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.

Catalytic Properties of Flavocytochrome c Sulfide Dehydrogenase from Haloalkaliphilic Bacterium Thioalkalivibrio paradoxus.

Flavocytochrome c sulfide dehydrogenase (FCC) is one of the central enzymes of the respiratory chain in sulfur-oxidizing bacteria. FCC catalyzes oxidation of sulfide and polysulfide ions to elemental sulfur accompanied by electron transfer to cytochrome c. The catalytically active form of the enzyme is a non-covalently linked heterodimer composed of flavin- and heme-binding subunits. The Thioalkalivibrio paradoxus ARh1 genome contains five copies of genes encoding homologous FCCs with an amino acid sequence identity from 36 to 54%. When growing on thiocyanate or thiosulfate as the main energy source, the bacterium synthesizes products of different copies of FCC genes. In this work, we isolated and characterized FCC synthesized during the growth of Tv. paradoxus on thiocyanate. FCC was shown to oxidize exclusively sulfide but not other reduced sulfur compounds, such as thiosulfate, sulfite, tetrathionate, and sulfur, and it also does not catalyze the reverse reaction of sulfur reduction to sulfide. Kinetic parameters of the sulfide oxidation reaction are characterized.

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.

Trinuclear copper biocatalytic center forms an active site of thiocyanate dehydrogenase.

Biocatalytic copper centers are generally involved in the activation and reduction of dioxygen, with only few exceptions known. Here we report the discovery and characterization of a previously undescribed copper center that forms the active site of a copper-containing enzyme thiocyanate dehydrogenase (suggested EC 1.8.2.7) that was purified from the haloalkaliphilic sulfur-oxidizing bacterium of the genus Thioalkalivibrio ubiquitous in saline alkaline soda lakes. The copper cluster is formed by three copper ions located at the corners of a near-isosceles triangle and facilitates a direct thiocyanate conversion into cyanate, elemental sulfur, and two reducing equivalents without involvement of molecular oxygen. A molecular mechanism of catalysis is suggested based on high-resolution three-dimensional structures, electron paramagnetic resonance (EPR) spectroscopy, quantum mechanics/molecular mechanics (QM/MM) simulations, kinetic studies, and the results of site-directed mutagenesis.

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.

Comparative Genomics of Thiohalobacter thiocyanaticus HRh1T and Guyparkeria sp. SCN-R1, Halophilic Chemolithoautotrophic Sulfur-Oxidizing Gammaproteobacteria Capable of Using Thiocyanate as Energy Source.

The genomes of Thiohalobacter thiocyanaticus and Guyparkeria (formerly known as Halothiobacillus) sp. SCN-R1, two gammaproteobacterial halophilic sulfur-oxidizing bacteria (SOB) capable of thiocyanate oxidation via the "cyanate pathway", have been analyzed with a particular focus on their thiocyanate-oxidizing potential and sulfur oxidation pathways. Both genomes encode homologs of the enzyme thiocyanate dehydrogenase (TcDH) that oxidizes thiocyanate via the "cyanate pathway" in members of the haloalkaliphilic SOB of the genus Thioalkalivibrio. However, despite the presence of conservative motives indicative of TcDH, the putative TcDH of the halophilic SOB have a low overall amino acid similarity to the Thioalkalivibrio enzyme, and also the surrounding genes in the TcDH locus were different. In particular, an alternative copper transport system Cus is present instead of Cop and a putative zero-valent sulfur acceptor protein gene appears just before TcDH. Moreover, in contrast to the thiocyanate-oxidizing Thioalkalivibrio species, both genomes of the halophilic SOB contained a gene encoding the enzyme cyanate hydratase. The sulfur-oxidizing pathway in the genome of Thiohalobacter includes a Fcc type of sulfide dehydrogenase, a rDsr complex/AprAB/Sat for oxidation of zero-valent sulfur to sulfate, and an incomplete Sox pathway, lacking SoxCD. The sulfur oxidation pathway reconstructed from the genome of Guyparkeria sp. SCN-R1 was more similar to that of members of the Thiomicrospira-Hydrogenovibrio group, including a Fcc type of sulfide dehydrogenase and a complete Sox complex. One of the outstanding properties of Thiohalobacter is the presence of a Na+-dependent ATP synthase, which is rarely found in aerobic Prokaryotes.Overall, the results showed that, despite an obvious difference in the general sulfur-oxidation pathways, halophilic and haloalkaliphilic SOB belonging to different genera within the Gammaproteobacteria developed a similar unique thiocyanate-degrading mechanism based on the direct oxidative attack on the sulfane atom of thiocyanate.

