Effects of ethanol and acetaldehyde on iron-sulfure centers in the mitochondrial respiratory chain.

Incubation of submitochondrial particles with relatively low concentrations of ethanol (20–100 mm) or acetaldehyde (1–10 mm) produces alterations in the electron paramagnetic resonance spectra of the iron-sulfur centers in the NADH dehydrogenase segments of the respiratory chain. The iron-sulfur centers in the NADH dehydrogenase region are most sensitive to both ethanol and acetaldehyde, in comparison to the iron-sulfur centers in succinate dehydrogenase and the cytochrome b-c region. Centers N-3, 4, N-5, 6 and N-1b are affected after relatively short incubation periods (3–30 min) while center N-2 shows considerable sensitivity over somewhat longer incubations (20–90 min). The most ethanol-sensitive center in the succinate dehydrogenase region of the respiratory chain is high potential iron-sulfur protein-type center S-3. Potentiometric analysis shows that these alterations are not due to simple changes in the redox state caused by addition of dissolved oxygen. Changes in the electron paramagnetic resonance spectra can be correlated with decreased rates of oxidation of NADH and, to a lesser extent, succinate in both ethanol- and acetaldehyde-treated submitochondrial particles.     

Studies on the electron transfer pathway, topography of iron-sulfur centers, and site of coupling in NADH-Q oxidoreductase

Electron transfer activities and steady state reduction levels of Fe-S centers of NADH-Q oxidoreductase were measured in mitochondria, submitochondrial particles (ETPH), and complex I after treatment with various reagents. p-Chloromercuribenzenesulfonate destroyed the signal from center N-4 (gx = 1.88) in ETPH but not in mitochondria, showing that N-4 is accessible only from the matrix side of the inner membrane. N-Bromosuccinimide also destroyed the signal from N-4 but without inhibiting rotenone-sensitive electron transfer to quinone, suggesting a branched pathway for electron transfer. Diethylpyrocarbonate caused oxidation of N-3 and N-4 in the steady state without changing N-1, suggesting N-1 is before N-3 and N-4. Difluorodinitrobenzene and dicyclohexylcarbodiimide inhibited oxidation of all Fe-S centers and tetranitromethane inhibited reduction of all Fe-S centers. Titrations of the rate of superoxide (O2-) generation in rotenone-treated submitochondrial particles were similar with the ratio [NADH]/[NAD] and that of 3-acetyl pyridine adenine nucleotide in spite of different midpoint potentials of the two couples. On reaction with inhibitors the inhibition of O2- formation was similar to that of ferricyanide reductase rather than quinone reductase. The rate of O2- formation during ATP-driven reverse electron transfer was 16% of the rate observed with NADH. The presence of NAD increased the rate to 83%. The results suggest that bound, reduced nucleotide, probably E-NAD., is the main source of O2- in NADH dehydrogenase. The effect of ATP on the reduction levels of Fe-S centers in well-coupled ETPH was measured by equilibrating with either NADH/NAD or succinate/fumarate redox couples. With NADH/NAD none of the Fe-S centers showed ATP induced changes, but with succinate/fumarate all centers showed ATP-driven reduction with or without NAD present. The effect on N-2 was smaller than that on N-1, N-3, and N-4. These observations indicate that the major coupling interaction is between N-2 and the low potential centers, N-1, N-3, and N-4. Possible schemes of coupling in this segment are discussed.     

Studies on electron paramagnetic resonance spectra manifested by a respiratory chain hydrogen carrier.

The high-potential iron-sulfur protein (HiPIP) center of succinate dehydrogenase has an electron paramagnetic resonance (epr) signal in the oxidized form, centered at g = 2.01, and under certain conditions this epr signal is accompanied by absorbances at g = 2.04, g = 1.99, and g = 1.96. These absorbances have been attributed to a spin-spin interaction of paramagnetic species, the semiquinone form of ubiquinone being involved (Ruzicka et al., Proc. Nat. Acad. Sci. USA72, 2886). In the present work this magnetic interaction is studied further; it is concluded that of the three possible species (HiPIP, Flavin H and UQ?H (ubiquinone)) which may interact with UQ?H; a second UQ? most likely partner for the interaction. Nonetheless, the HiPIP center of succinate dehydrogenase also plays a role in the interaction by acting as a “magnetic relaxer” of one or both of the interacting UQ?Hs. The physiological reaction of that part of the ubiquinone pool associated with the succinate dehydrogenase (on the matrix side of the inner mitochondrial membrane) is UQH₂ ? UQ?H + H⁺ + e^?. This is in line with recent postulates of the mechanism of ubiquinone mediation in electron transfer.     

