Assessing heterogeneity of peroxisomes: isolation of two subpopulations from rat liver.

Peroxisomes exhibit a heterogeneous morphological appearance in rat liver tissue. In this respect, the isolation and subsequent biochemical characterization of peroxisome species from different subcellular prefractions should help to solve the question of whether peroxisomes indeed diverge into functionally specialized subgroups in one tissue. As a means to address this question, we provide a detailed separation protocol for the isolation of peroxisomes from both the light (LM-Po) and the heavy (HM-Po) mitochondrial prefraction for their subsequent comparative analysis. Both isolation strategies rely on centrifugation in individually adapted Optiprep gradients. In case of the heavy mitochondrial fraction, free flow electrophoresis is appended as an additional separation step to yield peroxisomes of sufficient purity. In view of their morphology, peroxisomes isolated from both fractions are surrounded by a continuous single membrane and contain a gray-opaque inner matrix. However, beyond this overall similar appearance, HM-Po exhibit a smaller average diameter, float at lower density, and show a more negative average membrane charge when compared to LM-Po.

[Obtaining homogenous preparations of succinate dehydrogenase isoforms from the D-507 strain of Sphaerotilus natans].

Enzymatic preparations of two isoforms of succinate dehydrogenase (SDG) with specific activity of 22.00 E/mg of protein were obtained from the colorless sulfur bacterium Sphaerotilus natans D-507 cultured organotrophically. Both SDG forms were shown to be heteromers with subunit molecular masses of 70.8, 35.0, 31.8, and 16.2 kDa. The K(m) values for the first and the second forms of SDG were evaluated as 0.615 and 0.531 mM, respectively, with an optimal pH value of 7.2. It was found that the Cl- ion has an activating effect on the SDG activity that can be explained by the specific chemical modification of the enzyme molecule. The results suggest that the isolated enzyme forms are included in different multienzyme complexes, which provide the functioning of the tricarboxylic acid cycle, and SDG preparations can be used for the investigation of other enzyme systems or in vitro modeling of supramolecular cellular structures.

Periplasmic electron transfer via the c-type cytochromes MtrA and FccA of Shewanella oneidensis MR-1.

Dissimilatory microbial reduction of insoluble Fe(III) oxides is a geochemically and ecologically important process which involves the transfer of cellular, respiratory electrons from the cytoplasmic membrane to insoluble, extracellular, mineral-phase electron acceptors. In this paper evidence is provided for the function of the periplasmic fumarate reductase FccA and the decaheme c-type cytochrome MtrA in periplasmic electron transfer reactions in the gammaproteobacterium Shewanella oneidensis. Both proteins are abundant in the periplasm of ferric citrate-reducing S. oneidensis cells. In vitro fumarate reductase FccA and c-type cytochrome MtrA were reduced by the cytoplasmic membrane-bound protein CymA. Electron transfer between CymA and MtrA was 1.4-fold faster than the CymA-catalyzed reduction of FccA. Further experiments showing a bidirectional electron transfer between FccA and MtrA provided evidence for an electron transfer network in the periplasmic space of S. oneidensis. Hence, FccA could function in both the electron transport to fumarate and via MtrA to mineral-phase Fe(III). Growth experiments with a DeltafccA deletion mutant suggest a role of FccA as a transient electron storage protein.

Crystallization of mitochondrial rhodoquinol-fumarate reductase from the parasitic nematode Ascaris suum with the specific inhibitor flutolanil.

In adult Ascaris suum (roundworm) mitochondrial membrane-bound complex II acts as a rhodoquinol-fumarate reductase, which is the reverse reaction to that of mammalian complex II (succinate-ubiquinone reductase). The adult A. suum rhodoquinol-fumarate reductase was crystallized in the presence of octaethyleneglycol monododecyl ether and n-dodecyl-beta-D-maltopyranoside in a 3:2 weight ratio. The crystals belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 123.75, b = 129.08, c = 221.12 A, and diffracted to 2.8 A resolution using synchrotron radiation. The presence of two molecules in the asymmetric unit (120 kDa x 2) gives a crystal volume per protein mass (V(M)) of 3.6 A(3) Da(-1).

The bacterial flagellar switch complex is getting more complex.

