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PubMed1999

Hymenolepis diminuta: catalysis of transmembrane proton translocation by mitochondrial NADPH-->NAD transhydrogenase.

The mitochondrial, inner-membrane-associated, reversible NADPH-->NAD transhydrogenase of adult Hymenolepis diminuta physiologically couples matrix-localized, NADP-specific "malic" enzyme with NADH-dependent anaerobic electron transport. Employing submitochondrial particles (SMP) as the source of enzyme activity and both spectrophotometric and fluorometric assessments, the present study made evident that in its catalysis of transhydrogenation between NADPH and NAD, the cestode enzyme engages in the concomitant transmembrane translocation of protons. As assessed spectrophotometrically, the catalysis of NADPH-dependent NAD reduction by H. diminuta SMP was stimulated significantly by carbonyl cyanide 3-chlorophenylhydrazone (CCCP), carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), as well as by the protonophoric anthelmintic, niclosamide. In addition, N,N'-dicyclohexylcarbodiimide (DCCD) markedly diminished SMP-catalyzed hydride ion transfer between NADPH and NAD. The catalysis by SMP of concomitant, transhydrogenase-mediated proton translocation was evaluated more directly via fluorometric assays using 8-anilino-1-napthalenesulfonic acid (ANS) as the probe. These latter evaluations revealed a transhydrogenase-dependent enhancement of ANS fluorescence in accord with an intravesicular accumulation of protons. ANS fluorescence was quenched rapidly when the assay system was supplemented with CCCP, FCCP, or niclosamide. Consistent with the helminth transhydrogenase acting as a proton pump, transhydrogenase-mediated enhanced fluorescence also was inhibited by DCCD. Considered collectively, these data indicated, apparently for the first time for any invertebrate system, that the transhydrogenase, in catalyzing the NADPH-->NAD reaction, acts in the translocation of protons from the matrix to the intermembrane space mitochondrial compartment.

Anilino Naphthalenesulfonates↗
PubMed1987

Studies on the mechanism of oxidative phosphorylation. Flow-force relationships in mitochondrial energy-linked reactions.

The relationship between the steady-state level of membrane potential (delta psi) and the rates of energy production and consumption has been studied in mitochondria and submitochondrial particles. The energy-linked reactions investigated were oxidative phosphorylation (with NADH, succinate, and beta-hydroxybutyrate as respiratory substrates) and nucleoside triphosphate-driven transhydrogenation from NADH to NADP and uphill electron transfer from succinate to NAD. Results have shown the following. 1) Attenuation of the rates of the energy-producing reactions results in a parallel change in the rates of the energy-consuming reactions with little or no change in the magnitude of steady-state delta psi. 2) At low rates of energy production and consumption, steady-state delta psi decreases. However, this is due largely to the energy leak of the system which lowers static-head delta psi when the rate of energy production is slow. 3) When the rate of energy production and static-head delta psi are held constant, and the rate of energy consumption is diminished by partial inhibition or the use of suboptimal conditions (e.g. subsaturating substrate concentrations), then even a small decrease in the rate of energy consumption results in an upward adjustment of the level of steady-state delta psi. The lower the rate of energy input, the greater the upward adjustment of steady-state delta psi upon suppression of the rate of energy consumption. 4) The above results have been discussed with regard to the role of bulk-phase delta mu H+ or delta psi in the mitochondrial energy transfer reactions.

Animals↗
PubMed1993

Alkoxyl and methyl radical formation during cleavage of tert-butyl hydroperoxide by a mitochondrial membrane-bound, redox active copper pool: an EPR study.

