Structure of Escherichia coli Succinate:Quinone Oxidoreductase with an Occupied and Empty Quinone-binding Site

Three new structures of Escherichia coli succinate-quinone oxidoreductase (SQR) have been solved. One with the specific quinone-binding site (Q-site) inhibitor carboxin present has been solved at 2.4 Å resolution and reveals how carboxin inhibits the Q-site. The other new structures are with the Q-site inhibitor pentachlorophenol and with an empty Q-site. These structures reveal important details unresolved in earlier structures. Comparison of the new SQR structures shows how subtle rearrangements of the quinone-binding site accommodate the different inhibitors. The position of conserved water molecules near the quinone binding pocket leads to a reassessment of possible water-mediated proton uptake networks that complete reduction of ubiquinone. The dicarboxylate-binding site in the soluble domain of SQR is highly similar to that seen in high resolution structures of avian SQR (PDB 2H88) and soluble flavocytochrome c (PDB 1QJD) showing mechanistically significant structural features conserved across prokaryotic and eukaryotic SQRs.Succinate:quinone oxidoreductase (SQR,⁴ succinate dehydrogenase) and menaquinol:fumarate oxidoreductase (QFR, fumarate reductase), members of the Complex II family, are homologous integral membrane proteins which couple the interconversion of succinate and fumarate with quinone and quinol (1–4). SQR is a key enzyme in the Krebs cycle, oxidizing succinate to fumarate during aerobic growth and reducing quinone to quinol and, thus, acts as a direct link between the Krebs cycle and the respiratory chain. QFR is found in anaerobic or facultative bacteria and lower eukaryotes, where it couples the oxidation of reduced quinones to the reduction of fumarate (1, 4). Escherichia coli SQR has four subunits, two hydrophilic subunits exposed to the cytoplasm (SdhA and SdhB), which interact with two hydrophobic membrane-intrinsic subunits (SdhC and SdhD) (5). SdhA contains the dicarboxylate-binding site and a covalently bound FAD cofactor which cycles between FAD and FADH₂ redox states during succinate oxidation (6). The electrons from succinate oxidation are sequentially transferred via a [2Fe-2S], a [4Fe-4S], and a [3Fe-4S] iron-sulfur cluster relay system in SdhB to a quinone-binding site (Q_P) located at the interface of the SdhB, SdhC, and SdhD subunits. SdhC and SdhD are both composed of three transmembrane helices and coordinate a low spin b-type heme via His residues contributed by each subunit (7, 8).The first structural information about members of the Complex II family came from x-ray structures of the QFR enzymes from E. coli at 3.3 Å resolution (9) and Wolinella succinogenes at 2.2 Å resolution (10). These structures revealed details of the overall architecture of the subunits, the position of key redox cofactors, the electron transfer pathway, and the quinone-binding sites. At around the same time, the structures of soluble fumarate reductases found in anaerobic and microaerophilic bacteria and structurally homologous to the flavoprotein subunit of Complex II were solved by x-ray crystallography (1). Analysis of these soluble fumarate reductases has proven particularly informative in describing the mechanism of fumarate reduction and succinate oxidation at the dicarboxylate-binding site (11–14).Structures of SQRs lagged behind those of the QFRs until the structure of the E. coli enzyme was solved at 2.6 Å (15). This structure, solved in space group R32, revealed that the E. coli enzyme is packed as a trimer. The structures of the SdhA and SdhB subunits were highly similar to those of E. coli and W. succinogenes QFRs, but the transmembrane SdhC and SdhD subunits showed differences compared with their QFR counterparts. The structure revealed the position of the redox sites and the dicarboxylate- and quinone-binding (Q) sites. The heme b molecule was shown to lie away from the electron transfer pathway, suggesting electrons are preferentially transferred from the [3Fe-4S] cluster to ubiquinone, on the grounds of the edge-to-edge distances