Hydrogen bonding rearrangement by a mitochondrial disease mutation in cytochrome bc1 perturbs heme bH redox potential and spin state.

Hemes are common elements of biological redox cofactor chains involved in rapid electron transfer. While the redox properties of hemes and the stability of the spin state are recognized as key determinants of their function, understanding the molecular basis of control of these properties is challenging. Here, benefiting from the effects of one mitochondrial disease-related point mutation in cytochrome b, we identify a dual role of hydrogen bonding (H-bond) to the propionate group of heme bH of cytochrome bc1, a common component of energy-conserving systems. We found that replacing conserved glycine with serine in the vicinity of heme bH caused stabilization of this bond, which not only increased the redox potential of the heme but also induced structural and energetic changes in interactions between Fe ion and axial histidine ligands. The latter led to a reversible spin conversion of the oxidized Fe from 1/2 to 5/2, an effect that potentially reduces the electron transfer rate between the heme and its redox partners. We thus propose that H-bond to the propionate group and heme-protein packing contribute to the fine-tuning of the redox potential of heme and maintaining its proper spin state. A subtle balance is needed between these two contributions: While increasing the H-bond stability raises the heme potential, the extent of increase must be limited to maintain the low spin and diamagnetic form of heme. This principle might apply to other native heme proteins and can be exploited in engineering of artificial heme-containing protein maquettes.

Changing the Electron Acceptor Specificity of Rhodobacter capsulatus Formate Dehydrogenase from NAD+ to NADP.

Formate dehydrogenases catalyze the reversible oxidation of formate to carbon dioxide. These enzymes play an important role in CO2 reduction and serve as nicotinamide cofactor recycling enzymes. More recently, the CO2-reducing activity of formate dehydrogenases, especially metal-containing formate dehydrogenases, has been further explored for efficient atmospheric CO2 capture. Here, we investigate the nicotinamide binding site of formate dehydrogenase from Rhodobacter capsulatus for its specificity toward NAD+ vs. NADP+ reduction. Starting from the NAD+-specific wild-type RcFDH, key residues were exchanged to enable NADP+ binding on the basis of the NAD+-bound cryo-EM structure (PDB-ID: 6TG9). It has been observed that the lysine at position 157 (Lys157) in the ß-subunit of the enzyme is essential for the binding of NAD+. RcFDH variants that had Glu259 exchanged for either a positively charged or uncharged amino acid had additional activity with NADP+. The FdsBL279R and FdsBK276A variants also showed activity with NADP+. Kinetic parameters for all the variants were determined and tested for activity in CO2 reduction. The variants were able to reduce CO2 using NADPH as an electron donor in a coupled assay with phosphite dehydrogenase (PTDH), which regenerates NADPH. This makes the enzyme suitable for applications where it can be coupled with other enzymes that use NADPH.

The cbb3-type cytochrome oxidase assembly factor CcoG is a widely distributed cupric reductase.

Copper (Cu)-containing proteins execute essential functions in prokaryotic and eukaryotic cells, but their biogenesis is challenged by high Cu toxicity and the preferential presence of Cu(II) under aerobic conditions, while Cu(I) is the preferred substrate for Cu chaperones and Cu-transport proteins. These proteins form a coordinated network that prevents Cu accumulation, which would lead to toxic effects such as Fenton-like reactions and mismetalation of other metalloproteins. Simultaneously, Cu-transport proteins and Cu chaperones sustain Cu(I) supply for cuproprotein biogenesis and are therefore essential for the biogenesis of Cu-containing proteins. In eukaryotes, Cu(I) is supplied for import and trafficking by cell-surface exposed metalloreductases, but specific cupric reductases have not been identified in bacteria. It was generally assumed that the reducing environment of the bacterial cytoplasm would suffice to provide sufficient Cu(I) for detoxification and cuproprotein synthesis. Here, we identify the proposed cbb3-type cytochrome c oxidase (cbb3-Cox) assembly factor CcoG as a cupric reductase that binds Cu via conserved cysteine motifs and contains 2 low-potential [4Fe-4S] clusters required for Cu(II) reduction. Deletion of ccoG or mutation of the cysteine residues results in defective cbb3-Cox assembly and Cu sensitivity. Furthermore, anaerobically purified CcoG catalyzes Cu(II) but not Fe(III) reduction in vitro using an artificial electron donor. Thus, CcoG is a bacterial cupric reductase and a founding member of a widespread class of enzymes that generate Cu(I) in the bacterial cytosol by using [4Fe-4S] clusters.

