Applied Chemistry

Cesium-phosphorous-silicon mixed oxide (Cs-P-Si oxide), a typical acid-base bifunctional catalyst, efficiently catalyzes propylene carbonate synthesis from CO 2 and propylene oxide under supercritical conditions (8-10 MPa). This catalyst contains no halogens and requires no additional solvents. A portion of the catalyst was eluted into the product solution during catalysis. It was also found that Cs 3 PO 4 , one of the species that may be eluted from the Cs-P-Si oxide, is an effective non-halogen homogeneous catalyst. #

Lipophilic alkaline earth metal complexes, prepared from the corresponding metal hydroxides and phosphoric acid diesters, are quite effective for the transport of some monosaccharides, especially ribose, across a chloroform liquid membrane under neutral conditions.

Bismuth bromide was found to work efficiently as a versatile catalyst for the cyanation and allylation of carbonyl compounds and acetals with organosilicon reagents, affording the corresponding alcohols and ethers in high yields. © 1997 Elsevier Science Ltd.

Analysis of the atp operon from the thermoalkaliphilic Bacillus sp. TA2.A1 and comparison with other atp operons from alkaliphilic bacteria reveals the presence of a conserved lysine residue at position 180 (Bacillus sp. TA2.A1 numbering) within the a subunit of these F 1 F o -ATP synthases. We hypothesize that the basic nature of this residue is ideally suited to capture protons from the bulk phase at high pH. To test this hypothesis, a heterologous expression system for the ATP synthase from Bacillus sp. TA2.A1 (TA2F 1 F o ) was developed in Escherichia coli DK8 (⌬atp). Amino acid substitutions were made in the a subunit of TA2F 1 F o at position 180. Lysine (aK180) was substituted for the basic residues histidine (aK180H) or arginine (aK180R), and the uncharged residue glycine (aK180G). ATP synthesis experiments were performed in ADP plus P i -loaded right-side-out membrane vesicles energized by ascorbate-phenazine methosulfate. When these enzyme complexes were examined for their ability to perform ATP synthesis over the pH range from 7.0 to 10.0, TA2F 1 F o and aK180R showed a similar pH profile having optimum ATP synthesis rates at pH 9.0 -9.5 with no measurable ATP synthesis at pH 7.5. Conversely, aK180H and aK180G showed maximal ATP synthesis at pH values 8.0 and 7.5, respectively. ATP synthesis under these conditions for all enzyme forms was sensitive to DCCD. These data strongly imply that amino acid residue Lys 180 is a specific adaptation within the a subunit of TA2F 1 F o to facilitate proton capture at high pH. At pH values near the pK a of Lys 180 , the trapped protons readily dissociate to reach the subunit c binding sites, but this dissociation is impeded at neutral pH values causing either a blocking of the proposed H ؉ channel and/or mechanism of proton translocation, and hence ATP synthesis is inhibited. The abbreviations used are: ⌬H ϩ , electrochemical proton gradient; ⌬, transmembrane electrical potential; ⌬pH, transmembrane pH gradient; TA2F 1 F o , F 1 F o -ATP synthase from Bacillus sp. TA2.A1 (recombinant TA2F 1 F o investigated in the present study has a hexa-histidine tag at the N-terminus of the ␤ subunit); ECF 1 F o , E. coli F 1 F o -ATP synthase; aK180H, aK180G, and aK180R, recombinant TA2F 1 F o where Lys 180 in the a subunit was sub-

Caldalkalibacillus thermarum strain TA2.A1 grew in pH-controlled batch culture containing a fermentable growth substrate (i.e., sucrose) from pH 7.5 to 10.0 with no significant change in the specific growth rate, suggesting that this bacterium was a facultative alkaliphile. However, when strain TA2.A1 was grown on a nonfermentable carbon source, such as succinate or malate, no growth was observed until the external pH was >9.0, suggesting that this bacterium was an obligate alkaliphile. Succinate transport and sucrose transport by strain TA2.A1 showed pH profiles similar to that of growth on these carbon sources, and the molar growth yield on sucrose was higher at pH 9.5 than at pH 7.5, despite the increased energy demands on the cell for intracellular pH regulation. Succinate transport, succinate-dependent oxygen consumption, and succinate dehydrogenase and F1Fo-ATPase specific activities were all significantly lower in cultures of strain TA2.A1 grown at pH 7.5 than in those cultured at pH 9.5. No significant ATP synthesis via the F1Fo-ATP synthase was detected until the external pH was >8.5. On the basis of these results, we propose that nonfermentative thermoalkaliphilic growth is specialized to function at high pH values, but not at pH values near neutral pH.

Figure 3. (a, b) Fluorescence micrographs of ATTO565-labeled CymA in the nested trap after application of a field for (a) 15 min and (b) 17 h. (cÀe) Plots of the relative average concentration (C/C 0 ) (black squares) for regions 1, 2 and 3, respectively, showing a clear increase in protein concentration toward the center. Green lines show the applied field. The initial CymA concentration C 0 was ∼0.25 wt %.

An enigma in the bioenergetics of methanogens is how the generation of proton and sodium gradients are used to synthesize ATP. Results: Purified methanogen ATP synthase was stimulated by sodium ions that also provided pH-dependent protection against DCCD.

springs in Armenia), which was grown anaerobically at pH 7.0 under light, from succinate using different amino acids (glutamate, alanine, tyrosine, and glycine) as nitrogen sources have been studied. The maximal cell growth rate and H 2 production by R. sphaeroides were obtained when glutamate was used as nitrogen source. The H 2 production rate, in the presence of alanine and tyrosine, in comparison with glutamate, decreased about 3-fold. In the presence of glycine the bacterial growth, but not H 2 production was observed. This process was suppressed at the presence of the N,N′-dicyclohexylcarbodiimide (DCCD), the F O F 1 -ATPase inhibitor. The addition of DCCD (0.5 mM) in succinate-glutamate, succinate-tyrosine or succinate-alanine medium caused a decrease of H 2 production rates (about 50%).