and redox potentials of the relevant groups. The structure revealed density in the Q-site that was interpreted as ubiquinone, and the position of the binding site was confirmed by the structure of the E. coli enzyme co-crystallized with the Q-site inhibitor 2-(1-methyl-hexyl)-4,6-dinitrophenol (DNP-17, PDB code 1NEN (15)). The E. coli enzyme was subsequently co-crystallized with the Q-site inhibitor Atpenin A5 (AA5) (PDB code 2ACZ (16)). This inhibitor was bound deeper into the quinone-binding site than ubiquinone or DNP-17, suggesting that there are two binding positions for ubiquinone in its binding site. The structure also identified a water-mediated proton pathway, proposed to deliver protons to the quinone-binding site. The first structure of a mitochondrial SQR was from porcine heart at 2.4 Å resolution (PDB code 1ZOY (17). This structure revealed a monomer in the asymmetric unit, suggesting that mitochondrial SQRs were likely to function as monomers. Superposition of the porcine and E. coli SQR structures revealed the high structural similarity of the SdhA and SdhB subunits and the conservation in position of the redox cofactors. Larger divergences were observed in the transmembrane subunits.Further structural information about SQRs was obtained by analysis of structures of avian SQR crystallized with oxaloacetate (2.2 Å resolution, PDB code 1YQ3), with 3-nitropropionate (2.4 Å resolution, PDB code 1YQ4), and with the Q-site inhibitor carboxin (2.1 Å resolution, PDB code 2FBW) (18). These structures revealed important differences in the position of key residues in the dicarboxylate-binding site compared with the E. coli and porcine structures. Arg-297 (equivalent to Arg-298 in porcine and Arg-286 in E. coli SQRs) was ideally located to act as a general base catalyst, accepting a proton during dehydrogenation of succinate, as in the soluble Shewanella flavocytochrome c3 (PDB code 1QJD) (11), suggesting conservation of mechanism between these distantly related enzymes. An unusual cis-serine peptide bond was proposed to position another arginine residue for binding dicarboxylates. Density for the dicarboxylate in 1YQ3 and 2FBW was shown to be distinctly non-planar and could be modeled by the “malate-like intermediate” seen in 1QJD. The nature of the ligand in the dicarboxylate site was further analyzed in a 1.74 Å resolution structure of avian SQR (PDB code 2H88), confirming the high structural similarity of the ligand and binding site residues in the SQR and flavocytochrome c3 structures (11, 12, 14).Despite the structural information described above, there are still unresolved issues regarding the structure and function of SQRs and QFRs. These include the location of conserved waters, which may form a channel involved in protonation of quinone, and the ability of the Q-site to accommodate different quinones and inhibitors. To further address these issues, we pursued structure-function studies of E. coli SQR. We developed alternative crystallization conditions that provided crystals more reproducibly and diffracting to higher resolution. By exchanging the enzyme into decyl-β-d-maltoside (DM) during purification, it was possible to crystallize the enzyme in the orthorhombic P2₁2₁2₁ space group. These crystals routinely diffracted in the 3–3.5 Å resolution range. Co-crystallization with the biochemically well characterized Q-site inhibitor carboxin improved diffraction to 2.1–2.8 Å. This structure shows new features related to the dicarboxylate-binding site of E. coli SQR including a rare cis-peptide bond in SdhA, as found in avian SQR (14), which helps shape the geometry of the active site. Comparisons of the structure with those of SQR binding PCP and SQR with an empty Q-site show how subtle rearrangements of the Q-site accommodate the different inhibitors. The orientation of carboxin in the Q-site differs with computational predictions (16) and with that seen in avian SQR (2FBW). The position of conserved water molecules around the Q-site suggests a new water-mediated proton uptake pathway consistent with recent mutational and biophysical studies (19).     