The cleavage of tert-butyl hydroperoxide by submitochondrial particles yielded two distinctive radicals, alkoxyl and methyl radicals, detected by the electron paramagnetic resonance technique in conjunction with the spin trap 5,5'-dimethyl-1-pyrrolyne-N-oxide. Free radical formation was partly sensitive to bathocuproine disulfonate and was augmented upon supplementation of the mitochondrial membranes with copper, thus suggesting that a redox active copper pool in mitochondrial membranes participated actively in the cleavage of the O-O bond of tert-butyl hydroperoxide. This view was experimentally substantiated by the following: first, observation of the maximal EPR signal intensity required the presence of exogenous electron donors, such as succinate or reduced nicotinamide adenine dinucleotide (NADH). Second, copper reduction was accomplished partly by a superoxide radical-dependent mechanism as indicated by the sensitivity of the electron paramagnetic resonance (EPR) signal to superoxide dismutase. Third, the enhancing effect of the respiratory chain inhibitors, antimycin A and rotenone, on free radical yield assessed in the presence of superoxide dismutase pointed to the occurrence of two potential loci in the respiratory chain involved in direct electron transfer to membrane-bound copper: one located between NADH and the rotenone-sensitive site and another, quantitatively less important, between the rotenone- and antimycin-sensitive sites. These results support the notion that a redox pool of copper tightly bound to the mitochondrial membrane contributes significantly to the reductive cleavage of organic peroxides associated with free radical production.

Animals↗
PubMed1998

Topological and functional relationship of subunits F1-gamma and F0I-PVP(b) in the mitochondrial H+-ATP synthase.

Diamide treatment of the F0F1-ATP synthase in "inside out" submitochondrial particles (ESMP) in the absence of a respiratory Delta mu H+ as well as of isolated Fo reconstituted with F1 or F1-gamma subunit results in direct disulfide cross-linking between cysteine 197 in the carboxy-terminal region of the F0I-PVP(b) subunit and cysteine 91 at the carboxyl end of a small alpha-helix of subunit F1-gamma, both located in the stalk. The F0I-PVP(b) and F1-gamma cross-linking cause dramatic enhancement of oligomycin-sensitive decay of Delta mu H+. In ESMP and MgATP particles the cross-linking is accompanied by decoupling of respiratory ATP synthesis. These effects are consistent with the view that F0I-PVP(b) and F1-gamma are components of the stator and rotor of the proposed rotary motor, respectively. The fact that the carboxy-terminal region of F0I-PVP(b) and the short alpha-helix of F1-gamma can form a direct disulfide bridge shows that these two protein domains are, at least in the resting state of the enzyme, in direct contact. In isolated F0, diamide also induces cross-linking of OSCP with another subunit of F0, but this has no significant effect on proton conduction. When ESMP are treated with diamide in the presence of Delta mu H+ generated by respiration, neither cross-linking between F0I-PVP(b) and F1-gamma subunits nor the associated effects on proton conduction and ATP synthesis is observed. Cross-linking is restored in respiring ESMP by Delta mu H+ collapsing agents as well as by DCCD or oligomycin. These observations indicate that the torque generated by Delta mu H+ decay through Fo induces a relative motion and/or a separation of the F0I-PVP(b) subunit and F1-gamma which places the single cysteine residues, present in each of the two subunits, at a distance at which they cannot be engaged in disulfide bridging.

Adenosine Triphosphate↗
PubMed1989

An atypical heme-binding structure of cytochrome c1 of Euglena gracilis mitochondrial complex III.

Complex III was purified from submitochondrial particles prepared from Euglena gracilis. The purified complex consisted of 10 subunits and lost antimycin sensitivity. The Euglena complex III showed an atypical difference absorption spectrum for cytochrome c1 with its alpha-band maximum at 561 nm. The pyridine ferrohemochrome prepared from covalently bound heme in the Euglena complex III had an alpha-peak at 553 nm. This wavelength is the same as that of pyridine ferrohemochrome prepared from Euglena mitochondrial cytochrome c (c-558), the heme of which is linked to only a single cysteine residue through a thioether bond. Cytochrome c1 which was a heme-stained subunit with a molecular mass of 32.5 kDa was isolated from the purified complex III and its N-terminal sequence of 46 amino acids was determined. On the basis of apparent homologies to cytochromes c1 from other sources, this sequence included the heme-binding region. However, the amino acid at position 36, corresponding to the first cysteine involved in heme linkage in other cytochromes c1, was phenylalanine. Position 39, corresponding to the second cysteine, was not identified despite the treatment for removal of the heme and carboxymethylation of the expected cysteine. The unidentified amino acid is assumed to be a derivative of cysteine to which the heme is linked through a single thioether bond. The histidine-40 corresponding to the probable fifth ligand for heme iron was conserved in Euglena cytochrome c1.