Characterization of iron-sulfur clusters in rat liver submitochondrial particles by electron paramagnetic resonance spectroscopy. Alterations produced by chronic ethanol consumption

Iron-sulfur clusters present in rat liver submitochondrial particles were characterized by ESR at temperatures between 30 and 5.5 K combined with potentiometric titrations. The spectral and thermodynamic characteristics of the iron-sulfur clusters were generally similar to those previously reported for pigeon or bovine heart submitochondrial particles. Clusters N-1a, N-1b, N-2, N-3 and N-4 of NADH dehydrogenase had midpoint oxidation-reduction potentials at pH 7.5 of ?425, ?265, ?85, ?240 and ?260 mV, respectively. Clusters S-1 and S-3 of succinate dehydrogenase had midpoint potentials of 0 and +65 mV, respectively. The iron-sulfur cluster of electron-transferring flavoprotein-ubiquinone oxidoreductase exhibited the g_z signal at g = 2.08 and had a midpoint potential of +30 mV. This signal was relatively prominent in rat liver compared to pigeon or bovine heart.Submitochondrial particles from rats chronically treated with ethanol (36% of total calories, 40 days) showed decreases of 20–30% in amplitudes of signals due to clusters N-2, N-3 and N-4 compared to those from pair-fed control rats. Signals from clusters N-1b, S-1, S-3 and electron-transferring flavoprotein-ubiquinone oxidoreductase were unaffected. Microwave power-saturation behavior was similar for both submitochondrial particle preparations, suggesting that the lower signal amplitudes reflected a lower content of these particular clusters. NADH dehydrogenase activity was significantly decreased (46%), whilst succinate dehydrogenase activity was elevated (25%), following chronic ethanol consumption. The results indicate that chronic ethanol treatment leads to an alteration of the structure and function of the NADH dehydrogenase segment of the electron transfer chain. This alteration is one of the factors contributing to the lower respiration rates observed following chronic ethanol administration.     

Glucagon treatment of rats activates the respiratory chain of liver mitochondria at more than one site

The rate of reduction of ferricyanide in the presence and absence of antimycin and ubiquinone-1 was measured using liver mitochondria from control and glucagon treated rats. Glucagon treatment was shown to increase electron flow from both NADH and succinate to ubiquinone, and from ubiquinone to cytochrome c. 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) was shown to inhibit the oxidation of glutamate + malate to a much greater extent than that of succinate or duroquinol. Spectral and kinetic studies confirmed that electron flow between NADH and ubiquinone was the primary site of action but that the interaction of the ubiquinone pool with complex 3 was also affected. The effects of various respiratory chain inhibitors on the rate of uncoupled oxidation of succinate and glutamate + malate by control and glucagon treated mitochondria were studied. The stimulation of respiration seen in the mitochondria from glucagon treated rats was maintained or increased as respiration was progressively inhibited with DCMU, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), 2-heptyl-4-hydroxyquinoline-n-oxide (HQNO) and colletotrichin, but greatly reduced when inhibition was produced with malonate or antimycin. These data were also shown to support the conclusion that glucagon treatment may cause some stimulation of electron flow through NADH dehydrogenase, succinate dehydrogenase and through the bc1 complex, probably at the point of interaction of the complexes with the ubiquinone pool. The effects of glucagon treatment on duroquinol oxidation and the inhibitor titrations could not be mimicked by increasing the matrix volume, nor totally reversed by aging of mitochondria. These are both processes that have been suggested as the means by which glucagon exerts its effects on the respiratory chain (Armston, A.E., Halestrap, A.P. and Scott, R.D., 1982, Biochim. Biophys. Acta 681, 429-439). It is concluded that an additional mechanism for regulating electron flow must exist and a change in lipid peroxidation of the inner mitochondrial membrane is suggested.     