Serum disposition of bovine lactoferrin after oral and anal administration and its proteolytic cleavage by gastric transit in rainbow trout (Oncorhynchus mykiss W.)

Several studies have shown an immunomodulatory effect of orally administered bovine lactoferrin (LF) in fish, but the process of digestion was not characterized.In the present study, we investigated the fate of bovine LF after oral and anal administration, and studied the appearance of intact LF in the bloodstream and its proteolytic attack during the gastric transit in rainbow trout (Oncorhynchus mykiss) held at 9 °C and 18 °C.Data obtained showed the presence of intact bovine LF in the bloodstream only after anal administration in fish held at 18 °C and the presence of several peptides derived from bovine LF in the gastric content. Immunoblotting analysis showed that only a part of bovine LF-derived peptides reacted with the applied anti-bovine LF antibody. The concentration of intact bovine LF, after 30 min of administration, in the gastric content of fish reared at 18 °C, being extremely low, if any, led us to suspect that the immunoregulatory effect of dietary bovine LF shown in fish by several authors is not due to the intact form but to bioactive fragments, originated by the proteolytic attack during the gastric transit, as demonstrated in higher vertebrates.     

α‐tocopherol affects neuronal plasticity in adult rat dentate gyrus: The possible role of PKCδ

Hippocampus dentate gyrus (DG) is characterized by neuronal plasticity processes in adulthood, and polysialylation of NCAM promotes neuronal plasticity. In previous investigations we found that α‐tocopherol increased the PSA‐NCAM‐positive granule cell number in adult rat DG, suggesting that α‐tocopherol may enhance neuronal plasticity. To verify this hypothesis, in the present study, structural remodeling in adult rat DG was investigated under α‐tocopherol supplementation conditions. PSA‐NCAM expression was evaluated by Western blotting, evaluation of PSA‐NCAM‐positive granule cell density, and morphometric analysis of PSA‐NCAM‐positive processes. In addition, the optical density of synaptophysin immunoreactivity and the synaptic profile density, examined by electron microscopy, were evaluated. Moreover, considering that PSA‐NCAM expression has been found to be related to PKCδ activity and α‐tocopherol has been shown to inhibit PKC activity in vitro, Western blotting and immunohistochemistry followed by densitometry were used to analyze PKC. Our results demonstrated that an increase in PSA‐NCAM expression and optical density of DG molecular layer synaptophysin immunoreactivity occurred in α‐tocopherol‐treated rats. Electron microscopy analysis showed that the increase in synaptophysin expression was related to an increase in synaptic profile density. In addition, Western blotting revealed a decrease in phospho‐PKC Pan and phospho‐PKCδ, demonstrating that α‐tocopherol is also able to inhibit PKC activity in vivo. Likewise, immunoreactivity for the active form of PKCδ was lower in α‐tocopherol‐treated rats than in controls, while no changes were found in PKCδ expression. These results demonstrate that α‐tocopherol is an exogenous factor affecting neuronal plasticity in adult rat DG, possibly through PKCδ inhibition. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006     

Use of heterologous antigens for the immunodiagnosis of abdominal angiostrongyliasis by an enzyme-linked immunosorbent assay

Angiostrongylus costaricensis has a broad geographic distribution spanning from North to South America and the infections of vertebrates with this nematode can result in abdominal complications. Human infections are diagnosed by histological or serological methods because the isolation of larvae from feces is not feasible, as most parasites become trapped in intestinal tissues due to intense eosinophilic inflammation. Because A. costaricensis is difficult to maintain in the laboratory, an immunodiagnostic IgG enzyme-linked immunosorbent assay (ELISA) using antigens from the congeneric Angiostrongylus cantonensis species was evaluated against a panel of serum samples from patients who were histologically diagnosed with A. costaricensis infections. Sera from uninfected individuals and individuals infected with other parasites were used as controls. The sensitivity and specificity of the assay were estimated at 88.4% and 78.7%, respectively. Because the use of purified or cloned antigens has not been established as a reliable diagnostic tool, the use of heterologous antigens may provide a viable alternative for the development of an ELISA-based immunodetection system for the diagnosis of abdominal angiostrongyliasis.     

Observations on Haemagogus janthinomys Dyar (Diptera: Culicidae) and other mosquito populations within tree holes in a gallery forest in the northwestern region of Sao Paulo state, Brazil

In 2000, an outbreak of sylvatic yellow fever possibly occurred in gallery forests of the Grande river in the Paraná basin in the northwestern region of S?o Paulo state. The aim of this study was to obtain information on the bionomics of Haemagogus and other mosquitoes inside tree holes in that area. Eighteen open tree holes were sampled for immature specimens. Adults were collected twice a month in the forest in Santa Albertina county from July 2000 to June 2001. The seasonal frequency of fourth instars was obtained by the Williams geometric mean (Mw), while the adult frequency was estimated either by hourly arithmetic or the Williams' means. Cole's index was applied to evaluate larval inter-specific associations. Among the ten mosquito species identified, the most abundant was Aedes terrens Walker followed by Sabethes tridentatus Cerqueira and Haemagogus janthinomys Dyar. Larval and adult abundance of these species was higher in summer than in winter. Although larval abundance of Hg. janthinomys peaked in the rainy season, correlation with rainfall was not significant. Six groups of larval associations were distinguished, one of which the most positively stable. The Hg. janthinomys and Ae. terrens association was significant, and Limatus durhamii Theobald was the species with most negative associations.     

Sugarcane molasses and yeast powder used in the Fructooligosaccharides production by Aspergillus japonicus-FCL 119T and Aspergillus niger ATCC 20611

Different concentrations of sucrose (3–25% w/v) and peptone (2–5% w/v) were studied in the formulation of media during the cultivation of Aspergillus japonicus-FCL 119T and Aspergillus niger ATCC 20611. Moreover, cane molasses (3.5–17.5% w/v total sugar) and yeast powder (1.5–5% w/v) were used as alternative nutrients for both strains’ cultivation. These media were formulated for analysis of cellular growth, β-Fructosyltransferase and Fructooligosaccharides (FOS) production. Transfructosylating activity (U _t ) and FOS production were analyzed by HPLC. The highest enzyme production by both the strains was 3% (w/v) sucrose and 3% (w/v) peptone, or 3.5% (w/v) total sugars present in cane molasses and 1.5% (w/v) yeast powder. Cane molasses and yeast powder were as good as sucrose and peptone in the enzyme and FOS (around 60% w/w) production by studied strains.     