Structure of the flavocytochrome c sulfide dehydrogenase associated with the copper-binding protein CopC from the haloalkaliphilic sulfur-oxidizing bacterium Thioalkalivibrio paradoxusARh 1.

Flavocytochrome c sulfide dehydrogenase from Thioalkalivibrio paradoxus (TpFCC) is a heterodimeric protein consisting of flavin- and monohaem c-binding subunits. TpFCC was co-purified and co-crystallized with the dimeric copper-binding protein TpCopC. The structure of the TpFCC-(TpCopC)2 complex was determined by X-ray diffraction at 2.6 Šresolution. The flavin-binding subunit of TpFCC is structurally similar to those determined previously, and the structure of the haem-binding subunit is similar to that of the N-terminal domain of dihaem FCCs. According to classification based on amino-acid sequence, TpCopC belongs to a high-affinity CopC subfamily characterized by the presence of a conserved His1-Xxx-His3 motif at the N-terminus. Apparently, a unique α-helix which is present in each monomer of TpCopC at the interface with TpFCC plays a key role in complex formation. The structure of the copper-binding site in TpCopC is similar to those in other known CopC structures. His3 is not involved in binding to the copper ion and is 6-7 Šaway from this ion. Therefore, the His1-Xxx-His3 motif cannot be considered to be a key factor in the high affinity of CopC for copper(II) ions. It is suggested that the TpFCC-(TpCopC)2 heterotetramer may be a component of a large periplasmic complex that is responsible for thiocyanate metabolism.

Evolving stability and pH-dependent activity of the high redox potential Botrytis aclada laccase for enzymatic fuel cells.

Fungal high redox potential laccases are proposed as cathodic biocatalysts in implantable enzymatic fuel cells to generate high cell voltages. Their application is limited mainly through their acidic pH optimum and chloride inhibition. This work investigates evolutionary and engineering strategies to increase the pH optimum of a chloride-tolerant, high redox potential laccase from the ascomycete Botrytis aclada. The laccase was subjected to two rounds of directed evolution and the clones screened for increased stability and activity at pH 6.5. Beneficial mutation sites were investigated by semi-rational and combinatorial mutagenesis. Fourteen variants were characterised in detail to evaluate changes of the kinetic constants. Mutations increasing thermostability were distributed over the entire structure. Among them, T383I showed a 2.6-fold increased half-life by preventing the loss of the T2 copper through unfolding of a loop. Mutations affecting the pH-dependence cluster around the T1 copper and categorise in three types of altered pH profiles: pH-type I changes the monotonic decreasing pH profile into a bell-shaped profile, pH-type II describes increased specific activity below pH 6.5, and pH-type III increased specific activity above pH 6.5. Specific activities of the best variants were up to 5-fold higher (13 U mg-1) than BaL WT at pH 7.5.

Structural adaptations of octaheme nitrite reductases from haloalkaliphilic Thioalkalivibrio bacteria to alkaline pH and high salinity.

Bacteria Tv. nitratireducens and Tv. paradoxus from soda lakes grow optimally in sodium carbonate/NaCl brines at pH range from 9.5 to 10 and salinity from 0.5 to 1.5 M Na+. Octaheme nitrite reductases (ONRs) from haloalkaliphilic bacteria of genus Thioalkalivibrio are stable and active in a wide range of pH (up to 11) and salinity (up to 1 M NaCl). To establish adaptation mechanisms of ONRs from haloalkaliphilic bacteria a comparative analysis of amino acid sequences and structures of ONRs from haloalkaliphilic bacteria and their homologues from non-halophilic neutrophilic bacteria was performed. The following adaptation strategies were observed: (1) strategies specific for halophilic and alkaliphilic proteins (an increase in the number of aspartate and glutamate residues and a decrease in the number of lysine residues on the protein surface), (2) strategies specific for halophilic proteins (an increase in the arginine content and a decrease in the number of hydrophobic residues on the solvent-accessible protein surface), (3) strategies specific for alkaliphilic proteins (an increase in the area of intersubunit hydrophobic contacts). Unique adaptation mechanism inherent in the ONRs from bacteria of genus Thioalkalivibrio was revealed (an increase in the core in the number of tryptophan and phenylalanine residues, and an increase in the number of small side chain residues, such as alanine and valine, in the core).

