Respiratory Chain-linked NADH Dehydrogenase Research Articles

Respiratory chain-linked NADH dehydrogenase. Mechanisms of assembly.

The assembly of mitochondrially and cytoplasmically translated subunits of NADH dehydrogenase in the inner mitochondrial membrane was studied in rat hepatoma cultures. A polyclonal antibody to the purified bovine heart holoenzyme, which reacted with comigrating proteins of both rat liver and hepatoma mitochondria on immunoblots, precipitated 25-30 [35S]methionine-labeled proteins from hepatoma cell lysates. Six of these were sensitive to an inhibitor of mitochondrial translation (chloramphenicol), resistant to an inhibitor of cytosolic translation (cycloheximide), and were not present in cytochrome oxidase. By these criteria, six NADH dehydrogenase subunits are identified as being translated on mitochondrial ribosomes. The metabolic properties of the three most prominent of these at 51, 43, and 11 kDa were studied in more detail. Mitochondrial and nuclear-coded polypeptides assemble into NADH dehydrogenase at different rates as measured by incorporation of pulse-labeled proteins into immunoprecipitable enzyme. Nuclear-coded, imported polypeptides appear immediately after a pulse with [35S]methionine and retain constant stoichiometry. Mitochondrially coded proteins, although rapidly translated, appear at peak levels at different times between 0 and 12 h of chase in the immunoprecipitated enzyme. Ongoing synthesis and import of nuclear-coded proteins is necessary for mitochondrially coded proteins to be assembled. Excess, unassembled mitochondrially translated subunits are degraded in an oligomycin-sensitive manner. These data are consistent with a model in which a scaffold of imported proteins forms the inner core of the enzyme, and later arriving mitochondrially translated proteins attach to the scaffold in a time-dependent manner.

No unique mitochondrial translation products in respiratory chain-linked NADH dehydrogenase

Antibody prepared against beef heart mitochondrial NADH dehydrogenase immunoprecipitated 26 polypeptides from detergent solubilized beef heart mitochondria. All 26 polypeptides co-migrated with those present in the dehydrogenase antigen when resolved side by side on sodium dodecyl sulfateurea polyacrylamide gels. From mixed rat liver-[ 35S]methionine pulsed hepatoma mitochondria the antibody immunoprecipitated 24 stained liver polypeptides and 19 radio-labelled hepatoma polypeptides. The translation of three of the labelled polypeptides was resistent to inhibition by cycloheximide, indicating these are translated on mitochondrial ribosomes. These same polypeptides, however, wre previously identified as cytochrome c oxidase subunits; and, apparently, non-specifically co-precipitate with dehydrogenase associated polypeptides. We conclude that there are no mitochondrially translated polypeptides specifically associated with NADH dehydrogenase.

Structural identification of iron-sulfur clusters of the respiratory chain-linked NADH dehydrogenase.

The structural nature of the iron-sulfur clusters of NADH dehydrogenase from beef heart mitochondria has been studied by the cluster extrusion technique. Enzyme samples were unfolded anaerobically in 80% (v/v) hexamethylphosphoramide/aqueous buffer in the presence of o-xylyl-alpha,alpha'-dithiol as the displacing agent and the extruded clusters were then reacted with p-trifluoromethylbenzenethiol and analyzed by Fourier transform 19F NMR at 339 MHz. Whenever extrusion was nearly complete, both binuclear and tetranuclear clusters were found at a mole ratio of approximately 2:1. Thus, the dehydrogenase, with 16 g atoms of non-heme iron present/mol of FMN, contains most likely four [2Fe-2S] and two [4Fe-4S] clusters. Because the enzyme contains four or, at the most five, EPR-detectable iron-sulfur centers, it appears that one or more of the clusters are EPR-silent.