The CckA-ChpT-CtrA Phosphorelay Controlling Rhodobacter capsulatus Gene Transfer Agent Production Is Bidirectional and Regulated by Cyclic di-GMP.

Protein phosphorylation is a universal mechanism for transducing cellular signals in prokaryotes and eukaryotes. The histidine kinase CckA, the histidine phosphotransferase ChpT, and the response regulator CtrA are conserved throughout the alphaproteobacteria. In Rhodobacter capsulatus, these proteins are key regulators of the gene transfer agent (RcGTA), which is present in several alphaproteobacteria. Using purified recombinant R. capsulatus proteins, we show in vitro autophosphorylation of CckA protein, and phosphotransfer to ChpT and thence to CtrA, to demonstrate biochemically that they form a phosphorelay. The secondary messenger cyclic di-GMP changed CckA from a kinase to a phosphatase, resulting in reversal of the phosphotransfer flow in the relay. The substitutions of two residues in CckA greatly affected the kinase or phosphatase activity of the protein in vitro, and production of mutant CckA proteins in vivo confirmed the importance of kinase but not phosphatase activity for the lytic release of RcGTA. However, phosphatase activity was needed to produce functional RcGTA particles. The binding of cyclic di-GMP to the wild-type and mutant CckA proteins was evaluated directly using a pulldown assay based on biotinylated cyclic di-GMP and streptavidin-linked beads.IMPORTANCE The CckA, ChpT, and CtrA phosphorelay proteins are widespread in the alphaproteobacteria, and there are two groups of organisms that differ in terms of whether this pathway is essential for cell viability. Little is known about the biochemical function of these proteins in organisms where the pathway is not essential, a group that includes Rhodobacter capsulatus This work demonstrates biochemically that CckA, ChpT, and CtrA also form a functional phosphorelay in the latter group and that the direction of phosphotransfer is reversed by cyclic di-GMP. It is important to improve understanding of more representatives of this pathway in order to obtain deeper insight into the function, composition, and evolutionary significance of a wider range of bacterial regulatory networks.

Repressor Activity of SqrR, a Master Regulator of Persulfide-Responsive Genes, Is Regulated by Heme Coordination.

Reactive sulfur species (RSS) are involved in bioactive regulation via persulfidation of proteins. However, how cells regulate RSS-based signaling and RSS metabolism is poorly understood, despite the importance of universal regulation systems in biology. We previously showed that the persulfide-responsive transcriptional factor SqrR acts as a master regulator of sulfide-dependent photosynthesis in proteobacteria. Here, we demonstrated that SqrR also binds heme at a near one-to-one ratio with a binding constant similar to other heme-binding proteins. Heme does not change the DNA-binding pattern of SqrR to the target gene promoter region; however, DNA-binding affinity of SqrR is reduced by the binding of heme, altering its regulatory activity. Circular dichroism spectroscopy clearly showed secondary structural changes in SqrR by the heme binding. Incremental change in the intracellular heme concentration is associated with small, but significant reduction in the transcriptional repression by SqrR. Overall, these results indicate that SqrR has an ability to bind heme to modulate its DNA-binding activity, which may be important for the precise regulation of RSS metabolism in vivo.

Mg-protoporphyrin IX monomethyl ester cyclase from Rhodobacter capsulatus: radical SAM-dependent synthesis of the isocyclic ring of bacteriochlorophylls.