Inhibition of electron transfer from ferrocytochrome b to ubiquinone, cytochrome c1 and duroquinone by antimycin.

The effect of antimycin on (i) the respiratory activity of the KCN-insensitive pathway of mitochondria of Neurospora grown on chloramphenicol (chloramphenicol-grown) with durohydroquinone and succinate or NADH as substrate, (ii) the electron transfer from the b-type cytochromes to ubiquinone with durohydroquinone as electron donor as well as (iii) the electron transfer from the b-type cytochromes to duroquinone with succinate as electron donor in chloramphenicol-grown Neurospora and beef heart submitochondrial particles was studied. All experiments were performed in the uncoupled state. 1. The respiratory chain of chloramphenicol-grown Neurospora mitochondria branches at ubiquinone into two pathways. Besides the cytochrome oxidase-dependent pathway, a KCN-insensitive branch equiped with a salicylhydroxamate-sensitive oxidase exists. Durohydroquinone, succinate or NADH are oxidized via both pathways. The durohydroquinone oxidation via the KCN-insensitive pathway is inhibited by antimycin, wheras the succinate or NADH oxidation is not. The titer for ful inhibition is one mol antimycin per mol cytochrome b-563 or cytochrome b-557. 2. The electron transfer from durohydroquinone to ubiquinone, which takes place in the KCN-inhibited state, does not occur in the antimycin-inhibited state. 3. The reduction of duroquinone by succinate in the presence of KCN is inhibited by antimycin. The titer for full inhibition is one mol antimycin per mol cytochrome b-566 or cytochrome b-562 for beef heart (or cytochrome b-563 or cytochrome b-557 for Neurospora). 4. When electron transfer from the b-type cytochromes to cytochrome C1, ubiquinone and duroquinone is inhibited by antimycin, the hemes of cytochrome b-566 and cytochrome b-562 (or cytochrome b-563 and cytochrome b-557) are in the reduced state. 5. The experimental results suggest that the two b-type cytochromes form a binary complex the electron transferring activity of which is inhibited by antimycin, the titer for full inhibition being one mol of antimycin per mol of complex. The electron transfer from the b-type cytochromes to ubiquinone is inhibited in a non-linear fashion.     

EPR characterization of the iron-sulfur clusters in the NADH: ubiquinone oxidoreductase segment of the respiratory chain in Paracoccus denitrificans

The physicochemical properties of the iron-sulfur clusters present in the NADH:ubiquinone oxidoreductase of Paracoccus denitrificans have been examined in the cytoplasmic membrane particles by redox potentiometry and EPR spectroscopy. Analogous to the iron-sulfur clusters present in the mitochondrial NADH: ubiquinone oxidoreductase, we have found two binuclear and three tetranuclear EPR detectable iron-sulfur clusters, namely, N-1a, N-1b, N-2, N-3, and N-4. In the bacterial system, the two binuclear clusters differ in line shape and in Em values; the cluster with more rhombic symmetry (gx,y,z = 1.918, 1.937, 2.029) has the Em7.0 value of -150 while the almost axial one (gx,y,z = 1.929, 1.941, 2.019) has Em7.0 of -270 mV. The Em of the former cluster is pH dependent (-60 mV/pH) as in the case of mammalian N-1a while the latter is pH independent as is the mammalian cluster N-1b. The pH-dependent P. denitrificans [2Fe-2S] cluster, which we have labeled N-1a, has an Em7.0 as high as that of N-2, in contrast to the mammalian N-1a. Thus N-1a is reducible with a physiological reductant, NADH in this bacterial system. The Em of the cluster N-2 is also pH dependent (Em7.0 = -130 mV) with a pK value near 7.7. The Em values of all other clusters exhibit no pH dependence as in the case of their mammalian counterparts. We have found that the cluster N-1a is the most labile component among the five iron-sulfur clusters and may give rise to variable relative spin concentrations and extremely low Em values due to the facile modifications of the microenvironment of the cluster. The P. denitrificans NADH:ubiquinone oxidoreductase provides a unique and useful site I model system where redox composition is similar to the mitochondrial enzyme but with fewer numbers of polypeptides (Yagi, T. (1986) Arch. Biochem. Biophys. 250, 302-311).     