Effect of the L499M mutation of the ascomycetous Botrytis aclada laccase on redox potential and catalytic properties.

Laccases are members of a large family of multicopper oxidases that catalyze the oxidation of a wide range of organic and inorganic substrates accompanied by the reduction of dioxygen to water. These enzymes contain four Cu atoms per molecule organized into three sites: T1, T2 and T3. In all laccases, the T1 copper ion is coordinated by two histidines and one cysteine in the equatorial plane and is covered by the side chains of hydrophobic residues in the axial positions. The redox potential of the T1 copper ion influences the enzymatic reaction and is determined by the nature of the axial ligands and the structure of the second coordination sphere. In this work, the laccase from the ascomycete Botrytis aclada was studied, which contains conserved Ile491 and nonconserved Leu499 residues in the axial positions. The three-dimensional structures of the wild-type enzyme and the L499M mutant were determined by X-ray crystallography at 1.7 Šresolution. Crystals suitable for X-ray analysis could only be grown after deglycosylation. Both structures did not contain the T2 copper ion. The catalytic properties of the enzyme were characterized and the redox potentials of both enzyme forms were determined: E0 = 720 and 580 mV for the wild-type enzyme and the mutant, respectively. Since the structures of the wild-type and mutant forms are very similar, the change in the redox potential can be related to the L499M mutation in the T1 site of the enzyme.

Contrasting catalytic profiles of multiheme nitrite reductases containing CxxCK heme-binding motifs.

The multiheme cytochromes from Thioalkalivibrio nitratireducens (TvNiR) and Escherichia coli (EcNrfA) reduce nitrite to ammonium. Both enzymes contain His/His-ligated hemes to deliver electrons to their active sites, where a Lys-ligated heme has a distal pocket containing a catalytic triad of His, Tyr, and Arg residues. Protein-film electrochemistry reveals significant differences in the catalytic properties of these enzymes. TvNiR, but not EcNrfA, requires reductive activation. Spectroelectrochemistry implicates reduction of His/His-ligated heme(s) as being key to this process, which restricts the rate of hydroxide binding to the ferric form of the active-site heme. The K M describing nitrite reduction by EcNrfA varies with pH in a sigmoidal manner that is consistent with its modulation by (de)protonation of a residue with pK a ≈ 7.6. This residue is proposed to be the catalytic His in the distal pocket. By contrast, the K M for nitrite reduction by TvNiR decreases approximately linearly with increase of pH such that different features of the mechanism define this parameter for TvNiR. In other regards the catalytic properties of TvNiR and EcNrfA are similar, namely, the pH dependence of V max and the nitrite dependence of the catalytic current-potential profiles resolved by cyclic voltammetry, such that the determinants of these properties appear to be conserved.

Comparative structural and functional analysis of two octaheme nitrite reductases from closely related Thioalkalivibrio species.

UNLABELLED: Octaheme nitrite reductase from the haloalkaliphilic bacterium Thioalkalivibrio paradoxus was isolated and characterized. A comparative structural and functional analysis of two homologous octaheme nitrite reductases from closely related Thioalkalivibrio species was performed. It was shown that both enzymes have similar catalytic properties, owing to high structural similarity. Both enzymes are characterized by specific structural features distinguishing them from pentaheme cytochrome c nitrite reductases, such as the Tyr-Cys bond in the active site, the hexameric structure resulting in the formation of a void space inside the hexamer, and the product channel that opens into the void interior space of the hexamer. It is suggested that these specific structural features are responsible for the higher nitrite reductase activity, the greater preference for nitrite than for sulfite as a substrate, and the wider pH range of the catalytic activity of octaheme nitrite reductases than of pentaheme homologs. DATABASE: Nucleotide sequence data are available in the GenBank database under the accession number HQ665012.1. Structural data are available in the RCSB Protein Data Bank database under the accession numbers 3SXQ and 3TTB STRUCTURED DIGITAL ABSTRACT: TvPaR and TvPaR bind by x-ray crystallography (View interaction).