Amino Acid Sequence↗
PubMed1979

[Free fatty acids as inhibitors of the respiratory chain in testicular homogenates].

The supernatants of the 440 000 . g . min centrifugation of homogenates of rat, bull and boar testicles and sperm inhibit the NADH-oxidase activity of non-phosphorylating submitochondrial particles (ETP). Whereas no inhibitory activity was observed with young rats (150 g), a marked inhibition was detected with heavier animals. The inhibitory activity of testicles was located in the microsomal fraction. The reaction of the testicular inhibitor with the ETP is initiated by an instant reversible binding followed by a slow irreversible inhibition of the electron transport. The reason of the time-dependence is neither an interaction between the enzymes of the ETP and those of the microsomal electron transport nor a slow degradation of the ETP by microsomal phospholipases. Some observations indicate an indirect involvement of phospholipase via the formation of free fatty acids (FFA). The inhibitory fraction could be solubilized from the microsomes both by sodium cholate treatment and by ethanol extraction. After separation of the lipid classes by chromatography on silica gel and gas-chromatographic analysis the inhibitory fraction was identified as a mixture of free fatty acids (FFA) of different chain lengths and degree of saturation. Thus a new effect of FFA on the mitochondrial electron transport has been detected which is different from other actions known up till now. The degradation of the phospholipids of the endoplasmic reticulum in the spermatozoa may be the source of the enhanced formation of FFA. An inhibition of the cell respiration presumably does not occur in vivo. The high FFA level in the testicular homogenates of sexually mature animals is a consequence of an intensive FFA metabolism, especially of high phospholipase activity.

Animals↗
PubMed1997

Modification of membrane-bound F1 by p-fluorosulfonylbenzoyl-5'-adenosine: sites of binding and effect on activity.

Bovine heart submitochondrial particles (smp) were incubated with p-fluorosulfonylbenzoyl-5'-adenosine (FSBA) in order to study the binding of this ligand and its effect on ATP synthesis and ATP hydrolysis in smp and to compare the results with those obtained with isolated F1. The binding was measured with the 14C-labeled compound. ATP hydrolysis was in all cases as much inhibited as succinate-driven ATP synthesis and ITP hydrolysis was more inhibited than ATP hydrolysis. The binding experiments show that modification of three nucleotide binding sites results in nearly complete inhibition of ATPase activity. In the presence of pyrophosphate up to 6 mol [14C]SBA/mol F1 can be bound. FSBA preferentially modifies amino acids of the alpha-subunits but also beta-subunits are modified. It is concluded that modification of both subunits results in inhibition of activity. The results are very well comparable with the results obtained with isolated F1, which indicates that our preparation of F1 is a good model for F1 in the intact system. Furthermore it is concluded that each alpha-subunit of F1 in smp, just like in the isolated form, contains two pockets where adenosine moieties can bind, one located above the P-loop, modifying alpha-Tyr-244 and alpha-Tyr-300 and the other one located below the P-loop where also the adenosine moiety of AD(T)P binds, modifying beta-Tyr-368.

Adenosine↗
PubMed1989

Uncoupling of oxidative phosphorylation: different effects of lipophilic weak acids and electrogenic ionophores on the kinetics of ATP synthesis.