Absence of NADH channeling in coupled reaction of mitochondrial malate dehydrogenase and complex I in alamethicin-permeabilized rat liver mitochondria

A simple in situ model of alamethicin-permeabilized isolated rat liver mitochondria was used to investigate the channeling of NADH between mitochondrial malate dehydrogenase (MDH) and NADH:ubiquinone oxidoreductase (complex I). Alamethicin-induced pores in the mitochondrial inner membrane allow effective transport of low molecular mass components such as NAD+/NADH but not soluble proteins. Permeabilized mitochondria demonstrate high rates of respiration in the presence of malate/glutamate and NAD+ due to coupled reaction between MDH and complex I. In the presence of pyruvate and lactate dehydrogenase, an extramitochondrial competitive NADH utilizing system, respiration of permeabilized mitochondria with malate/glutamate and NAD+ was completely abolished. These data are in agreement with the free diffusion of NADH and do not support the suggestion of direct channeling of NADH from MDH to complex I.     

Genotypic fidelity of micropropagated pineapple (Ananas comosus) plantlets assessed by isozyme and RAPD markers

In the present work, pineapple plantlets (cv. `Amarelinho') micropropagated by stationary (S) and temporary immersion (T) systems were evaluated in terms of genotypic fidelity by isozyme and RAPD markers. Neither isozymes (average 0.67%) nor RAPDs (average 7.5%) alone detected significant differences between the two micropropagated systems. However, when combined isozymes and RAPDs data more somaclonal variants were detected in S than T, with RAPDs revealing more variation than isozymes. In the T system, the treatment with PBZ 6.0 M gave the greatest occurrence of variants (8.9%), although significant statistical differences (; p>0.05) between presence and absence of PBZ or GA were not detected. Temporary immersion in comparison with the stationary system resulted in the lowest proportion of somaclonal variants (1.9% vs. 3.9%). Although it does not represent an in depth genome analysis, ours is the first work that has attempted to evaluate the genotypic fidelity of micropropagated pineapple plantlets.     

Enhanced heart rate variability and baroreflex index after stress and cholinesterase inhibition in mice

Experiments tested the effect of stress coupled with cholinesterase inhibition on blood pressure, heart rate, baroreflex index, and variability in time and frequency domain in conscious mice. The objective was to determine whether cholinergic systems interact with stress to alter cardiovascular responses. Male C57BL/6J mice with arterial catheters were exposed to 3-day treatments: 1) intermittent shaker stress, 2) pyridostigmine (10 mg.kg(-1).day(-1)); or 3) combined pyridostigmine and stress. Pyridostigmine reduced blood cholinesterase (-33%) with no added effects of stress. Twenty-four-hour blood pressure recordings showed that there were no differences in blood pressure and heart rate with the treatments. Pulse interval standard deviation was greatly increased in the pyridostigmine/stress group compared with stress or pyridostigmine groups (11.0 +/- 1.4, 5.0 +/- 0.9, and 7.5 +/- 0.9 ms, respectively). Spectral analysis showed two distinct components for pulse interval variability (low and high frequency). Variability in the low-frequency range was greatly enhanced in the pyridostigmine/stress group, seen as a doubling of the power (9.5 +/- 1.7, 3.3 +/- 0.9, and 5.0 +/- 0.6 ms for pyridostigmine/stress, stress and pyridostigmine groups, respectively). Baroreflex sensitivity was also increased in the pyridostigmine/stress group (3.6 +/- 0.5 compared with 1.8 +/- 0.3 and 1.7 +/- 0.5 ms/mmHg in the stress and pyridostigmine groups, respectively). There was no difference in blood pressure variability or its spectral components. Results demonstrate that there are potent interactions between a mild stressor and cholinesterase inhibition seen as an accentuation of low-frequency variability in pulse interval time series, probably associated with baroreflex input and autonomic drive.     

Synthesis and secretion of transferrin by isolated ciliary epithelium of rabbit

It has been shown that the vitreous contains several intrinsic glycoproteins whose origin remains to be clarified. Isolated ciliary epithelium (CE) was assayed to verify its role in the synthesis and secretion of transferrin for the vitreous body. It was cultured in the presence of [35S]methionine and the incubation medium was processed for immunoprecipitation. Total RNA from CE was processed for RT-PCR and the amplification products were sequenced. Also, whole preparations of isolated CE were processed for immunolocalization of transferrin. From the incubation assays, a labeled peptide of about 80 kDa was immunopurified that is the expected size of transferrin. The RT-PCR and sequencing experiments detected the presence of transferrin mRNA. Both layers of the CE exhibited transferrin reactivity, following immunohistochemical processing. Taken altogether, these results indicate the CE as one of the possible sources of vitreous intrinsic transferrin.