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The Caucasus Region belongs among the most remarkable biodiversity hotspots globally. Local mountain floodplain forests represent highly endangered ecosystems and possess the status of protective forests. However, they have not been extensively phytosociologically assessed using Braun-Blanquet methods to date. Here, we present a novel dataset of vegetation plots recorded in two remarkable areas of forest plant diversity in this region. They were Enguri (Black Sea watershed) and Aragvi (Caspian Sea watershed) River basins in the Western and Eastern Greater Caucasus, respectively. Based on 49 relevés, sampled on a broad elevation gradient (980-1830 m), we distinguished three vegetation types using unsupervised classification. Main gradients in their species composition reflect biogeographical and climatic differences. Alnus incana dominated two types. The Enguri type (Brunnero macrophyllae-Alnetum incanae) was characterized by numerous species typical of the western part of the Greater Caucasus, including tall forbs and mountain species. The Aragvi type (Veronico filiformis-Alnetum incanae) showed a diagnostic combination of common Caucasian species and relatively thermophilous forest species. The subdivision of this unit included three subtypes according to grazing intensity. The last type (Sedo stoloniferi-Alnetum barbatae) was significant by a putative Tertiary relict Alnus glutinosa subsp. barbata as a canopy dominant. It occupied lower elevations of the Enguri Region, significantly influenced by the nearby Colchic Region. To provide a regional context of the sampled vegetation, we compiled an expanded dataset of alluvial alder forests from the Caucasus and its surroundings. Their joint classification highlighted the uniqueness of the recorded communities with A. incana and clearly distinguished them from the Euxinian alliance Alnion barbatae delimited by relict species typical of the Colchic refugium (e.g. Diospyros lotus, Pterocarya fraxinifolia). A comparison of the Caucasian A. incana forests with the alluvial forests of the boreonemoral alliance Alnion incanae supported the individuality of the Caucasian stands in terms of floristic composition and allowed us to describe them as a new alliance (Veronico filiformis-Alnion incanae all. nova). It is characterized by the dominance of A. incana accompanied by numerous geographically restricted species (e.g. Senecio propinquus, Symphytum asperum) coupled with boreo-montane forest species (e.g. Oxalis acetosella, Polygonatum verticillatum). Caucasian alluvial forests face many ongoing threats, including constructing of new water reservoirs and hydropower plants, overgrazing and illegal cutting. [ABSTRACT FROM AUTHOR]

Ferric reductase B (FerB) is a flavin mononucleotide (FMN)‐containing NAD(P)H:acceptor oxidoreductase structurally close to the Gluconacetobacter hansenii chromate reductase (ChrR). The crystal structure of ChrR was previously determined with a chloride bound proximal to FMN in the vicinity of Arg101, and the authors suggested that the anionic electron acceptors, chromate and uranyl tricarbonate, bind similarly. Here, we identify the corresponding arginine residue in FerB (Arg95) as being important for the reaction of FerB with superoxide. Four mutants at position 95 were prepared and found kinetically to have impaired capacity for superoxide binding. Stopped‐flow data for the flavin cofactor showed that the oxidative step is rate limiting for catalytic turnover. The findings are consistent with a role for FerB as a superoxide scavenging contributor. [ABSTRACT FROM AUTHOR]

FerB from Paracoccus denitrificans is a soluble cytoplasmic flavoprotein that accepts redox equivalents from NADH or NADPH and transfers them to various acceptors such as quinones, ferric complexes and chromate. The crystal structure and small-angle X-ray scattering measurements in solution reported here reveal a head-to-tail dimer with two flavin mononucleotide groups bound at the opposite sides of the subunit interface. The dimers tend to self-associate to a tetrameric form at higher protein concentrations. Amino acid residues important for the binding of FMN and NADH and for the catalytic activity are identified and verified by site-directed mutagenesis. In particular, we show that Glu77 anchors a conserved water molecule in close proximity to the O2 of FMN, with the probable role of facilitating flavin reduction. Hydride transfer is shown to occur from the 4-pro-S position of NADH to the solvent-accessible si side of the flavin ring. When using deuterated NADH, this process exhibits a kinetic isotope effect of about 6 just as does the NADH-dependent quinone reductase activity of FerB; the first, reductive half-reaction of flavin cofactor is thus rate-limiting. Replacing the bulky Arg95 in the vicinity of the active site with alanine substantially enhances the activity towards external flavins that obeys the standard bi-bi ping-pong reaction mechanism. The new evidence for a cryptic flavin reductase activity of FerB justifies the previous inclusion of this enzyme in the protein family of NADPH-dependent FMN reductases. [ABSTRACT FROM AUTHOR]

