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.

Homooligomeric LonA proteases are the key components of the protein quality control system in bacteria and eukaryotes. Domain organization of the common pool of LonA proteases is determined by the...

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.

Copper-containing enzymes catalyze a broad spectrum of redox reactions. Thiocyanate dehydrogenase (TcDH) from Thioalkalivibrio paradoxus Arh1 enables the bacterium to use thiocyanate as a unique source of energy and nitrogen. Oxidation of thiocyanate takes place in the trinuclear copper center of TcDH with peculiar organization. Despite the TcDH crystal structure being established, a role of some residues in the enzyme active site has yet to be obscured. F436 residue is located in the enzyme active site and conserved among a number of TcDH homologs, however, its role in the copper center formation or the catalytic process is still not clear. To address this question, a mutant form of the enzyme with F436Q substitution (TcDH^F436Q) was obtained, biochemically characterized, and its crystal structure was determined. The TcDH^F436Q had an unaltered protein fold but did not possess enzymatic activity, whereas it contained all three copper ions, according to ICP-MS data. The structural data showed that the F436Q substitution resulted in a disturbance of hydrophobic interactions within the active site crucial for a correct transition between open/closed forms of the enzyme–substrate channel. Thus, we demonstrated that F436 does not participate in copper ion binding, but rather possesses a structural role in the TcDH active site.