Purification of the reduced nicotinamide adenine dinucleotide dehydrogenase from membranes of Acholeplasma laidlawii

The ethanol-extracted respiratory chain-linked NADH dehydrogenase of Acholeplasma laidlawii has been purified 25–35-fold. This purification involved delipidation of the ethanol-extracted minute non-sedimentable membrane fragments by detergent treatment and gel filtration on Bio-Gel P-200. Sodium deoxycholate-sucrose density gradient centrifugation was followed by dialysis of the active NADH dehydrogenase fractions which caused flocculation of 60% of the membrane proteins while the NADH dehydrogenase remained suspended. Poylacrylamide gel electrophoresis of the purified NADH dehydrogenase gave one major and two minor bands after staining with Coomassie Blue. The purified enzyme gave straight line kinetics in Lineweaver-Burk plots and a K m = 0.510 mM and V = 0.236 μmol/min. Fatty acid supplementation of A. laidlawii membranes had negligible effect on the membrane-bound or ethanol-extracted dehydrogenase, but substantiated the values of the K m and V. Purification, however, altered the constants by 2–4-fold, suggesting that alteration of the microenvironment or fragmentation of the dehydrogenase was significant. The purified dehydrogenase was very susceptible to a rapid inhibition with heavy metal ions. Mg 2+ and Ca 2+ inhibited the enzyme but this inhibition was much slower (90 min) and less complete. Consideration of published purification procedures of NADH dehydrogenase strongly suggested that the purified A. laidlawii respiratory chain-linked NADH dehydrogenase was over 90% pure and certainly one of the most purified respiratory chainlinked bacterial NADH dehydrogenases.

A protective function of superoxide dismutase during respiratory chain activity

1. (1) Aerobic incubation of heart muscle submitochondrial particles in phosphate buffer after treatment with NADH causes a progressive and substantial inhibition of the NADH oxidation system. Succinate oxidation remains almost unaffected by NADH treatment. 2. (2) The loss of NADH oxidase activity is due to an inhibition of the respiratory chain-linked NADH dehydrogenase. This inhibition of the enzyme is very similar to that caused by combination of the organic mercurial mersalyl with NADH dehydrogenase. 3. (3) The inhibition of NADH oxidation is largely prevented by compounds that are known to react with superoxide ions (O 2 −., including superoxide dismutase, cytochrome c, tiron and Mn 2+. EDTA also has a protective effect, but a number of other metal chelating agents, and several proteins, including catalase, are without effect. 4. (4) It is concluded that the inhibition of NADH oxidation is due to the action of superoxide ions formed during respiratory chain activity. 5. (5) The inhibition of NADH oxidation by superoxide ions or by mersalyl is reversible and is therefore not due to the loss of oxidoreduction components from the respiratory chain or to an irreversible change in protein conformation. 6. (6) The function of mitochondrial superoxide dismutase is discussed in relation to the key role of NADH dehydrogenase in energy-conserving reactions and the formation of hydrogen peroxide during mitochondrial oxidations.

On the Reaction of Piericidin A with the Reduced Nicotinamide Adenine Dinucleotide Dehydrogenase of Candida utilis

Abstract The characteristics of the inhibition of the respiratory chain-linked NADH dehydrogenase of Candida utilis by piericidin A and the binding of this inhibitor have been studied in inner membrane preparations with the aid of [14C]piericidin A. As in beef heart preparations, the binding of piericidin by the particles is linear and extends considerably beyond the range of maximal inhibition of NADH oxidation, suggesting the existence of unspecific binding sites. Washing of the particles with bovine serum albumin, following inhibition with piericidin, or titrating with piericidin in the presence of bovine serum albumin, however, does not distinguish the specific binding sites involved in the inhibition from unspecific ones, as in the case of beef heart preparations, because bovine serum albumin dissociates the inhibitor from all sites and causes virtually complete release of the inhibition. The apparently lower affinity for piericidin binding of sites concerned with inhibition of NADH oxidation prevented accurate determination of their number per mole of NADH dehydrogenase. However, from the amount of piericidin required for maximal inhibition of NADH oxidase and from Hill plots, it is clear that more than one binding site is involved in complete inhibition and available evidence suggests that there may be two sites, as in animal tissues. Spectrophotometric studies of the oxidation-reduction cycle of NADH dehydrogenase in C. utilis gave results comparable to those reported for beef heart preparations. These showed, in accord with the effect of piericidin on the reaction of the enzyme with various electron acceptors, that this inhibitor blocks electron flux in Candida between iron-sulfur Center 2 of the enzyme (Site 1 of energy conservation) and the coenzyme Q pool, as in animal tissues.