Background: CymA is the central menaquinol-7 dehydrogenase in anaerobic respiration of Shewanella sp. Results: CymA uses menaquinone-7 as a cofactor. Conclusion: CymA has one cofactor site that is specific for menaquinone-7 and one low affinity Q/QH 2 site that is in equilibrium with the quinone pool. Significance: The function of quinones needs to be reevaluated and crystallographically determined quinone binding pockets might not be the site of quinone conversion.

CymA (tetrahaem cytochrome c) is a member of the NapC/NirT family of quinol dehydrogenases. Essential for the anaerobic respiratory flexibility of shewanellae, CymA transfers electrons from menaquinol to various dedicated systems for the reduction of terminal electron acceptors including fumarate and insoluble minerals of Fe(III). Spectroscopic characterization of CymA from Shewanella oneidensis strain MR-1 identifies three lowspin His/His co-ordinated c-haems and a single high-spin chaem with His/H 2 O co-ordination lying adjacent to the quinolbinding site. At pH 7, binding of the menaquinol analogue, 2-heptyl-4-hydroxyquinoline-N-oxide, does not alter the midpoint potentials of the high-spin (approximately − 240 mV) and low-spin (approximately − 110, − 190 and − 265 mV) haems that appear biased to transfer electrons from the high-to lowspin centres following quinol oxidation. CymA is reduced with menadiol (E m = − 80 mV) in the presence of NADH (E m = − 320 mV) and an NADH-menadione (2-methyl-1,4-naphthoquinone) oxidoreductase, but not by menadiol alone. In cytoplasmic membranes reduction of CymA may then require the thermodynamic driving force from NADH, formate or H 2 oxidation as the redox poise of the menaquinol pool in isolation is insufficient. Spectroscopic studies suggest that CymA requires a non-haem co-factor for quinol oxidation and that the reduced enzyme forms a 1:1 complex with its redox partner Fcc 3 (flavocytochrome c 3 fumarate reductase). The implications for CymA supporting the respiratory flexibility of shewanellae are discussed.

It is well established that the structural details of electrodes and their interaction with adsorbed enzyme influences the interfacial electron transfer rate. However, for nanostructured electrodes, it is likely that the structure also impacts on substrate flux near the adsorbed enzymes and thus catalytic activity. Furthermore, for enzymes converting macro-molecular substrates it is possible that the enzyme orientation determines the nature of interactions between the adsorbed enzyme and substrate and therefore catalytic rates. In essence the electrode may impede substrate access to the active site of the enzyme. We have tested these possibilities through studies of the catalytic performance of two enzymes adsorbed on topologically distinct electrode materials. Escherichia coli NrfA, a nitrite reductase, was adsorbed on mesoporous, nanocrystalline SnO2 electrodes. CymA from Shewanella oneidensis MR-1 reduces menaquinone-7 within 200 nm sized liposomes and this reaction was studied with the enzyme adsorbed on SAM modified ultra-flat gold electrodes.

Protein−protein interactions are well-known to regulate enzyme activity in cell signaling and metabolism. Here, we show that protein−protein interactions regulate the activity of a respiratory-chain enzyme, CymA, by changing the direction or bias of catalysis. CymA, a member of the widespread NapC/NirT superfamily, is a menaquinol-7 (MQ-7) dehydrogenase that donates electrons to several distinct terminal reductases in the versatile respiratory network of Shewanella oneidensis. We report the incorporation of CymA within solid-supported membranes that mimic the inner membrane architecture of S. oneidensis. Quartz-crystal microbalance with dissipation (QCM-D) resolved the formation of a stable complex between CymA and one of its native redox partners, flavocytochrome c 3 (Fcc 3 ) fumarate reductase. Cyclic voltammetry revealed that CymA alone could only reduce MQ-7, while the CymA-Fcc 3 complex catalyzed the reaction required to support anaerobic respiration, the oxidation of MQ-7. We propose that MQ-7 oxidation in CymA is limited by electron transfer to the hemes and that complex formation with Fcc 3 facilitates the electron-transfer rate along the heme redox chain. These results reveal a yet unexplored mechanism by which bacteria can regulate multibranched respiratory networks through protein−protein interactions.

BACKGROUND: Tumour cell lysates are an excellent source of many defined and undefined tumour antigens and have been used clinically in immunotherapeutic regimes but with limited success. METHODS: We conjugated Mel888 melanoma lysates to rabbit haemorrhagic disease virus virus-like particles (VLP), which can act as vehicles to deliver multiple tumour epitopes to dendritic cells (DC) to effectively activate antitumour responses. RESULTS: Virus-like particles did not stimulate the phenotypic maturation of DC although, the conjugation of lysates to VLP (VLP-lysate) did overcome lysate-induced suppression of DC activation. Lysate-conjugated VLP enhanced delivery of antigenic proteins to DC, while the co-delivery of VLP-lysates with OK432 resulted in cross-priming of naïve T cells, with expansion of a MART1 þ population of CD8 þ T cells and generation of a specific cytotoxic response against Mel888 tumour cell targets. The responses generated with VLPlysate and OK432 were superior to those stimulated by unconjugated lysate with OK432. CONCLUSION: Collectively, these results show that the combination of VLP-lysate with OK432 delivered to DC overcomes the suppressive effects of lysates, and enables priming of naïve T cells with superior ability to specifically kill their target tumour cells.