Farnesylacetone, a sesquiterpenic hormone of Crustacea, inhibits electron transport in isolated rat liver mitochondria

Farnesylacetone (C18 H30 0) is a male hormone extracted from the androgenic gland of crab, Carcinus maenas. Appropriate enzymatic assays, as well as spectrophotometric studies, indicate that micromolar concentrations of farnesylacetone interact with the electron transport pathway of rat liver mitochondria. By the use of artificial electron donors and electron acceptors, it is shown that farnesylacetone immediately inhibits the electron transfer within complex I (NADH ubiquinone reductase activity) and complex II (succinate ubiquinone reductase activity). It is proposed that farneylacetone could interact with these two complexes of the respiratory chain at the level of the iron-sulfur centers implicated in the dehydrogenase activities. These observations are compared with the results obtained with terpenic molecules which interact with mitochondrial respiration.     

Fourier transform infrared spectroscopic study on the conformational reorganization in Escherichia coli complex I due to redox-driven proton translocation

The proton-pumping NADH:ubiquinone oxidoreductase (complex I) couples the transfer of electrons from NADH to ubiquinone with the translocation of protons across the membrane. Electron transfer is accomplished by flavin mononucleotide (FMN) and a series of iron-sulfur (Fe/S) clusters. A novel mechanism has been proposed wherein the electron transfer reaction induces conformational changes that subsequently lead to the translocation of protons. Redox-induced Fourier transform infrared difference spectra have been obtained, showing strong conformational changes in the amide I region. The amplitude of the signal is pH dependent, as expected for an energy coupling step in the enzymes reaction. Furthermore, pH-dependent protonation events and quinone binding were detected.     

Catalytic importance of acidic amino acids on subunit NuoB of the Escherichia coli NADH:ubiquinone oxidoreductase (complex I)

The NADH:ubiquinone oxidoreductase (complex I) from Escherichia coli is composed of 13 subunits called NuoA through NuoN and contains one FMN and 9 iron-sulfur clusters as redox groups. Electron transfer from NADH to ubiquinone is coupled with the translocation of protons across the membrane by a yet unknown mechanism. Redox-induced Fourier transform infrared difference spectroscopy showed that the oxidation of iron-sulfur cluster N2 located on NuoB is accompanied by the protonation of acidic amino acid(s). Here, we describe the effect of mutating the conserved acidic amino acids on NuoB. The complex was assembled in all mutants but the electron transfer activity was completely abolished in the mutants E67Q, D77N, and D94N. The complex isolated from these mutants contained N2 although in diminished amounts. The protonation of acidic amino acid(s) coupled with the oxidation of N2 was not detectable in the complex from the mutant E67Q. However, the conservative mutations E67D and D77E did not disturb the enzymatic activity, and the signals because of the protonation of acidic amino acid(s) were detectable in the E67D mutant. We discuss the possible participation of Glu(67) in a proton pathway coupled with the redox reaction of N2.     

Function of conserved acidic residues in the PSST homologue of complex I (NADH:ubiquinone oxidoreductase) from Yarrowia lipolytica

Proton-translocating NADH:ubiquinone oxidoreductase (complex I) is the largest and least understood enzyme of the respiratory chain. Complex I from bovine mitochondria consists of more than forty different polypeptides. Subunit PSST has been suggested to carry iron-sulfur center N-2 and has more recently been shown to be involved in inhibitor binding. Due to its pH-dependent midpoint potential, N-2 has been proposed to play a central role both in ubiquinone reduction and proton pumping. To obtain more insight into the functional role of PSST, we have analyzed site-directed mutants of conserved acidic residues in the PSST homologous subunit of the obligate aerobic yeast Yarrowia lipolytica. Mutations D136N and E140Q provided functional evidence that conserved acidic residues in PSST play a central role in the proton translocating mechanism of complex I and also in the interaction with the substrate ubiquinone. When Glu(89), the residue that has been suggested to be the fourth ligand of iron-sulfur center N-2 was changed to glutamine, alanine, or cysteine, the EPR spectrum revealed an unchanged amount of this redox center but was shifted and broadened in the g(z) region. This indicates that Glu(89) is not a ligand of N-2. The results are discussedin the light of structural similarities to the homologous [NiFe] hydrogenases.     