Previous studies from this laboratory have shown that the kinetics of ATP synthesis by bovine heart submitochondrial particles (SMP) are modulated by the coupled rate of respiration between two extremes of Vmax and apparent Km's for ADP and Pi [Matsuno-Yagi, A., & Hatefi, Y. (1986) J. Biol. Chem. 261, 14031-14038; Hekman, C., Matsuno-Yagi, A., & Hatefi, Y. (1988) Biochemistry 27, 7559-7565]. Thus, with ADP as the variable substrate, ATP synthesis occurred with Vmax = 200 nmol of ATP min-1 (mg of protein)-1 at 30 degrees C and an apparent KmADP = 2-4 microM at low rates of respiration, and with Vmax = 11,000 nmol of ATP min-1 (mg of protein)-1 at 30 degrees C and an apparent KmADP = 120-160 microM at high rates of respiration. At intermediate respiration rates, it was necessary to introduce a third intermediate KmADP for best fit of the kinetic data, indicating that transition from one kinetic extreme to the other is not abrupt and involves intermediate kinetic states of the ATP synthase complexes. The present paper shows that uncouplers affect the kinetics of ATP synthesis by SMP in two ways. When used at moderate concentrations, electrogenic ionophores such as gramicidin D or valinomycin plus nigericin decreased the Vmax for ATP synthesis without changing the contributions of the low, intermediate, and high KmADP to the overall rate of ATP synthesis. By contrast, potent lipophilic weak acid uncouplers, such as FCCP, CCCP, S-13, and SF6847, decreased Vmax and converted the kinetics of ATP synthesis toward high KmADP.(ABSTRACT TRUNCATED AT 250 WORDS)

Acids↗
PubMed2003

The ND5 subunit was labeled by a photoaffinity analogue of fenpyroximate in bovine mitochondrial complex I.

Fenpyroximate is a potent inhibitor of the mitochondrial proton-translocating NADH-quinone oxidoreductase (complex I). We synthesized its photoaffinity analogue [(3)H](trifluoromethyl)phenyldiazirinylfenpyroximate ([(3)H]TDF). When bovine heart submitochondrial particles (SMP) were illuminated with UV light in the presence of [(3)H]TDF, radioactivity was mostly incorporated into a 50 kDa band. There was a good correlation between radioactivity labeling of the 50 kDa band and inhibition of the NADH oxidase activity, indicating that a 50 kDa protein is responsible for the inactivation of complex I. Blue native gel electrophoresis of the [(3)H]TDF-labeled SMP revealed that the majority of radioactivity was found in complex I. Analysis of the complex I band on an SDS gel showed a major peak of radioactivity at approximately 50 kDa. There are three subunits in complex I that migrate in this region: FP51K, IP49K, and ND5. Further analysis using the 2D gel electrophoresis implied that the labeled protein was the ND5 subunit. Labeling of the ND5 subunit was stimulated by NADH/NADPH but was prevented by various complex I inhibitors. Amiloride derivatives that are known to be inhibitors of Na(+)/H(+) antiporters also diminished the labeling. In agreement with the protective effect, we observed that the amiloride derivatives inhibited NADH-ubiquinone-1 reductase activity but not NADH-K(3)Fe(CN)(6) reductase activity in bovine SMP. These results suggest that the ND5 subunit is involved in construction of the inhibitor- and quinone-binding site(s). Furthermore, it seems likely that the ND5 subunit may participate in H(+)(Na(+)) translocation in coupling site 1.

Amino Acid Sequence↗
PubMed1991

Mitochondrial energy-linked nicotinamide nucleotide transhydrogenase. Membrane topography of the bovine enzyme.

