Molecular mechanism of thiocyanate dehydrogenase at atomic resolution.

Some sulfur-oxidizing bacteria playing an important role in global geochemical cycles utilize thiocyanate as the sole source of energy and nitrogen. In these bacteria the process of thiocyanate into cyanate conversion is mediated by thiocyanate dehydrogenases - a recently discovered family of copper-containing enzymes with the three­copper active site unique among the other copper proteins. To get a deeper insight into the structure and molecular mechanism of action of thiocyanate dehydrogenases we isolated, purified, and comprehensively characterized an enzyme from the bacterium Pelomicrobium methylotrophicum. High-resolution crystal structures of the thiocyanate dehydrogenase in the free state and in the complexes with the transition state analog, thiourea, and the closest substrate analog, selenocyanate, unveiled the fine details of molecular events occurring at the enzyme active site. During the reaction thiocyanate dehydrogenase undergoes profound conformational change that affects the position of the constituent copper ions and results in the activation of the attacking water molecule. The structure of the enzyme complex with the selenium atom bridged in-between two copper ions was obtained representing an important transient intermediate. Structures of the complexes with inhibitors supplemented with quantum chemical calculations clarify the role of copper ions and refine molecular mechanism of catalysis by thiocyanate dehydrogenase.

Trichlorobacter ammonificans, a dedicated acetate-dependent ammonifier with a novel module for dissimilatory nitrate reduction to ammonia.

Dissimilatory nitrate reduction to ammonia (DNRA) is a common biochemical process in the nitrogen cycle in natural and man-made habitats, but its significance in wastewater treatment plants is not well understood. Several ammonifying Trichlorobacter strains (former Geobacter) were previously enriched from activated sludge in nitrate-limited chemostats with acetate as electron (e) donor, demonstrating their presence in these systems. Here, we isolated and characterized the new species Trichlorobacter ammonificans strain G1 using a combination of low redox potential and copper-depleted conditions. This allowed purification of this DNRA organism from competing denitrifiers. T. ammonificans is an extremely specialized ammonifier, actively growing only with acetate as e-donor and carbon source and nitrate as e-acceptor, but H2 can be used as an additional e-donor. The genome of G1 does not encode the classical ammonifying modules NrfAH/NrfABCD. Instead, we identified a locus encoding a periplasmic nitrate reductase immediately followed by an octaheme cytochrome c that is conserved in many Geobacteraceae species. We purified this octaheme cytochrome c protein (TaNiR), which is a highly active dissimilatory ammonifying nitrite reductase loosely associated with the cytoplasmic membrane. It presumably interacts with two ferredoxin subunits (NapGH) that donate electrons from the menaquinol pool to the periplasmic nitrate reductase (NapAB) and TaNiR. Thus, the Nap-TaNiR complex represents a novel type of highly functional DNRA module. Our results indicate that DNRA catalyzed by octaheme nitrite reductases is a metabolic feature of many Geobacteraceae, representing important community members in various anaerobic systems, such as rice paddy soil and wastewater treatment facilities.

Extracellular Fe(III) reductase structure reveals a modular organization enabling S-layer insertion and electron transfer to insoluble substrates.

The thermophilic anaerobic Gram-positive bacterium Carboxydothermus ferrireducens utilizes insoluble Fe(III) oxides as electron acceptors in respiratory processes using an extracellular 11-heme cytochrome c OmhA as a terminal reductase. OmhA is able to transfer electrons to soluble and insoluble Fe(III) compounds, substrates of multiheme oxidoreductases, and soluble electron shuttles. The crystal structure of OmhA at 2.5 Å resolution shows that it consists of two functionally distinct parts: the cytochrome с electron transfer and the S-layer binding domains. Nonaheme C-terminal subdomain of the cytochrome с domain is structurally similar to the extracellular multiheme cytochrome OcwA from the metal-reducing Gram-positive bacterium "Thermincola potens." S-layer binding domain of OmhA is responsible for interaction with the S-layer that surrounds the Carboxydothermus ferrireducens cell envelope. The structural foundations enabling the embedding of extracellular multiheme cytochromes to the S-layer of a Gram-positive-type cell wall and putative electron transfer pathways to insoluble minerals are discussed.

Unusual Cytochrome c552 from Thioalkalivibrio paradoxus: Solution NMR Structure and Interaction with Thiocyanate Dehydrogenase.

The search of a putative physiological electron acceptor for thiocyanate dehydrogenase (TcDH) newly discovered in the thiocyanate-oxidizing bacteria Thioalkalivibrio paradoxus revealed an unusually large, single-heme cytochrome c (CytC552), which was co-purified with TcDH from the periplasm. Recombinant CytC552, produced in Escherichia coli as a mature protein without a signal peptide, has spectral properties similar to the endogenous protein and serves as an in vitro electron acceptor in the TcDH-catalyzed reaction. The CytC552 structure determined by NMR spectroscopy reveals significant differences compared to those of the typical class I bacterial cytochromes c: a high solvent accessible surface area for the heme group and so-called "intrinsically disordered" nature of the histidine-rich N- and C-terminal regions. Comparison of the signal splitting in the heteronuclear NMR spectra of oxidized, reduced, and TcDH-bound CytC552 reveals the heme axial methionine fluxionality. The TcDH binding site on the CytC552 surface was mapped using NMR chemical shift perturbations. Putative TcDH-CytC552 complexes were reconstructed by the information-driven docking approach and used for the analysis of effective electron transfer pathways. The best pathway includes the electron hopping through His528 and Tyr164 of TcDH, and His83 of CytC552 to the heme group in accordance with pH-dependence of TcDH activity with CytC552.