The mechanism of function of the bacterial flagellar switch, which determines the direction of flagellar rotation and is essential for chemotaxis, has remained an enigma for many years. Here we show that the switch complex associates with the membrane-bound respiratory protein fumarate reductase (FRD). We provide evidence that FRD binds to preparations of isolated switch complexes, forms a 1:1 complex with the switch protein FliG, and that this interaction is required for both flagellar assembly and switching the direction of flagellar rotation. We further show that fumarate, known to be a clockwise/switch factor, affects the direction of flagellar rotation through FRD. These results not only uncover a new component important for switching and flagellar assembly, but they also reveal that FRD, an enzyme known to be primarily expressed and functional under anaerobic conditions in Escherichia coli, nonetheless, has important, unexpected functions under aerobic conditions.

Isolation and characterization of mammalian D-aspartyl endopeptidase.

The accumulation of D-isomers of aspartic acid (D-Asp) in proteins during aging has been implicated in the pathogenesis of Alzheimer's disease (AD), cataracts and arteriosclerosis. Here, we identified a specific lactacystin-sensitive endopeptidase that cleaves the D-Asp-containing protein and named it D-aspartyl endopeptidase (DAEP). DAEP has a multi-complex structure (MW: 600 kDa) and is localized in the inner mitochondrial membrane. However, DAEP activity was not detected in E. coli, S. cerevisiae, and C. elegans. A specific inhibitor for DAEP, i-DAEP: (benzoyl-L-Arg-L-His-[D-Asp]-CH(2)Cl; MW: 563.01), was newly synthesized and inhibited DAEP activity (IC(50), 3 microM), a factor of ten greater than lactacystin on DAEP. On the other hand, i-DAEP did not inhibit either the 20S or 26S proteasome. And we identified succinate dehydrogenase and glutamate dehydrogenase 1 as components of DAEP by affinity label using biotinylated i-DAEP. In the long life span of mammals, DAEP may serve as a scavenger against accumulation of racemized proteins in aging. Insights into DAEP will provide the foundation for developing treatments of diseases, such as AD, in which accumulation of D-Asp-containing proteins are implicated.

Experimental evidence for proton motive force-dependent catalysis by the diheme-containing succinate:menaquinone oxidoreductase from the Gram-positive bacterium Bacillus licheniformis.

In Gram-positive bacteria and other prokaryotes containing succinate:menaquinone reductases, it has previously been shown that the succinate oxidase and succinate:menaquinone reductase activities are lost when the transmembrane electrochemical proton potential, Deltap, is abolished by the rupture of the bacteria or by the addition of a protonophore. It has been proposed that the endergonic reduction of menaquinone by succinate is driven by the electrochemical proton potential. Opposite sides of the cytoplasmic membrane were envisaged to be separately involved in the binding of protons upon the reduction of menaquinone and their release upon succinate oxidation, with the two reactions linked by the transfer of two electrons through the enzyme. However, it has previously been argued that the observed Deltap dependence is not associated specifically with the succinate:menaquinone reductase. Definitive insight into the mechanism of catalysis of this reaction requires a corresponding functional characterization of an isolated, membrane-bound succinate:menaquinone reductase from a Gram-positive bacterium. Here, we describe the purification, reconstitution into proteoliposomes, and functional characterization of the diheme-containing succinate:menaquinone reductase from the Gram-positive bacterium Bacillus licheniformis and, with the help of the design, synthesis, and characterization of quinones with finely tuned oxidation/reduction potentials, provide unequivocal evidence for Deltap-dependent catalysis of succinate oxidation by quinone as well as for Deltap generation upon catalysis of fumarate reduction by quinol.

Proteomic analysis of succinate dehydrogenase and ubiquinol-cytochrome c reductase (Complex II and III) isolated by immunoprecipitation from bovine and mouse heart mitochondria.