FerB from Paracoccus denitrificans is a soluble cytoplasmic flavoprotein that accepts redox equivalents from NADH or NADPH and transfers them to various acceptors such as quinones, ferric complexes and chromate. The crystal structure and small-angle X-ray scattering measurements in solution reported here reveal a head-to-tail dimer with two flavin mononucleotide groups bound at the opposite sides of the subunit interface. The dimers tend to self-associate to a tetrameric form at higher protein concentrations. Amino acid residues important for the binding of FMN and NADH and for the catalytic activity are identified and verified by site-directed mutagenesis. In particular, we show that Glu77 anchors a conserved water molecule in close proximity to the O2 of FMN, with the probable role of facilitating flavin reduction. Hydride transfer is shown to occur from the 4-pro-S position of NADH to the solvent-accessible si side of the flavin ring. When using deuterated NADH, this process exhibits a kinetic isotope effect of about 6 just as does the NADH-dependent quinone reductase activity of FerB; the first, reductive half-reaction of flavin cofactor is thus rate-limiting. Replacing the bulky Arg95 in the vicinity of the active site with alanine substantially enhances the activity towards external flavins that obeys the standard bi-bi ping-pong reaction mechanism. The new evidence for a cryptic flavin reductase activity of FerB justifies the previous inclusion of this enzyme in the protein family of NADPH-dependent FMN reductases. [ABSTRACT FROM AUTHOR]

The flavin-dependent enzyme FerB from Paracoccus denitrificans reduces a broad range of compounds, including ferric complexes, chromate and most notably quinones, at the expense of the reduced nicotinamide adenine dinucleotide cofactors NADH or NADPH. Recombinant unmodified and SeMet-substituted FerB were crystallized under similar conditions by the hanging-drop vapour-diffusion method with microseeding using PEG 4000 as the precipitant. FerB crystallized in several different crystal forms, some of which diffracted to approximately 1.8 Å resolution. The crystals of native FerB belonged to space group P21, with unit-cell parameters a = 61.6, b = 110.1, c = 65.2 Å, β = 118.2° and four protein molecules in the asymmetric unit, whilst the SeMet-substituted form crystallized in space group P21212, with unit-cell parameters a = 61.2, b = 89.2, c = 71.5 Å and two protein molecules in the asymmetric unit. Structure determination by the three-wavelength MAD/MRSAD method is now in progress. [ABSTRACT FROM AUTHOR]

Paracoccus denitrificans has a branched electron transport chain with three terminal oxidases transferring electrons to molecular oxygen, namely aa 3 -type and cbb 3 -type cytochrome c oxidases and ba 3 -type ubiquinol oxidase. In the present study, we focused on strains expressing only one of these enzymes. The competition experiments showed that possession of cbb 3 -type oxidase confers significant fitness advantage during oxygen-limited growth and supports the biofilm lifestyle. The aa 3 -type oxidase was shown to allow rapid aerobic growth at a high oxygen supply. Activity of the denitrification pathway that had been expressed in cells grown anaerobically with nitrate was fully inhibitable by oxygen only in wild-type and cbb 3 strains, while in strains aa 3 and ba 3 dinitrogen production from nitrate and oxygen consumption occurred simultaneously. Together, the results highlight the importance of the cbb 3 -type oxidase for the denitrification phenotype and suggest a way of obtaining novel bacterial strains capable of aerobic denitrification.