Two aspartic acid residues in the PSST-homologous NUKM subunit of complex I from Yarrowia lipolytica are essential for catalytic activity

Mitochondrial proton-translocating NADH:ubiquinone oxidoreductase (complex I) couples the transfer of two electrons from NADH to ubiquinone to the translocation of four protons across the mitochondrial inner membrane. Subunit PSST is the most likely carrier of iron-sulfur cluster N2, which has been proposed to play a crucial role in ubiquinone reduction and proton pumping. To explore the function of this subunit we have generated site-directed mutants of all eight highly conserved acidic residues in the Yarrowia lipolytica homologue, the NUKM protein. Mutants D99N and D115N had only 5 and 8% of the wild type catalytic activity, respectively. In both cases complex I was stably assembled but electron paramagnetic resonance spectra of the purified enzyme showed a reduced N2 signal (about 50%). In terms of complex I catalytic activity, almost identical results were obtained when the aspartates were individually changed to glutamates or to glycines. Mutations of other conserved acidic residues had less dramatic effects on catalytic activity and did not prevent assembly of iron-sulfur cluster N2. This excludes all conserved acidic residues in the PSST subunit as fourth ligands of this redox center. The results are discussed in the light of the structural similarities to the homologous small subunit of water-soluble [NiFe] hydrogenases.     

Ubiquinone reduction pattern in pigeon heart mitochondria. Identification of three distinct ubiquinone pools.

Intact pigeon heart mitochondria showed 10-30% ubiquinone reduction in the absence of substrates. This reduction could not be ascribed to endogenous substrates, as judged by lack of effect of inhibitors and uncouplers and by the very low endogenous respiratory rate. Addition of NADH in the presence of antimycin caused further reduction of about 10% ubiquinone, apparently coupled to the rotenone- and antimycin-sensitive exo-NADH oxidase system [Rasmussen (1969) FEBS Lett. 2, 157-162]. Citric acid cycle substrates reduced most of the remaining ubiquinone in the presence of antimycin; 15-20% of the total ubiquinone content was still in the oxidized form under the most reducing conditions. Three pools of ubiquinone therefore appeared to be present in heart mitochondria: a metabolically inactive pool consisting of reduced as well as oxidized ubiquinone, a pool coupled to oxidation of added (cytoplasmic) NADH, and the well-known pool coupled to citric acid cycle oxidations. Ferricyanide selectively oxidized the ubiquinol reduced by added NADH, indicating that this pool is situated on the outer surface of the mitochondrial inner membrane. Ubiquinone reduction levels were determined with a new method, which is described in detail.     

Characterization by electron paramagnetic resonance and studies on subunit location and assembly of the iron-sulfur clusters of Bacillus subtilis succinate dehydrogenase

Succinate dehydrogenase is a conserved membrane-bound enzyme consisting of two nonidentical subunits: a flavo iron-sulfur protein (Fp) subunit, containing a covalently bound flavin, and an iron-sulfur protein (Ip) subunit. Bacillus subtilis succinate dehydrogenase in wild type bacteria and 12 well characterized succinate dehydrogenase-defective mutants were examined by low temperature EPR spectroscopy to characterize the enzyme and study subunit location and biosynthesis of its iron-sulfur clusters. The wild type B. subtilis enzyme contains iron-sulfur clusters which are analogous to clusters S-1 and S-3 of bovine heart succinate dehydrogenase but with slightly different EPR characteristics. Spins from cluster S-2 were not detectable as in the case of the intact form of bovine heart succinate dehydrogenase. However, dithionite reduction of the B. subtilis enzyme greatly enhanced spin relaxation of the ferredoxin-type cluster S-1, indicating the presence of the cluster S-2. Iron-sulfur cluster S-1 was found to be assembled in soluble succinate dehydrogenase subunits in the cytoplasm, but only if full-length Fp polypeptides and relatively large fragments of Ip polypeptides were present. Cluster S-1 was not detected in mutants with soluble mutated Fp polypeptides or in a mutant totally lacking Ip subunit polypeptide. Iron-sulfur clusters S-1, S-2, and S-3 were assembled also when the covalently bound flavin in the Fp subunit was absent. Clusters S-1 and S-3 in the membrane-bound flavin-deficient succinate dehydrogenase were not reduced by succinate but could be reduced by electron transfer from NADH dehydrogenase via the menaquinone pool.     