The oxidative phosphorylation system (OXPHOS) consists of five multi-enzyme complexes, Complexes I-V, and is a key component of mitochondrial function relating to energy production, oxidative stress, cell signaling and apoptosis. Defects or a reduction in activity in various components that make up the OXPHOS enzymes can cause serious diseases, including neurodegenerative disease and various metabolic disorders. Our goal is to develop techniques that are capable of rapid and in-depth analysis of all five OXPHOS complexes. Here, we describe a mild, micro-scale immunoisolation and mass spectrometric/proteomic method for the characterization of Complex II (succinate dehydrogenase) and Complex III (ubiquinol-cytochrome c reductase) from bovine and rodent heart mitochondria. Extensive protein sequence coverage was obtained after immunocapture, 1D SDS PAGE separation and mass spectrometric analysis for a majority of the 4 and 11 subunits, respectively, that make up Complexes II and III. The identification of several posttranslational modifications, including the covalent FAD modification of flavoprotein subunit 1 from Complex II, was possible due to high mass spectrometric sequence coverage.

Multiprotein complex containing succinate dehydrogenase confers mitochondrial ATP-sensitive K+ channel activity.

The mitochondrial ATP-sensitive K(+) (mitoK(ATP)) channel plays a central role in protection of cardiac and neuronal cells against ischemia and apoptosis, but its molecular structure is unknown. Succinate dehydrogenase (SDH) is inhibited by mitoK(ATP) activators, fueling the contrary view that SDH, rather than mitoK(ATP), is the target of cardioprotective drugs. Here, we report that SDH forms part of mitoK(ATP) functionally and structurally. Four mitochondrial proteins [mitochondrial ATP-binding cassette protein 1 (mABC1), phosphate carrier, adenine nucleotide translocator, and ATP synthase] associate with SDH. A purified IM fraction containing these proteins was reconstituted into proteoliposomes and lipid bilayers and shown to confer mitoK(ATP) channel activity. This channel activity is sensitive not only to mitoK(ATP) activators and blockers but also to SDH inhibitors. These results reconcile the controversy over the basis of ischemic preconditioning by demonstrating that SDH is a component of mitoK(ATP) as part of a macromolecular supercomplex. The findings also provide a tangible clue as to the structural basis of mitoK(ATP) channels.

Superoxide destroys the [2Fe-2S]2+ cluster of FNR from Escherichia coli.

The oxygen sensing ability of the transcription factor FNR depends on the presence of a [4Fe-4S]2+ cluster. In the presence of O2, conversion of the [4Fe-4S]2+ cluster to a [2Fe-2S]2+ cluster inactivates FNR, but the fate of the [2Fe-2S]2+ cluster in cells grown under aerobic conditions is unknown. The present study shows that the predominant form of FNR in aerobic cells is apo-FNR (cluster-less FNR) indicating that the [2Fe-2S]2+ cluster, like the [4Fe-4S]2+ cluster, is not stable under these conditions. By quantifying the amount of [2Fe-2S]2+ cluster in 2Fe-FNR in vitro in the presence of various reductants and oxidants (GSH, DTT, cysteine, O2, hydrogen peroxide, and superoxide), we found that superoxide, a byproduct of aerobic metabolism, significantly destabilized the [2Fe-2S]2+ cluster. Mössbauer spectroscopy was used to monitor the effects of superoxide on 2Fe-FNR in vivo; under cellular conditions that favored superoxide production, we observed the disappearance of the signal representative of the [2Fe-2S]2+ cluster. We conclude that the [2Fe-2S]2+ cluster of FNR is labile to superoxide both in vitro and in vivo. This lability may explain the absence of the [2Fe-2S]2+ cluster form of FNR under aerobic growth conditions.

Complex II from phototrophic purple bacterium Rhodoferax fermentans displays rhodoquinol-fumarate reductase activity.

It has long been accepted that bacterial quinol-fumarate reductase (QFR) generally uses a low-redox-potential naphthoquinone, menaquinone (MK), as the electron donor, whereas mitochondrial QFR from facultative and anaerobic eukaryotes uses a low-redox-potential benzoquinone, rhodoquinone (RQ), as the substrate. In the present study, we purified novel complex II from the RQ-containing phototrophic purple bacterium, Rhodoferax fermentans that exhibited high rhodoquinol-fumarate reductase activity in addition to succinate-ubiquinone reductase activity. SDS/PAGE indicated that the purified R. fermentans complex II comprises four subunits of 64.0, 28.6, 18.7 and 17.5 kDa and contains 1.3 nmol heme per mg protein. Phylogenetic analysis and comparison of the deduced amino acid sequences of R. fermentans complex II with pro/eukaryotic complex II indicate that the structure and the evolutional origins of R. fermentans complex II are closer to bacterial SQR than to mitochondrial rhodoquinol-fumarate reductase. The results strongly indicate that R. fermentans complex II and mitochondrial QFR might have evolved independently, although they both utilize RQ for fumarate reduction.