Oxidation of Glycerol 3-Phosphate by the Perfused Rat Liver

Abstract Detritiation of [2-3H]glycerol-3-P by isolated liver mitochondria can be used to assay the activity of mitochondrial glycerol-3-P dehydrogenase. The method differs from measurements of dihydroxyacetone-3-P formation only by the isotope effect on the reaction rate. Under assay conditions liver mitochondria of hyperthyroid rats detritiated [2-3H]glycerol-3-P 16 times faster than those of normal controls. However, the value of K0.5s (the concentration of glycerol-3-P giving 0.5 Vmax) for the oxidation of glycerol-3-P by mitochondria of hyperthyroid rats was 5 times higher than for the reaction catalyzed by mitochondria of normal animals. From the data given by studies with isolated mitochondria, the expected rates of detritiation of [2-3H[glycerol-3-P by the intact liver could be estimated. Experiments then carried out on perfused livers of hypothyroid, normal, and hyperthyroid rats revealed that the rates of the reaction occurring in the intact organ exceeded by far the assayed capabilities of the mitochondrial glycerol-3-P dehydrogenase system. These results indicated that the rate of detritiation of [2-3H]-glycerol-3-P by the liver is not determined by the activity of the mitochondrial enzyme. Although livers of hyperthyroid rats were found to detritiate [2-3H]glycerol-3-P twice as rapidly as those of normal controls, the activity of mitochondrial glycerol-3-P dehydrogenase was far too low to account for the increase. These findings led to a test of the role of the NADH dehydrogenase system in the reaction. Infusion of rotenone into the liver of a normal rat resulted in a marked decrease in the rate of detritiation of [2-3H]glycerol-3-P, the rotenone-insensitive pathway accounting for only 20% of the over-all rate. The results described in this communication do not support the concept of a glycerol-3-P cycle in liver regulated by thyroid hormones but indicate that the pathway of oxidation of extramitochondrial hydrogen leads through respiratory chain-linked NADH dehydrogenase.

Studies on the Respiratory Chain-linked Nicotinamide Adenine Dinucleotide Dehydrogenase: XXII. RHEIN, A COMPETITIVE INHIBITOR OF THE DEHYDROGENASE

Abstract Rhein (4,5-dihydroxyanthraquinone-2-carboxylic acid) inhibits the mitochondrial oxidation of reduced nicotamide adenine dinucleotide (NADH) but not of succinate. The inhibition is competitive with respect to substrate and involves a block between NADH and the flavin of NADH dehydrogenase. The Ki is about 2 µm at 30°. Evidence for this localization has come from kinetic analysis of the effect of rhein on the transhydrogenation and oxidation of NADH by purified NADH dehydrogenase. Rhein inhibits at similar concentrations the NADH-ferricyanide reaction and energy-linked NAD reduction in submitochondrial particles, but in these instances a secondary, noncompetitive inhibition is also apparent. Rhein has a pronounced tendency to combine with proteins, resulting in shifts of its absorption spectrum. These spectral changes may be used to monitor the combination of rhein with submitochondrial particles and the release of rhein from NADH dehydrogenase by NAD. The displacement of rhein by NAD suggests that this inhibitor may be used as a selective probe of the active site of NADH dehydrogenase. Combination of rhein with bovine serum albumin may be used to prevent or reverse the inhibition of NADH dehydrogenase. Combination with other proteins may mask the inhibition of respiratory chain-linked NADH dehydrogenase in tissues or mitochondria having a relatively low content of NADH dehydrogenase, e.g. liver. Rhein is not entirely specific for NADH dehydrogenase; at similar concentrations it also inhibits mitochondrial transhydrogenation and DT-diaphorase and at somewhat higher concentrations lactate and malate dehydrogenases from heart muscle but is without effect on a number of other pyridine nucleotide-linked enzymes, flavoproteins, and membrane-bound enzymes tested.