During bacteriochlorophyll a biosynthesis, the oxygen-independent conversion of Mg-protoporphyrin IX monomethyl ester (Mg-PME) to protochlorophyllide (Pchlide) is catalyzed by the anaerobic Mg-PME cyclase termed BchE. Bioinformatics analyses in combination with pigment studies of cobalamin-requiring Rhodobacter capsulatus mutants indicated an unusual radical S-adenosylmethionine (SAM) and cobalamin-dependent BchE catalysis. However, in vitro biosynthesis of the isocyclic ring moiety of bacteriochlorophyll using purified recombinant BchE has never been demonstrated. We established a spectroscopic in vitro activity assay which was subsequently validated by HPLC analyses and H218O isotope label transfer onto the carbonyl-group (C-131-oxo) of the isocyclic ring of Pchlide. The reaction product was further converted to chlorophyllide in the presence of light-dependent Pchlide reductase. BchE activity was stimulated by increasing concentrations of NADPH or SAM, and inhibited by S-adenosylhomocysteine. Subcellular fractionation experiments revealed that membrane-localized BchE requires an additional, heat-sensitive cytosolic component for activity. BchE catalysis was not sustained in chimeric experiments when a cytosolic extract from E. coli was used as a substitute. Size-fractionation of the soluble R. capsulatus fraction indicated that enzymatic activity relies on a specific component with an estimated molecular mass between 3 and 10 kDa. A structure guided site-directed mutagenesis approach was performed on the basis of a three-dimensional homology model of BchE. A newly established in vivo complementation assay was used to investigate 24 BchE mutant proteins. Potential ligands of the [4Fe-4S] cluster (Cys204, Cys208, Cys211), of SAM (Phe210, Glu308 and Lys320) and of the proposed cobalamin cofactor (Asp248, Glu249, Leu29, Thr71, Val97) were identified.

Regulation of stringent factor by branched-chain amino acids.

When faced with amino acid starvation, prokaryotic cells induce a stringent response that modulates their physiology. The stringent response is manifested by production of signaling molecules guanosine 5'-diphosphate,3'-diphosphate (ppGpp) and guanosine 5'-triphosphate,3'-diphosphate (pppGpp) that are also called alarmones. In many species, alarmone levels are regulated by a multidomain bifunctional alarmone synthetase/hydrolase called Rel. In this enzyme, there is an ACT domain at the carboxyl region that has an unknown function; however, similar ACT domains are present in other enzymes that have roles in controlling amino acid metabolism. In many cases, these other ACT domains have been shown to allosterically regulate enzyme activity through the binding of amino acids. Here, we show that the ACT domain present in the Rhodobacter capsulatus Rel alarmone synthetase/hydrolase binds branched-chain amino acids valine and isoleucine. We further show that the binding of these amino acids stimulates alarmone hydrolase activity both in vitro and in vivo. Furthermore, we found that the ACT domain present in Rel proteins from many diverse species also binds branched-chain amino acids. These results indicate that the cellular concentration of amino acids can directly affect Rel alarmone synthetase/hydrolase activity, thus adding another layer of control to current models of cellular control of the stringent response.

Cyclic di-GMP-Mediated Regulation of Gene Transfer and Motility in Rhodobacter capsulatus.

Gene transfer agents (GTAs) are bacteriophage-like particles produced by several bacterial and archaeal lineages that contain small pieces of the producing cells' genomes that can be transferred to other cells in a process similar to transduction. One well-studied GTA is RcGTA, produced by the alphaproteobacterium Rhodobacter capsulatus RcGTA gene expression is regulated by several cellular regulatory systems, including the CckA-ChpT-CtrA phosphorelay. The transcription of multiple other regulator-encoding genes is affected by the response regulator CtrA, including genes encoding putative enzymes involved in the synthesis and hydrolysis of the second messenger bis-(3'-5')-cyclic dimeric GMP (c-di-GMP). To investigate whether c-di-GMP signaling plays a role in RcGTA production, we disrupted the CtrA-affected genes potentially involved in this process. We found that disruption of four of these genes affected RcGTA gene expression and production. We performed site-directed mutagenesis of key catalytic residues in the GGDEF and EAL domains responsible for diguanylate cyclase (DGC) and c-di-GMP phosphodiesterase (PDE) activities and analyzed the functions of the wild-type and mutant proteins. We also measured RcGTA production in R. capsulatus strains where intracellular levels of c-di-GMP were altered by the expression of either a heterologous DGC or a heterologous PDE. This adds c-di-GMP signaling to the collection of cellular regulatory systems controlling gene transfer in this bacterium. Furthermore, the heterologous gene expression and the four gene disruptions had similar effects on R. capsulatus flagellar motility as found for gene transfer, and we conclude that c-di-GMP inhibits both RcGTA production and flagellar motility in R. capsulatusIMPORTANCE Gene transfer agents (GTAs) are virus-like particles that move cellular DNA between cells. In the alphaproteobacterium Rhodobacter capsulatus, GTA production is affected by the activities of multiple cellular regulatory systems, to which we have now added signaling via the second messenger dinucleotide molecule bis-(3'-5')-cyclic dimeric GMP (c-di-GMP). Similar to the CtrA phosphorelay, c-di-GMP also affects R. capsulatus flagellar motility in addition to GTA production, with lower levels of intracellular c-di-GMP favoring increased flagellar motility and gene transfer. These findings further illustrate the interconnection of GTA production with global systems of regulation in R. capsulatus, providing additional support for the notion that the production of GTAs has been maintained in this and related bacteria because it provides a benefit to the producing organisms.