The mitochondrial energy-linked nicotinamide nucleotide transhydrogenase is a homodimer of monomer Mr = 109,228. Hydropathy analysis of its cDNA-deduced amino acid sequence (1043 residues) has indicated that the molecule is composed of 3 domains: a 430-residue-long hydrophilic N-terminal domain which binds NAD(H), a 200-residue-long hydrophilic C-terminal domain which binds NADP(H), and a 400-residue-long hydrophobic central domain which appears to be made up mainly of about 14 hydrophobic clusters of approximately 20 residues each. In this study, antibodies were raised to the hydrophilic N- and C-terminal domains cleaved from the isolated transhydrogenase by proteolytic digestion, and to a synthetic, hydrophilic pentadecapeptide, which corresponded to position 540-554 within the central hydrophobic domain. Immunochemical experiments with mitoplasts (mitochondria denuded of outer membrane) and submitochondrial particles (inside-out inner membrane vesicles) as sources of antigens showed that essentially the entire N- and C-terminal hydrophilic domains of the transhydrogenase, as well as epitopes from the central pentadecapeptide, protrude from the inner membrane into the mitochondrial matrix, where the N- and C-terminal domains would be expected to come together to form the enzyme's catalytic site. Treatment of mitoplasts with several proteolytic enzymes indicated that large protease-sensitive masses of the transhydrogenase are not exposed on the cytosolic side of the inner membrane, which agreed with the exception that the central highly hydrophobic domain of the molecule should be largely membrane-intercalated. Trypsin, alpha-chymotrypsin, and papain had little or no effect on the mitoplast-embedded transhydrogenase. Proteinase K, subtilisin (Nagarse), thermolysin, and pronase E each split the mitoplast-embedded enzyme into two fragments only, a fragment of approximately 70 kDa containing the N-terminal hydrophilic domain, and one of approximately 40 kDa bearing the C-terminal hydrophilic domain. The cleavage site of proteinase K was determined to be A690 -A691, which is located in a small hydrophilic segment within the central hydrophobic domain. This protease-sensitive loop appears to be exposed on the cytosolic side of the inner membrane. The proteinase K-nicked enzyme containing two peptides of 71 and 39 kDa was isolated from mitoplasts and shown to have high transhydrogenase activity.

Amino Acid Sequence↗
PubMed1994

The dynamics of proton transfer at the C side of the mitochondrial membrane: picosecond and microsecond measurements.

The excited-state proton emitter, pyranine (8-hydroxypyrene-1,3,6-trisulfonate), was introduced into the inner aqueous space of inside-out submitochondrial particles (SMP). Upon initiation of respiration, the dye recorded acidification of this space. Incorporation of high concentrations of the dye (approximately 100 nmol/mg of protein) had no effect on the respiratory functions of the vesicles, nor on their capacity to execute delta microH(+)-coupled reverse electron transfer. The respiratory control ratio (RCR) remained as high as RCR > 4. Pulse irradiation of the dye caused photodissociation of the proton from the 8-hydroxy position. The release of the proton and its reaction with the matrix of the inner space of SMP were monitored at two time intervals: nanosecond fluorimetry measured the dissociation of the proton from the excited dye molecule (phi OH*), while microsecond spectroscopy followed the reaction between the proton and the ground-state anion (phi O-). Numerical integration of the differential rate equations describes the diffusion of protons in the perturbed system. The nanosecond measurements yield the physical characteristics of the aqueous phase that dissolves the dye. The apparent dielectric constant of that space is rather low (epsilon = 20). The diffusion coefficient of the proton is 2.3 x 10(-5) cm2/s, and the activity of water is aH2O = 0.87. All of these values imply that a large fraction of the intervesicular aqueous phase is taken up by the hydration layer of the lipids and proteins of the C side of the membrane. The microsecond dynamics measurements indicate that the rates of proton binding to the membrane surface components reach an equilibrium within 60 microseconds.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Production of nitric oxide and other iron-containing metabolites during the reductive metabolism of nitroprusside by microsomes and by thiols.