Quinone interaction with the respiratory chain-linked NADH dehydrogenase of beef heart mitochondria I. Juglone reductase activity

1. Substituted benzoquinones and napthoquinones can function as electron acceptors for the NADH dehydrogenase segment of the mitochondrial electron transport system. Anthraquinones and several hydroxylated quinones are not reduced by the enzyme complex. 2. Piericidin A treatment at concentrations known to inhibit electron transport causes varying degrees of inhibition of quinone reductase activities. The pattern of piericidin A inhibition suggests that certain quinones are reduced at sites either before or after the piericidin A inhibition site. Reduction of quinones such as 5-hydroxy-1,4-napthoquinone (juglone) and 1,2-napthoquinone is inhibited only slightly. Reduction of ubiquinone 1 and 2 and 2,3,5,6-tetramethyl-1,4-benzoquinone (duroquinone) is almost completely inhibited following piericidin A treatment. 3. Comparison of juglone reductase activity with ferricyanide reductase activity suggests that these acceptors are reduced at nonequivalent sites in the NADH dehydrogenase. Juglone reductase activity is stimulated following mercurial treatment while ferricyanide reductase activity is slightly inhibited. Preincubation of NADH dehydrogenase with NADH followed by mercurial treatment causes almost complete inhibition of ferricyanide reductase activity. Activation of juglone reductase activity still occurs under these conditions. The activation is specific for preparations of NADH dehydrogenase, for mercurials, and for napthoquinone reductase activity.

Four quinone reduction sites in the NADH dehydrogenase complex

Quinones can be used as electron acceptors and donors in a new approach to the study and resolution of the respiratory chain-linked NADH dehydrogenase system of beef heart mitochondria. Juglone (5-hydroxynapthoquinone), ubiquinone, duroquinone (tetramethylbenzoquinone), and ferricyanide are found to react at four separate sites in the complex. The sites can be defined by chemical and enzymatic inhibition or by fractionation of the system. The reduced form of one quinone, duroquinol, has the unique property of being an electron donor for the energy-linked reduction of NAD +.

An electron-transport system associated with the outer membrane of liver mitochondria. A biochemical and morphological study.

Preparations of rat-liver mitochondria catalyze the oxidation of exogenous NADH by added cytochrome c or ferricyanide by a reaction that is insensitive to the respiratory chain inhibitors, antimycin A, amytal, and rotenone, and is not coupled to phosphorylation. Experiments with tritiated NADH are described which demonstrate that this "external" pathway of NADH oxidation resembles stereochemically the NADH-cytochrome c reductase system of liver microsomes, and differs from the respiratory chain-linked NADH dehydrogenase. Enzyme distributation data are presented which substantiate the conclusion that microsomal contamination cannot account for the rotenone-insensitive NADH-cytochrome c reductase activity observed with the mitochondria. A procedure is developed, based on swelling and shrinking of the mitochondria followed by sonication and density gradient centrifugation, which permits the separation of two particulate subfractions, one containing the bulk of the respiratory chain components, and the other the bulk of the rotenone-insensitive NADH-cytochrome c reductase system. Morphological evidence supports the conclusion that the former subfraction consists of mitochondria devoid of outer membrane, and that the latter represents derivatives of the outer membrane. The data indicate that the electron-transport system associated with the mitochondrial outer membrane involves catalytic components similar to, or identical with, the microsomal NADH-cytochrome b(5) reductase and cytochrome b(5).

Stereochemistry of hydrogen-transfer in the energy-linked pyridine nucleotide transhydrogenase and related reactions

The stereochemistry of hydrogen-transfer in the energy-linked and non-energy-linked pyridine nucleotide transhydrogenase, the respiratory chain-linked NADH dehydrogenase, and the DT diaphorase-catalyzed reactions have been investigated with tritiated pyridine nucleotides. Both the energy-linked and the non-energy-linked pyridine nucleotide transhydrogenase reactions, catalyzed by stibmitochondrial particles from beef heart, involve the 4A hydrogen atom of NADH, and the 4B hydrogen atom of NADPH. The reactions involve no intrinsic exchange of hydrogen atoms between the reduced pyridine nucleotides and water. The aerobic oxidation of NADH, and the energy-linked reduction of NAD+ by succinate, catalyzed by the same particles, involve the 4B hydrogen atom of NADH, and are connected with a rapid exchange of hydrogen atoms between the latter and water. The reaction catalyzed by purified rat-liver DT diaphorase involves the 4A hydrogen atom of either NADH or NADPH, and involves no exchange of hydrogen atoms between the reduced pyridine nucleotides and water. Studies with 14C-labelled NADH indicate that the energy-linked pyridine nucleotide transhydrogenase reaction proceeds without an exchange of the nicotinamide moieties of the two pyridine nucleotides. These results are discussed in relation to the mechanism of the energy-linked pyridine nucleotide transhydrogenase reaction.