Functional significance of conserved histidines and arginines in the 49-kDa subunit of mitochondrial complex I

We have studied the ubiquinone-reducing catalytic core of NADH:ubiquinone oxidoreductase (complex I) from Yarrowia lipolytica by a series of point mutations replacing conserved histidines and arginines in the 49-kDa subunit. Our results show that histidine 226 and arginine 141 probably do not ligate iron-sulfur cluster N2 but that exchanging these residues specifically influences the properties of this redox center. Histidines 91 and 95 were found to be essential for ubiquinone reductase activity of complex I. Mutations at the C-terminal arginine 466 affected ubiquinone affinity and inhibitor sensitivity but also destabilized complex I. These results provide further support for a high degree of structural conservation between the 49-kDa subunit of complex I and its ancestor, the large subunit of water-soluble [NiFe] hydrogenases. In several mutations of histidine 226, arginine 141, and arginine 466 the characteristic EPR signatures of iron-sulfur cluster N2 became undetectable, but specific, inhibitor-sensitive ubiquinone reductase activity was only moderately reduced. As we could not find spectroscopic indications for a modified cluster N2, we concluded that these complex I mutants were lacking most of this redox center but were still capable of catalyzing inhibitor-resistant ubiquinone reduction at near normal rates. We discuss that this at first surprising scenario may be explained by electron transfer theory; after removal of a single redox center in a chain, electron transfer rates are predicted to be still much faster than steady-state turnover of complex I. Our results question some of the central mechanistic functions that have been put forward for iron-sulfur cluster N2.     

Determination of the exchange integral in binuclear iron-sulfur clusters in proteins of varying complexity.

The coupling constants J between the iron atoms in ferredoxin type iron-sulfur proteins containing binuclear clusters were evaluated by two parallel methods. The temperature dependence of the EPR linewidths and integrated abosrption intensities are both related to the energy of the first excited state. The values of J obtained were: center S-1 in succinate dehydrogenase, 90 cm-1; Rieske's iron-sulfur center, 65 cm-1; adrenodoxin, 270 cm-1. The behavior of iron-sulfur center N-1a in NADH:UQ reductase was also examined; its similarity to that of center S-1 indicates that center N-1a is also a binuclear iron-sulfur center, with J = 90 cm-1. Greater rhombic distortion present in the EPR spectrum of a binuclear cluster was associated with smaller values of J.     

The role of iron-sulfur center 2 in electron transport and energy conservation in the NADH-ubiquinone segment of the respiratory chain in Paracoccus denitrificans.

1. The electron paramagnetic resonance spectra at 15 K of reduced membrane particles of Paracoccus denitrificans exhibit resonance signals with g values, line shapes and temperature profile which are similar to the signals of the iron-sulfur centers observed in the NADH-ubiquinone segment of mitochondrial respiratory chains. These iron-sulfur centers are reducible with NADH, NADPH as well as chemically with dithionite. 2. Sulphate-limited growth of Paracoccus denitrificans results in the loss of an electron paramagnetic resonance signal (gz approximately 2.05, gy approximately gx approximately 1.92) which has properties similar to those of iron-sulfur center 2 of the NADH dehydrogenase of mitochondrial origin. The loss of this signal is accompanied by a decrease in the NADH oxidase and NADH ferricyanide oxidoreductase activities to respectively 30 and 40% of the values found for succinate-limited growth conditions. In addition respiration in membrane particles from sulphate-limited cells loses its sensitivity to rotenone. 3. Since sulphate-limited growth of Paracoccus denitrificans induces loss of site I phosphorylation [Arch. Microbiol. (1977) 112, 25-34] these observations suggest a close correlation between site I phosphorylation, rotenone-sensitivity and the presence of an electron paramagnetic resonance signal with gz approximately 2.05 and gy approximately gx approximately 1.92.     