Sodium nitroprusside is used as a hypotensive agent because of its ability to produce nitric oxide (NO), although direct demonstration of this has not been reported in a biological system. Nitroprusside (NP) nitroxide radical anion, the first reduction product of NP generated in the presence of microsomes and NADPH, was found to undergo further metabolism. One of the products produced during this reductive metabolism was shown to be NO. By using N-methylglucamine dithiocarbamate-FeCl2 complex [(NMGD)2Fe(II)] as the NO trap, we have detected and characterized the mononitroso bis(N-methylglucamine dithiocarbamato) iron (II) complex (MNBI) (g = 2.040, and A(14N) = 13.3 G) as the product of NO trapping. The production of NO during the reductive metabolism of NP by submitochondrial particles and a human HepG2 hepatoblastoma cell line was also demonstrated using (NMGD)2Fe(II). In addition to MNBI, two other mononitrosyl iron complexes, the NP nitroxide radical anion and a second species designated as Fe(NO)(X)(Y) (g = 2.032, and A(14N) = 14.3 G), and additional unidentified paramagnetic products containing iron were also detected. Thiol compounds such as glutathione, cysteine, and cysteamine reduce NP to generate NP nitroxide radical anion and a paramagnetic species characterized as a dithiolated dinitroso iron complex (DDIC), Fe(NO)2(RS)2 g = 2.030, A(14N) = 2.2 G(2N), and A(1H) = 1.1 G(4H). At 77 K, DDIC generated from cysteine and NP has an axial symmetry, with g perpendicular = 2.040, and g parallel = 2.014. Two additional paramagnetic products, designated as species C (g = 2.020, linewidth = 4.8 G) and species D (g = 2.008, linewidth = 6.1 G), were also formed during NP reduction by thiol compounds. The characterization of these complexes has been hampered by the lack of hyperfine features in the ESR spectra. The production of NO during cysteamine reduction of NP was demonstrated by using (NMGD)2-Fe(II) as the spin-trap. These results directly demonstrate the production of NO during the reduction of NP by microsomes plus NADPH or by thiols. (NMGD)2-Fe(II) is a particularly useful spin-trap for the detection of NO in a strong reducing environment.

Animals↗
PubMed1990

Effects of chronic alcohol consumption on the steady-state kinetics properties of cytochrome oxidase in rat liver.

The effect of chronic alcohol consumption on steady-state kinetic characteristics of cytochrome oxidase in rat liver was studied using submitochondrial particles prepared from ethanol-fed and control rats. Preparations from both control and alcoholic rats had equivalent apparent Km values for cytochrome c of 13 microM in the presence of phenazine methosulfate or 19 microM with N,N,N',N'-tetramethylphenylene diamine as oxidation-reduction mediators at physiological ionic strength. Both preparations showed comparable stimulation (approx. 3-fold) of oxidase activity following detergent solubilization of the membrane and similar temperature dependence for oxidase activity. Under all conditions, preparations from alcohol-fed rats displayed 30 to 50% lower rats of cytochrome oxidase activity per unit membrane protein than those from control rats. The diminution in specific activity per mg protein was accompanied by a similar decline in heme aa3 content, as has been noted in previous studies. When expressed on a turnover number basis, the molecular activity of cytochrome oxidase (natoms O/min per nmol heme a) was equivalent in both alcoholic and control preparations. The results indicate that the intrinsic kinetic characteristics of cytochrome oxidase are not changed by alcohol consumption. The data suggest that the characteristic decline in heme aa3 content and cytochrome oxidase specific activity seen in ethanol-fed rats does not arise from alterations in the accessibility of the oxidase towards cytochrome c, or from changes in bulk phase lipid composition or physical properties. The results support the conclusion that ethanol consumption decreases the membrane content of functionally active oxidase molecules, but does not change the catalytic properties of these oxidase molecules.

Alcoholism↗
PubMed1984

Thiols in oxidative phosphorylation: inhibition and energy-potentiated uncoupling by monothiol and dithiol modifiers.