The Cu chaperone CopZ is required for Cu homeostasis in Rhodobacter capsulatus and influences cytochrome cbb3 oxidase assembly.

Cu homeostasis depends on a tightly regulated network of proteins that transport or sequester Cu, preventing the accumulation of this toxic metal while sustaining Cu supply for cuproproteins. In Rhodobacter capsulatus, Cu-detoxification and Cu delivery for cytochrome c oxidase (cbb3 -Cox) assembly depend on two distinct Cu-exporting P1B -type ATPases. The low-affinity CopA is suggested to export excess Cu and the high-affinity CcoI feeds Cu into a periplasmic Cu relay system required for cbb3 -Cox biogenesis. In most organisms, CopA-like ATPases receive Cu for export from small Cu chaperones like CopZ. However, whether these chaperones are also involved in Cu export via CcoI-like ATPases is unknown. Here we identified a CopZ-like chaperone in R. capsulatus, determined its cellular concentration and its Cu binding activity. Our data demonstrate that CopZ has a strong propensity to form redox-sensitive dimers via two conserved cysteine residues. A ΔcopZ strain, like a ΔcopA strain, is Cu-sensitive and accumulates intracellular Cu. In the absence of CopZ, cbb3 -Cox activity is reduced, suggesting that CopZ not only supplies Cu to P1B -type ATPases for detoxification but also for cuproprotein assembly via CcoI. This finding was further supported by the identification of a ~150 kDa CcoI-CopZ protein complex in native R. capsulatus membranes.

Sulfide-responsive transcriptional repressor SqrR functions as a master regulator of sulfide-dependent photosynthesis.

Sulfide was used as an electron donor early in the evolution of photosynthesis, with many extant photosynthetic bacteria still capable of using sulfur compounds such as hydrogen sulfide (H2S) as a photosynthetic electron donor. Although enzymes involved in H2S oxidation have been characterized, mechanisms of regulation of sulfide-dependent photosynthesis have not been elucidated. In this study, we have identified a sulfide-responsive transcriptional repressor, SqrR, that functions as a master regulator of sulfide-dependent gene expression in the purple photosynthetic bacterium Rhodobacter capsulatus SqrR has three cysteine residues, two of which, C41 and C107, are conserved in SqrR homologs from other bacteria. Analysis with liquid chromatography coupled with an electrospray-interface tandem-mass spectrometer reveals that SqrR forms an intramolecular tetrasulfide bond between C41 and C107 when incubated with the sulfur donor glutathione persulfide. SqrR is oxidized in sulfide-stressed cells, and tetrasulfide-cross-linked SqrR binds more weakly to a target promoter relative to unmodified SqrR. C41S and C107S R. capsulatus SqrRs lack the ability to respond to sulfide, and constitutively repress target gene expression in cells. These results establish that SqrR is a sensor of H2S-derived reactive sulfur species that maintain sulfide homeostasis in this photosynthetic bacterium and reveal the mechanism of sulfide-dependent transcriptional derepression of genes involved in sulfide metabolism.