Covalent modifications of the catalytic tyrosine in octahaem cytochrome c nitrite reductase and their effect on the enzyme activity.

Octahaem cytochrome c nitrite reductase from Thioalkalivibrio nitratireducens (TvNiR), like the previously characterized pentahaem nitrite reductases (NrfAs), catalyzes the six-electron reductions of nitrite to ammonia and of sulfite to sulfide. The active site of both TvNiR and NrfAs is formed by the lysine-coordinated haem and His, Tyr and Arg residues. The distinguishing structural feature of TvNiR is the presence of a covalent bond between the CE2 atom of the catalytic Tyr303 and the S atom of Cys305, which might be responsible for the higher nitrite reductase activity of TvNiR compared with NrfAs. In the present study, a new modified form of the enzyme (TvNiRb) that contains an additional covalent bond between Tyr303 CE1 and Gln360 CG is reported. Structures of TvNiRb in complexes with phosphate (1.45 Šresolution) and sulfite (1.8 Šresolution), the structure of TvNiR in a complex with nitrite (1.83 Šresolution) and several additional structures were determined. The formation of the second covalent bond by Tyr303 leads to a decrease in both the nitrite and sulfite reductase activities of the enzyme. Tyr303 is located at the exit from the putative proton-transport channel to the active site, which is absent in NrfAs. This is an additional argument in favour of the involvement of Tyr303 as a proton donor in catalysis. The changes in the activity of cytochrome c nitrite reductases owing to the formation of Tyr-Cys and Tyr-Gln bonds may be associated with changes in the pK(a) value of the catalytic tyrosine.

Structures of complexes of octahaem cytochrome c nitrite reductase from Thioalkalivibrio nitratireducens with sulfite and cyanide.

The structures of complexes of octahaem cytochrome c nitrite reductase from the bacterium Thioalkalivibrio nitratireducens (TvNiR) with the substrate sulfite (1.4 Å resolution; R(cryst) = 0.126) and the inhibitor cyanide (1.55 Å resolution; R(cryst) = 0.148) have been established. The complex with sulfite was prepared by the reduction of the protein crystal with sodium dithionite. The sulfite ion is bound to the iron ion of the catalytic haem through the S atom. The Fe-S distance is 2.24 Å. The structure of the cyanide complex with full occupancy of the ligand site was established for the first time for cytochrome c nitrite reductases. The cyanide ion is bound to the catalytic haem iron through the C atom. The Fe-C distance is 1.91 Å and the Fe-C-N angle is 171°. The sulfite reductase activity of TvNiR was measured at different pH values. The activity is 0.02 µmol of HS(-) per minute per milligram at pH 7.0; it decreases with increasing pH and is absent at pH 9.0.

High-resolution structural analysis of a novel octaheme cytochrome c nitrite reductase from the haloalkaliphilic bacterium Thioalkalivibrio nitratireducens.

Bacterial pentaheme cytochrome c nitrite reductases (NrfAs) are key enzymes involved in the terminal step of dissimilatory nitrite reduction of the nitrogen cycle. Their structure and functions are well studied. Recently, a novel octaheme cytochrome c nitrite reductase (TvNiR) has been isolated from the haloalkaliphilic bacterium Thioalkalivibrio nitratireducens. Here we present high-resolution crystal structures of the apoenzyme and its complexes with the substrate (nitrite) and the inhibitor (azide). Both in the crystalline state and in solution, TvNiR exists as a stable hexamer containing 48 hemes-the largest number of hemes accommodated within one protein molecule known to date. The subunit of TvNiR consists of two domains. The N-terminal domain has a unique fold and contains three hemes. The catalytic C-terminal domain hosts the remaining five hemes, their arrangement, including the catalytic heme, being identical to that found in NrfAs. The complete set of eight hemes forms a spatial pattern characteristic of other multiheme proteins, including structurally characterized octaheme cytochromes. The catalytic machinery of TvNiR resembles that of NrfAs. It comprises the lysine residue at the proximal position of the catalytic heme, the catalytic triad of tyrosine, histidine, and arginine at the distal side, channels for the substrate and product transport with a characteristic gradient of electrostatic potential, and, finally, two conserved Ca(2+)-binding sites. However, TvNiR has a number of special structural features, including a covalent bond between the catalytic tyrosine and the adjacent cysteine and the unusual topography of the product channels that open into the void interior space of the protein hexamer. The role of these characteristic structural features in the catalysis by this enzyme is discussed.