Three apparently different modifications of submitochondrial particles (SMP) or ATP synthase preparations (complex V) inhibit oxidative phosphorylation and ATP-32Pi exchange activities, all of which are reversible by addition of mono- or dithiols. (a) Triphenyltin chloride inhibits ATP synthesis and hydrolysis without uncoupling. The inhibition by triphenyltin chloride is reversible by addition of beta-mercaptoethanol, dithiothreitol, or dihydrolipoamide. (b) Factor B is a water-soluble protein of Mr (11-12) X 10(3), contains a vicinal dithiol, and is required for energy transfer to and from F1-ATPase when tested with SMP-rendered factor B deficient by extraction with ammonia-ethylenediaminetetraacetic acid (EDTA) (AE-SMP). Treatment of factor B with mono- and dithiol modifiers, such as p-(chloromercuri)benzenesulfonate (PCMPS), Cd2+, or diazenedicarboxylic acid bis(dimethylamide) (diamide), inhibits factor B. This inhibition is reversed by addition to modified factor B of appropriate mono- and dithiol compounds. Preparations of AE-SMP are partially F1 deficient and partially uncoupled. The uncoupling can be repaired completely by addition of factor B or low levels of oligomycin, or to a large extent by addition of F1-ATPase + oligomycin sensitivity conferring protein. (c) SMP, AE-SMP, and complex V can be completely uncoupled by treatment at 30 degrees C with phenylarsine oxide, Cd2+, diamide, PCMPS, monobromobimane, and mono- and bifunctional maleimides. The uncoupling by these reagents is potentiated by membrane energization. Uncoupling by diamide is greater than or equal to 80% reversed by dihydrolipoamide or beta-mercaptoethanol, the former being much more potent. Dithiothreitol and dithioerythritol are poorly effective.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Fo membrane domain of ATP synthase from bovine heart mitochondria: purification, subunit composition, and reconstitution with F1-ATPase.

The Fo membrane domain of the F1Fo-ATP synthase complex has been purified from bovine heart mitochondria. The purification procedure involves the removal of peripheral membrane proteins, including F1-ATPase, from submitochondrial particles with guanidine hydrochloride, followed by extraction of Fo and other membrane proteins from the stripped membranes in the presence of the detergent n-dodecyl beta-D-maltoside. Fo was then purified by ion-exchange and dye ligand chromatography in the presence of the same detergent. Approximately 15 mg of pure Fo was recovered from 1.8 g of mitochondrial membrane protein. The purified Fo is a complex of nine different polypeptides. They are subunits a, b, c, d, e, F6, and A6L characterized before in F1Fo-ATPase preparations, and two new hitherto undetected subunits, named f and g. The sequences of subunits f and g have been determined. They are not related significantly to any known protein, but subunit f appears to contain a membrane-spanning alpha-helix. Proteins f and g are also present in approximately stoichiometric amounts in a highly purified preparation of intact F1Fo-ATPase, and so it is concluded that they are authentic subunits of the bovine enzyme with unknown functions. Dibutyltin 3-hydroxyflavone, an inhibitor of F1Fo-ATPase, also binds to the purified Fo in detergent and competes for binding with venturicidin. In the presence of F1 and OSCP, the purified Fo was reassembled into the intact F1Fo-ATPase complex. Therefore, this procedure provides a relatively abundant source of pure and functional Fo that is suitable for structural analysis.

Adenosine Triphosphatases↗
PubMed1981

Modification of the spectral properties of cytochrome b in mutants of Saccharomyces cerevisiae resistant to 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Mapping at two distinct genetic loci of the split mitochondrial gene of cytochrome b.

The effects of five inhibitors of the cytochrome bc1 complex: 3-(3,4-dichlorophenyl)-1,1-dimethylurea (diuron), 2-n-heptyl-4-hydroxyquinoline-N-oxide (HpHOQnO), antimycin A, funiculosin and mucidin were measured in submitochondrial particles of strains of the yeast Saccharomyces cerevisiae belonging to two classes of diuron-resistant mutants Diu 1 and Diu 2 which are modified in different exons of the split mitochondrial gene of cytochrome b. 1. The oxidation of NADH and of cytochrome b-561 exhibits a similar resistance to diuron and HpHOQnO in Diu 1 and Diu 2 mutants. 2. No extra reduction of cytochrome b-561 and cytochrome b-565 is observed in the presence of diuron and HpHOQnO. 3. Both Diu 1 and Diu 2 mutants exhibit the red shift of cytochrome b-561 induced by concentrations of HpHOQno 2 -- 3-times higher than those required in the parental strains. 4. The spectral and respiratory effects of antimycin A, funiculosin and mucidin and generally similar in the diuron-resistant mutants and in their parental strains. However a cross-resistance between diuron and antimycin A is indicated in one Diu 2 mutant. 5. From the combined genetic and biochemical data it is concluded that the interaction of diuron and HpHOQnO with cytochrome b is mediated by at least two specific amino acids located apart in the central region of the apocytochrome b peptide coded by mitochondrial DNA. These two amino acids control tightly the extra reduction of cytochromes b-565 and b-561 as well as the flow of electrons through the bc1 complex. However the binding of HpHOQnO required for the expression of the red shift of cytochrome b-561 is only slightly affected by the diu-1 and diu-2 mutations.