Using rational screening and electron microscopy to optimize the crystallization of succinate:ubiquinone oxidoreductase from Escherichia coli.

The membrane-bound respiratory complex II, succinate:ubiquinone oxidoreductase (SQR) from Escherichia coli, has been anaerobically expressed, then purified and crystallized. The initial crystals obtained were small and diffracted poorly. In order to facilitate structure determination, rational screening and sample-quality analysis using electron microscopy was implemented. The crystals of SQR from E. coli belong to the trigonal space group R32, with unit-cell parameters a = b = 138.7, c = 521.9 A, and diffract to 2.6 A resolution. The optimization strategy used for obtaining well diffracting SQR crystals is applicable to a wide range of membrane proteins.

Characterization of the dihydroorotate dehydrogenase as a soluble fumarate reductase in Trypanosoma cruzi.

Trypanosoma cruzi, a protozoan causing Chagas' disease, excretes a considerable amount of succinate even though it uses the TCA cycle and the aerobic respiratory chain. For this reason, it was believed that unknown metabolic pathways participate in succinate production in this parasite. In the present study, we examined the molecular properties of dihydroorotate dehydrogenase (DHOD), the fourth enzyme of de novo pyrimidine biosynthetic pathway, as a soluble fumarate reductase (FRD) because our sequence analysis of pyr genes cluster showed that the amino acid sequence of T. cruzi DHOD is quite similar to that of type 1A DHOD of Saccharomyces cerevisiae, an enzyme that uses fumarate as an electron acceptor and produces succinate. Biochemical analyses of the cytosolic enzyme purified from the parasite and of the recombinant enzyme revealed that T. cruzi DHOD has methylviologen-fumarate reductase (MV-FRD) activity. In addition, T. cruzi DHOD was found to catalyze electron transfer from dihydroorotate to fumarate by a ping-pong Bi-Bi mechanism. The recombinant enzyme contained FMN as a prosthetic group. Dynamic light scattering analysis indicated that T. cruzi DHOD is a homodimer. These results clearly indicated that the cytosolic MV-FRD is attributable to T. cruzi DHOD. The DHOD may play an important role in succinate/fumarate metabolism as well as de novo pyrimidine biosynthesis in T. cruzi.

Purification, crystallisation and preliminary crystallographic studies of succinate:ubiquinone oxidoreductase from Escherichia coli.

A membrane protein complex, succinate dehydrogenase (SQR) from Escherichia coli has been purified and crystallised. This enzyme is composed of four subunits containing FAD, three iron-sulphur clusters and one haem b as prosthetic groups. The obtained crystals belong to the hexagonal space group P6(3) with the unit-cell dimensions of a=b=123.8 A and c=214.6 A. An asymmetric unit of the crystals contains one SQR monomer (M(r) 120 kDa). A data set is now available at 4.0 A resolution with 88.1% completeness and 0.106 R(merge). We have obtained a molecular replacement solution that shows sensible molecular packing, using the soluble domain of E. coli QFR (fumarate reductase) as a search model. The packing suggests that E. coli SQR is a crystallographic trimer rather than a dimer as observed for the E. coli QFR.

The succinate dehydrogenase from the thermohalophilic bacterium Rhodothermus marinus: redox-Bohr effect on heme bL.

The succinate dehydrogenase from the thermohalophilic bacterium Rhodothermus marinus is a member of the succinate:menaquinone oxidoreductases family. It is constituted by three subunits with apparent molecular masses of 70, 32, and 18 kDa. The optimum temperature for succinate dehydrogenase activity is 80 degrees C, higher than the optimum growth temperature of R. marinus, 65 degrees C. The enzyme shows a high affinity for both succinate (Km = 0.165 mM) and fumarate (Km = 0.10 mM). It contains the canonical iron-sulfur centers S1, S2, and S3, as well as two B-type hemes. In contrast to other succinate dehydrogenases, the S3 center has an unusually high reduction potential of +130 mV and is present in two different conformations, one of which presents an unusual EPR signal with g values at 2.035, 2.009, and 2.001. The apparent midpoint reduction potentials of the hemes, +75 and -65 mV at pH 7.5, are also higher than those reported for other enzymes. The heme with the lower potential (heme bL) presents a considerable dependence of the reduction potential with pH (redox-Bohr effect), having a pKa(OX) = 6.5 and a pKa(red) = 8.7. This behavior is consistent with the proposal that in these enzymes menaquinone reduction occurs close to heme bL, near to the periplasmic side of the membrane, and involving dissipation of the proton transmembrane gradient.