Thiocyanate hydrolase, the primary enzyme initiating thiocyanate degradation in the novel obligately chemolithoautotrophic halophilic sulfur-oxidizing bacterium Thiohalophilus thiocyanoxidans.

Thiohalophilus thiocyanoxidans is a first halophilic sulfur-oxidizing chemolithoautotrophic bacterium capable of growth with thiocyanate as an electron donor at salinity up to 4 M NaCl. The cells, grown with thiocyanate, but not with thiosulfate, contained an enzyme complex hydrolyzing thiocyanate to sulfide and ammonia under anaerobic conditions with carbonyl sulfide as an intermediate. Despite the fact of utilization of the <>, high cyanase activity was also detected in thiocyanate-induced cells. Three-stage column chromotography resulted in a highly purified thiocyanate-hydrolyzing protein with an apparent molecular mass of 140 kDa that consists of three subunits with masses 17, 19 and 29 kDa. The enzyme is a Co,Fe-containing protein resembling on its function and subunit composition the enzyme thiocyanate hydrolase from the Betaproteobacterium Thiobacillus thioparus. Cyanase, copurified with thiocyanate hydrolase, is a bisubstrate multisubunit enzyme with an apparent subunit molecular mass of 14 kDa. A possible role of cyanase in thiocyanate degradation by T. thiocyanoxidans is discussed.

Refined crystal structures of red and green fluorescent proteins from the button polyp Zoanthus.

The three-dimensional structures of the wild-type red (zRFP574) and green (zGFP506) fluorescent proteins (FP) from the button polyp Zoanthus have been determined at 1.51 and 2.2 A resolution, respectively. In addition, the crystal structures of a zGFP506 variant (zGFP506_N66D) with replacement of the first chromophore-forming residue (Asn66 to Asp) have been determined in the transitional 'green' and mature 'red' states at 2.4 and 2.2 A, respectively. The monomers of these proteins adopt the typical fold of the green fluorescent protein (GFP) family, consisting of an 11-stranded beta-barrel with a chromophore embedded in the middle of an internal alpha-helix directed along the beta-barrel axis. Post-translational modification of the chromophore-forming sequence Asn66-Tyr67-Gly68 within zGFP506 results in a typical GFP-like coplanar two-ring structure consisting of a five-membered imidazolinone heterocycle with the phenolic ring of Tyr67 in a cis orientation to the C(alpha)-N(67) bond. A novel post-translational modification of the chromophore-forming sequence Asp66-Tyr67-Gly68 in zRFP574 expands the protein maturation beyond the green-emitting form and results in decarboxylation of the Asp66 side chain. It is suggested that electrostatic conflict between the closely spaced negatively charged side chains of the chromophore Asp66 and the proximal catalytic Glu221 is most likely to be the trigger for the chain of reactions resulting in the observed decarboxylation. The chromophore structures of wild-type zGFP506 and of its mutant zGFP506_N66D in the 'green' and 'red' states support this suggestion. The beta-barrel frames of zRFP574 and zGFP506 reveal the presence of a water-filled pore leading to the chromophore Tyr67, similar to that observed previously in TurboGFP. An analysis of the residue composition at two inter-monomer interfaces in the tetrameric biological unit of zRFP574 and zGFP506, as well as of zYFP538 from the same species, has revealed a group of highly conserved residues that are apparently responsible for oligomerization. These residues present initial useful targets for rational mutagenesis aimed at designing monomeric forms of the fluorescent proteins, which are more suitable for practical applications.