Antimycin A↗
PubMed1990

Oxygenation of biological membranes by the pure reticulocyte lipoxygenase.

We find that the reticulocyte lipoxygenase can oxygenate rat liver mitochondrial membranes, beef heart submitochondrial particles, rat liver endoplasmic membranes, and erythrocyte plasma membranes (inside-out and right side-out ghosts) without prior action of a phospholipase. After alkaline hydrolysis of the ester lipids, the main products were identified as 15S-hydro(pero)xy-5Z,8Z,11Z,13E-eicosatetr aenoic acid, 17S-hydro(pero)xy-4Z,7Z,10Z,13Z,15E, 19Z,-docosahexaenoic acid, 13S-hydro(pero)xy-9Z,11E-octadecadienoic acid, 9(S/R)-hydro(pero)xy-10E,12Z-octadecadienoic acid as well as the two all-E hydro(pero)xy octadecadienoic acid isomers. At low membrane concentrations (1 mg of protein/ml), the enzyme maintains a high stereospecificity for the S-configuration, but at higher concentrations (20 mg/ml), the products were virtually racemic. Addition of the antioxidant 2,6-ditert-butyl-p-cresol counteracted this tendency to lose stereospecificity. During these enzyme-catalyzed reactions, substantially more oxygen is consumed than can be accounted for as the hydro(pero)xy products. This discrepancy is due to secondary reactions which lead to the decomposition of the primary oxygenation products, the hydroperoxy lipids, and to oxidative modifications of membrane proteins. These data indicate that the reticulocyte lipoxygenase can oxygenate polyenoic fatty acids in various types of biological membrane and that the oxidative modifications are not restricted to the membrane lipids. The results are discussed in terms of the proposed role of the enzyme in the breakdown of mitochondria and other intracellular organelles during the maturation of red blood cells.

Animals↗
PubMed1990

Inhibitor effects on redox-linked protonations of the b haems of the mitochondrial bc1 complex.

The effects of pH and inhibitors on the spectra and redox properties of the haems b of the bc1 complex of beef heart submitochondrial particles were investigated. The major findings were: (1) both haems have a weakly redox-linked protonatable group with pKox and pKred of around 6 and 8; (2) at pH values above 7, haem bH becomes heterogeneous in its redox behaviour. This heterogeneity is removed by the Qi site inhibitors antimycin A, funiculosin and HQNO, but not by the Qo site inhibitors myxothiazol or stigmatellin; (3) of all inhibitors tested only funiculosin had a large effect on the Em/pH profile of either haem b. In all cases where definite effects were found, the haem most affected was that thought to be closest to the site of inhibitor binding; (4) spectral shifts of haem groups caused by inhibitor binding were usually, but not always, of the haem group closest to the binding site; (5) titrations with succinate/fumarate were in reasonable agreement with redox-mediated data provided that strict anaerobiosis was maintained. Apparent large shifts of haem midpoint potentials with antimycin A and myxothiazol could be produced in aerobic succinate/fumarate titrations in the presence of cyanide, as already reported in the literature, but these were artefactual; (6) the heterogeneous haem bH titration behaviour can be simulated with a model similar to that proposed by Salerno et al. (J. Biol. Chem. (1989) 264, 15398-15403) in which there is redox interaction between haem bH and ubiquinone species bound at the Qi site. Simulations closely fit both the haem bH data and known semiquinone data only if it is assumed that semiquinone bound to oxidised haem bH is EPR-silent.

Animals↗