A comparison between Sypro Ruby and ruthenium II tris (bathophenanthroline disulfonate) as fluorescent stains for protein detection in gels.

A comparison between two fluorescent metal chelates for staining proteins separated by electrophoresis has been carried out. One of these chelates is ruthenium II tris (bathophenanthroline disulfonate) and the other is commercial Sypro Ruby. Both can be efficiently detected either with UV tables or with commercial laser fluorescence scanners. The sensitivity and homogeneity of the stains and the interference with mass spectrometry analysis have been investigated. It appears that both stains perform similarly for protein detection, while ruthenium II tris (bathophenanthroline disulfonate) performs better for mass spectrometry analyses and as cost-effectiveness ratio. However, Sypro Ruby is easier to use as a stain.

Quantitative cytochemical mapping of mitochondrial enzymes in rat cerebella.

Mitochondrial metabolic competence, defined as the organelle's capacity to provide adequate amounts of ATP in due time, appears to constitute an important determinant in several biological processes and pathological conditions. Thus, the assessment of the metabolic efficiency of the mitochondrial population in a given tissue area or cellular compartment may provide clues to identifying alterations of the cellular bioenergetic machinery, which may constitute a predisposing condition leading to impaired organ and system functions. In the cerebellar cortex of adult rats, the activities of the enzymes cytochrome oxidase (COX) and succinic dehydrogenase (SDH) were, respectively, evidenced by means of the diaminobenzidine and copper ferrocyanide preferential cytochemical techniques. At the electron microscope, the activities of these two key molecules of the respiratory chain were clearly visualised as dark precipitates at the inner mitochondrial membrane sites where COX and SDH are located. By means of the disector method, unbiased mitochondrial samplings were carried out to measure: the number of mitochondria/microm(3) of tissue (numeric density: Nv); the mitochondrial volume fraction/microm(3) of tissue (volume density: Vv) and the average mitochondrial volume (V) both on COX- and SDH-positive organelles in the cerebellar glomeruli and Purkinje cells, respectively. The ratio R (total area of the precipitates due either to COX or SDH activity within the single mitochondrion/area of the same organelle) was also evaluated to get information on the enzyme activity related to mitochondrial size.The documented accumulation of mutant mitochondrial DNA particularly in postmitotic cells results in a marked heteroplasmy (mixtures of normal and mutated genomes) at mitochondrial and cellular levels, thus the cellular potential for energy production is demanded to a mosaic of organelles with different functional capabilities. Assessment of the mitochondrial mosaic outline by means of quantitative cytochemistry of key enzymes of the respiratory chain, such as COX and SDH, may allow for the morphofunctional metabolic mapping of mitochondrial efficiency in discrete cellular or tissue compartments.

Autoantibodies to the flavoprotein subunit of succinate dehydrogenase: analysis of specificity in autoimmune thyroid disease.