Alternative Oxidase Activity and the Ubiquinone Redox Level in Soybean Cotyledon and Arum Spadix Mitochondria during NADH and Succinate Oxidation

In Arum and soybean (Glycine max L.) mitochondria, the dependence of the alternative oxidase activity on the redox level of ubiquinone, with NADH and succinate as substrates, was studied, using a voltametric procedure to measure the ubiquinone redox poise in the mitochondrial membrane. The results showed that when the enzyme was activated by pyruvate the relationship between the alternative oxidase rate and the redox state of the ubiquinone pool was the same for both NADH and succinate oxidations. In the absence of pyruvate the alternative oxidase had an apparent lower affinity for ubiquinol. This was more marked with NADH than with succinate and was possibly due to pyruvate production during succinate oxidation or to an activation of the alternative oxidase by succinate itself. In Arum spadix (unlike soybean cotyledon) mitochondria, succinate oxidation via the alternative oxidase maintained the ubiquinone pool in a partially reduced state (60%), whereas NADH oxidation kept it almost completely reduced. Previous data comparing mitochondria from thermogenic and nonthermogenic tissues have not examined the full range of ubiquinone redox levels in both tissues, leading to the suggestion that the activity of alternative oxidase for Arum was different from nonthermogenic tissues. When the complete range of redox states of ubiquinone is used and the oxidase is fully activated, the alternative oxidase from thermogenic tissue (Arum) behaves similarly to that of nonthermogenic tissue (soybean).     

Unidirectional effect of lauryl sulfate on the reversible NADH:ubiquinone oxidoreductase (Complex I)

Lauryl sulfate inhibits the Deltamu;(H)(+)-dependent reverse electron transfer reactions catalyzed by NADH:ubiquinone oxidoreductase (Complex I) in coupled bovine heart submitochondrial particles and in vesicles derived from Paracoccus denitrificans. The inhibitor affects neither NADH oxidase (coupled or uncoupled) nor NADH:ferricyanide reductase and succinate oxidase activities at the concentrations that selectively prevent the succinate-supported, rotenone-sensitive NAD(+) or ferricyanide reduction. Possible uncoupling effects of the inhibitor are ruled out: in contrast to oligomycin and gramicidin, which increases and decreases the rate of the reverse electron transfer, respectively, in parallel with their coupling and uncoupling effects, lauryl sulfate does not affect the respiratory control ratio. A mechanistic model for the unidirectional effect of lauryl sulfate on the Complex I catalyzed oxidoreduction is proposed.     

Purification of a reconstitutively active iron-sulfur protein (oxidation factor) from succinate . cytochrome c reductase complex of bovine heart mitochondria.

Oxidation factor, a protein required for electron transfer from succinate to cytochrome c in the mitochondrial respiratory chain, has been purified from isolated succinate . cytochrome c reductase complex. Purification of the protein has been followed by a reconstitution assay in which restoration of ubiquinol . cytochrome c reductase activity is proportional to the amount of oxidation factor added back to depleted reductase complex. The purified protein is a homogeneous polypeptide on acrylamide gel electrophoresis in sodium dodecyl sulfate and migrates with an apparent Mr = 24,500. Purified oxidation factor restores succinate . cytochrome c reductase and ubiquinol . cytochrome c reductase activities to depleted reductase complex. It is not required for succinate dehydrogenase nor for succinate . ubiquinone reductase activities of the reconstituted reductase complex. Oxidation factor co-electrophoreses with the iron-sulfur protein polypeptide of ubiquinol . cytochrome c reductase complex. The purified protein contains 56 nmol of nonheme iron and 36 nmol of acid-labile sulfide/mg of protein and possesses an EPR spectrum with the characteristic "g = 1.90" signal identical to that of the iron-sulfur protein of the cytochrome b . c1 complex. In addition, the optimal conditions for extraction of oxidation factor, including reduction with hydrosulfite and treatment of the b . c1 complex with antimycin, are identical to those which facilitate extraction of the iron-sulfur protein from the b . c1 complex. These results indicate that oxidation factor is a reconstitutively active form of the iron-sulfur protein of the cytochrome b . c1 complex first discovered by Rieske and co-workers (Rieske, J.S., Maclennan, D.H., and Coleman, R. (1964) Biochem. Biophys. Res. Commun. 15, 338-344) and thus demonstrate that this iron-sulfur protein is required for electron transfer from ubiquinol to cytochrome c in the mitochondrial respiratory chain.