upon the oxidation state of the dehydrogenase. This dependence may be due to changes in geometrical relationships within the system and in the conformation of the protein moiety which is also determined by the oxidation state of the prosthetic groups. These changes may in turn play an intrinsic role in the physiological action of the enzyme for electron transfer in the respiratory chain.

The preparation and properties of the membranal DPNH dehydrogenase from Escherichia coli

1. The membrane bound DPNH oxidase of Escherichia coli can reduce the artificial electron acceptors: ferricyanide, dichlorophenolindophenol (DCIP) and menadione. All three are reduced by the flavoprotein of DPNH oxidase, but at different sites of the enzyme. 2. Freeze-drying of the bacterial membranes causes a selective detachment of DPNH dehydrogenase (DPNH: (acceptor) oxidoreductase, EC 1.6.99.3) from the membranes. This solubilization is accompanied by a decrease of K m (K 3Fe(CN) 6) from 2.0 to 0.25 mM, while no change is detected in K m (DPNH). This enzyme is not the DPNH diaphorase found in the bacteria. 3. DPNH dehydrogenase of E. coli is a metalloflavoprotein, containing non-heme iron, labile sulfide, FMN and FAD. 4. Reduction of the enzyme with DPNH in the absence of electron acceptor (ferricyanide or DCIP) causes a rapid and irreversible change to a less active state, Form II. Form II is characterized by a higher K m (DPNH) and slower v max ., while the K m (K 3Fe(CN) 6) remains unchanged. 5. The transformation of the enzyme to Form II is accompanied by the reduction of the non-heme iron component. The role of non-heme iron in the enzymic reaction is discussed.

Oxidation-reduction components of a reduced diphosphopyridine nucleotide dehydrogenase

DPNH dehydrogenase, solubilized from a particulate DPNH oxidase by treatment of the latter with ethanol at pH 4.8 and 43 °, has been further purified by precipitation with ammonium sulfate at pH 7.8 and by filtration through Sephadex G-25. The enzyme migrates as a single band when subjected to electrophoresis on starch gel or polyacrylamide. Based upon a molecular weight of 70,000 (determined by filtration through Sephadex G-200), each molecule of protein contains 1 FMN, 2 nonheme irons, 1 labile sulfide group, and 7 thiol groups. The latter are determined by titration of the protein with DTNB in the presence of 4 M urea. When the enzyme is reduced by DPNH under anaerobic conditions and in the absence of an external electron acceptor, the flavin appears to be fully reduced rather than at the semiquinone level; under these conditions, each mole of enzyme oxidizes approximately 3 moles of DPNH. Removal of the bound FMN by passage of the enzyme through Florisil or Bio-Gel does not affect the DPNH-ferricyanide activity while completely abolishing activity with cytochrome c or 2,6-dichlorophenolindophenol; cytochrome c activity can be largely restored by the addition of FMN. p-Chloromercuribenzoate (pCMB) inhibits the dehydrogenation of DPNH coupled to ferricyanide, indophenol, or cytochrome c; inhibition of the DPNH-ferricyanide activity is a biphasic function of pCMB concentration. Preincubation of the enzyme with DPNH increases the sensitivity toward pCMB when ferricyanide and cytochrome c are used as acceptors.

Studies of the electron transport systems of heart muscle: Biochemical and antigenic properties of a soluble DPNH dehydrogenase

Highly purified DPNH dehydrogenase has been prepared from preparations of DPNH oxidase. The enzyme has an average molecular weight of 80,000 and contains flavin, nonheme iron, and labile sulfide groups in an approximate ratio of 1:2:2, respectively. In electron paramagnetic resonance studies the dehydrogenase showed a g = 1.93 signal upon reduction with hydrosulfite, but not with DPNH, which suggests that the iron is not active in the enzymic catalysis. Studies of immunodiffusion and of enzyme inhibition with specific antibodies suggest that the DPNH dehydrogenase has not been grossly modified during preparation, and that it and the dehydrogenases prepared by the methods of King and Howard and Cremona and Kearney are related to each other and to preparations of DPNH oxidase.

On the diversity of -SH groups in DPNH dehydrogenase and their tentative localization

On the diversity of -SH groups in DPNH dehydrogenase and their tentative localization

An experimental evaluation of a histochemical diagnosis of burn depth

<h2>Summary</h2> Preliminary findings are reported on the effects of linear burns on rat skin, using neotetrazolium chloride and reduced diphosphopyridine nucleotide as histochemical criteria for the detection of DPNH dehydrogenase activity in burned tissues. The appearance of a red band of cells in the injured dermis, immediately following thermal injury, as detected by this technique, may be of particular relevance to the rapid early diagnosis of burn depth. Above this band the dermal and epithelial tissue has been irreparably damaged and is destined to be sloughed; below the band, the dermal tissue unless further insulted will undergo repair. If epidermal elements (pilosebaceous units) are present below the band, resurfacing is greatly facilitated.