Octaheme nitrite reductase: The mechanism of intramolecular electron transfer and kinetics of nitrite bioelectroreduction.

Detailed impedance and voltammetric studies of hexameric octaheme nitrite reductase immobilized on carbon-based nanomaterials, specifically nanotubes and nanoparticles, were performed. Well-pronounced bioelectrocatalytic reduction of nitrite on enzyme-modified electrodes was obtained. Analysis of the impedance data indicated the absence of long-lived intermediates involved in the nitrite reduction. Cyclic voltammograms of biomodified electrodes had a bi-sigmoidal shape, which pointed to the presence of two enzyme orientations on carbon supports. The maximum (limiting) catalytic currents were determined and, by applying the correction by the mixed kinetics equation, the Tafel dependences were plotted for each catalytic wave/each enzyme orientation. Finally, two schemes for the rate-limiting processes during bioelectrocatalysis were proposed, viz. for low- and high-potential orientations.

Novel Extracellular Electron Transfer Channels in a Gram-Positive Thermophilic Bacterium.

Biogenic transformation of Fe minerals, associated with extracellular electron transfer (EET), allows microorganisms to exploit high-potential refractory electron acceptors for energy generation. EET-capable thermophiles are dominated by hyperthermophilic archaea and Gram-positive bacteria. Information on their EET pathways is sparse. Here, we describe EET channels in the thermophilic Gram-positive bacterium Carboxydothermus ferrireducens that drive exoelectrogenesis and rapid conversion of amorphous mineral ferrihydrite to large magnetite crystals. Microscopic studies indicated biocontrolled formation of unusual formicary-like ultrastructure of the magnetite crystals and revealed active colonization of anodes in bioelectrochemical systems (BESs) by C. ferrireducens. The internal structure of micron-scale biogenic magnetite crystals is reported for the first time. Genome analysis and expression profiling revealed three constitutive c-type multiheme cytochromes involved in electron exchange with ferrihydrite or an anode, sharing insignificant homology with previously described EET-related cytochromes thus representing novel determinants of EET. Our studies identify these cytochromes as extracellular and reveal potentially novel mechanisms of cell-to-mineral interactions in thermal environments.

Intracellular domains of the delta-subunits of Torpedo and rat acetylcholine receptors--expression, purification, and characterization.

There are quite detailed structural data on the extracellular ligand-binding domain and the intramembrane channel-forming domain of the nicotinic acetylcholine receptors (nAChR). However, the structure of the intracellular domain, which has variable amino acid sequences in different nAChR subunits, remains unknown. We expressed in Escherichia coli the intracellular loops (between transmembrane fragments TM3 and TM4) of the delta-subunits from the Torpedo californica and Rattus norvegicus muscle nAChRs. To facilitate purification, (His)6-tags were attached with or without linkers, and the effects of protein truncations at C- or N-termini were examined. The proteins were purified from inclusion bodies under denaturing conditions by Ni-NTA-chromatography. Molecular weight and peptide mass fingerprint was determined by MALDI mass spectrometry. Size-exclusion chromatography revealed that the Torpedo intracellular delta-loop refolded in an aqueous buffer was present in solution as a dimer. Phosphorylation of this protein with protein kinase A and tyrosine kinase (Abl) occurred at the same serine and tyrosine residues as in the native receptor. According to CD spectra, the secondary structure was not sensitive to phosphorylation. The rat intracellular loops could be solubilized only in the presence of non-ionic detergents or lipids. CD spectra indicate that the Torpedo and rat proteins have differences in their secondary structure. In the presence of dodecylphosphocholine, high concentrations (up to 6 mg/ml) of the Torpedo and rat intracellular loops were achieved. The results suggest that the spatial structure of the intracellular loops is dependent on environment and species, but is not changed significantly upon enzymatic phosphorylation.

Refolding of the Escherichia coli expressed extracellular domain of alpha 7 nicotinic acetylcholine receptor.

Heterologous expression of the extracellular domains (ECDs) of the nicotinic acetylcholine receptor (AChR) subunits may give large amounts of proteins for studying the functional and spatial characteristics of their ligand-binding sites. The ECD of the alpha 7 subunit of the homo-oligomeric alpha 7 neuronal AChR appears to be a more suitable object than the ECDs of other heteromeric neuronal or muscle-type AChRs. The rat alpha 7 ECDs (amino-acid residues approximately 1-210) were recently expressed in Escherichia coli as fusion proteins with maltose-binding protein [Fischer, M., Corringer, P., Schott, K., Bacher, A. & Changeux, J. (2001) Proc. Natl Acad. Sci. USA 98, 3567-3570] and glutathione S-transferase (GST) [Utkin, Y., Kukhtina, V., Kryukova, E., Chiodini, F., Bertrand, D., Methfessel, C. & Tsetlin, V. (2001) J. Biol. Chem. 276, 15810-15815]. However, these proteins exist in solution mostly as high-molecular mass aggregates rather than monomers or oligomers. In the present work it is found that refolding of GST-alpha 7-(1-208) protein in the presence of 0.1% SDS considerably decreases the formation of high-molecular mass aggregates. The C116S mutation in the alpha 7 moiety was found to further decrease the aggregation and to increase the stability of protein solutions. This mutation slightly increased the affinity of the protein for alpha-bungarotoxin (from Kd approximately 300 to 150 nm). Gel-permeation HPLC was used to isolate the monomeric form of the GST-alpha 7-(1-208) protein and its mutant almost devoid of SDS. CD spectra revealed that the C116S mutation considerably increased the content of beta structure and made it more stable under different conditions. The monomeric C116S mutant appears promising both for further structural studies and as a starting material for preparing the alpha 7 ECD in an oligomeric form.