OBJECTIVE: Thyroid-associated ophthalmopathy is a progressive eye disorder affecting the extraocular muscle and orbital connective tissue and is considered to have an autoimmune aetiology. A recent study reported a close relationship between serum antibodies against the flavoprotein subunit of succinate dehyhdrogenase (SDHFp) and active thyroid-associated ophthalmopathy involving eye muscle damage. The aim of the present study was to develop a sensitive and quantitative radiobinding assay for the detection of antibodies to the flavoprotein subunit of succinate dehydrogenase and to use this to determine the distribution of antibodies in different patient groups. DESIGN AND PATIENTS: Serum samples from the following patient groups were analysed: 20 systemic lupus erythematosus; 20 Addison's disease; 26 autoimmune hypothyroidism; 28 Graves' hyperthyroidism; 12 pretibial myxoedema; 25 thyroid-associated ophthalmopathy. Sera from 20 healthy subjects were used as controls. [35S]-labelled succinate dehydrogenase flavoprotein was produced in an in vitro transcription-translation system and subsequently used in immunoprecipitation experiments with sera from patient and control groups to test for the presence of antibodies to the flavoprotein. RESULTS: Succinate dehydrogenase flavoprotein antibodies were detected in five of the 20 (25%) patients with Addison's disease, six of the 20 (30%) with systemic lupus erythematosus, five of the 26 (19%) with autoimmune hypothyroidsm, six of the 28 (21%) with Graves' hyperthyroidism, two of the 12 (17%) with pretibial myxoedema and three of the 25 (12%) with thyroid-associated ophthalmopathy. The frequencies of flavoprotein antibodies were significantly greater than controls (P-value < 0.05) for patients with systemic lupus erythematosus (P = 0.02), but not for patients with either Addison's disease (P = 0.05), pretibial myxoedema (P = 0.13), Graves' hyperthyroidism (P = 0.07), autoimmune hypothyroidism (P = 0.06) or thyroid-associated ophthalmopathy (P = 0.24). For the patients with thyroid-associated ophthalmopathy, the frequency of SDHFp antibodies did not appear to be related to the length of time from diagnosis: the group containing samples taken less than one year from diagnosis showed no increased frequency of SDHFp antibodies when compared to controls (P = 0.10), with three of the 18 (17%) patients being positive. With respect to seven patients with thyroid-associated ophthalmopathy diagnosed for more than a year, SDHFp antibodies were not detected in any of their serum samples. In addition, the clinical severity of the disease, as recorded by the NOSPECS classification, did not correlate with the frequency of SDHFp antibodies: P = 0.13, 0.33 and 0.38, respectively, for patients with Grade II, III and IV ophthalmopathy. Similar results were also found in the case of patients with pretibial myxoedema and eye disease: P = 0.06 for patients with Grade III ophthalmopathy and, SDHFp antibodies were not detected in any of the sera taken from patients with Grade IV ophthalmopathy. In addition, no association was found between disease duration and the frequency of antibodies to the flavoprotein in this patient group. CONCLUSIONS: Our results indicate that succinate dehydrogenase flavoprotein antibodies are not a suitable marker for thyroid-associated ophthalmopathy, at least with the assay system used, as they can be found in patients who do not have eye disease and therefore lack the disease specificity required of a diagnostic tool.

Identification of the active site acid/base catalyst in a bacterial fumarate reductase: a kinetic and crystallographic study.

The active sites of respiratory fumarate reductases are highly conserved, indicating a common mechanism of action involving hydride and proton transfer. Evidence from the X-ray structures of substrate-bound fumarate reductases, including that for the enzyme from Shewanella frigidimarina [Taylor, P., Pealing, S. L., Reid, G. A., Chapman, S. K., and Walkinshaw, M. D. (1999) Nat. Struct. Biol. 6, 1108-1112], indicates that the substrate is well positioned to accept a hydride from N5 of the FAD. However, the identity of the proton donor has been the subject of recent debate and has been variously proposed to be (using numbering for the S. frigidimarina enzyme) His365, His504, and Arg402. We have used site-directed mutagenesis to examine the roles of these residues in the S. frigidimarina enzyme. The H365A and H504A mutant enzymes exhibited lower k(cat) values than the wild-type enzyme but only by factors of 3-15, depending on pH. This, coupled with the increase in K(m) observed for these enzymes, indicates that His365 and His504 are involved in Michaelis complex formation and are not essential catalytic residues. In fact, examination of the crystal structure of S. frigidimarina fumarate reductase has led to the proposal that Arg402 is the only plausible active site acid. Consistent with this proposal, we report that the R402A mutant enzyme has no detectable fumarate reductase activity. The crystal structure of the H365A mutant enzyme shows that, in addition to the replacement at position 365, there have been some adjustments in the positions of active site residues. In particular, the observed change in the orientation of the Arg402 side chain could account for the decrease in k(cat) seen with the H365A enzyme. These results demonstrate that an active site arginine and not a histidine residue is the proton donor for fumarate reduction.