Time-dependent enhancement of fluorescence from Rhodobacter capsulatus SB1003 and its critical dependence on concentration temperature and static magnetic field.

Continuous exposure of 395 nm light increases the fluorescence emission intensity of photosynthetic purple non-sulphur bacteria, Rhodobacter capsulatus (SB1003). We show that such an increase in fluorescence emission of extracellular pigment complexes (PC) from these photosynthetic bacteria depends on the concentration of the pigment and temperature and can also be modulated by the static magnetic field. The time-dependent enhanced emission disappears either at or below 300 K or below a threshold sample concentration (0.1 mg/ml). The enhanced emission reappears at this condition (T < 278 K) if a static magnetic field (395 mT) is introduced during fluorescence measurement. The time dependence of emission is expressed in terms of a first-order rate constant, k = dF/(Fdt). The sign of k shifts from positive to negative as PC concentration is lowered than a threshold value, implying onset of fluorescence decay (k < 0) rather than amplification (k > 0). At PC concentration higher than a threshold, k becomes negative if the temperature is lowered. But, surprisingly, at low temperature, a static magnetic field reverts the k value to positive. We explain the logical nature of k-switching and photo-dynamics of the aforesaid microbial fluorescence emission by aggregation of protoporphyrin rings present in the PC. While the simultaneous presence of decay in fluorescence and susceptibility to static magnetic field suggests the dominance of triplet states at low temperatures, the process is reversed by SMF-induced removal of spin degeneracy. At higher temperatures, the optical excitability and lack of magnetic response suggest the dominance of singlet states. We propose that the restructuring of the singlet-triplet distribution by intersystem crossing may be the basis of this logical behaviour. In context with microbial function, time-dependent enhancement of fluorescence also implies relay of red photons to the neighbouring microbes not directly exposed to the incident radiation, thus serving as an indirect photosynthetic regulator.

Characterization of a Glycyl Radical Enzyme Bacterial Microcompartment Pathway in Rhodobacter capsulatus.

Bacterial microcompartments (BMCs) are large (∼100-nm) protein shells that encapsulate enzymes, their substrates, and cofactors for the purposes of increasing metabolic reaction efficiency and protecting cells from toxic intermediates. The best-studied microcompartment is the carbon-fixing carboxysome that encapsulates ribulose-1,5-bisphosphate carboxylase and carbonic anhydrase. Other well-known BMCs include the Pdu and Eut BMCs, which metabolize 1,2-propanediol and ethanolamine, respectively, with vitamin B12-dependent diol dehydratase enzymes. Recent bioinformatic analyses identified a new prevalent type of BMC, hypothesized to utilize vitamin B12-independent glycyl radical enzymes to metabolize substrates. Here we use genetic and metabolic analyses to undertake in vivo characterization of the newly identified glycyl radical enzyme microcompartment 3 (GRM3) class of microcompartment clusters. Transcriptome sequencing analyses showed that the microcompartment gene cluster in the genome of the purple photosynthetic bacterium Rhodobacter capsulatus was expressed under dark anaerobic respiratory conditions in the presence of 1,2-propanediol. High-performance liquid chromatography and gas chromatography-mass spectrometry analyses showed that enzymes coded by this cluster metabolized 1,2-propanediol into propionaldehyde, propanol, and propionate. Surprisingly, the microcompartment pathway did not protect these cells from toxic propionaldehyde under the conditions used in this study, with buildup of this intermediate contributing to arrest of cell growth. We further show that expression of microcompartment genes is regulated by a two-component system located downstream of the microcompartment cluster.IMPORTANCE BMCs are protein shells that are designed to compartmentalize enzymatic reactions that require either sequestration of a substrate or the sequestration of toxic intermediates. Due to their ability to compartmentalize reactions, BMCs have also become attractive targets for bioengineering novel enzymatic reactions. Despite these useful features, little is known about the biochemistry of newly identified classes of BMCs. In this study, we have undertaken genetic and in vivo metabolic analyses of the newly identified GRM3 gene cluster.

Induction of Rhodobacter capsulatus Gene Transfer Agent Gene Expression Is a Bistable Stochastic Process Repressed by an Extracellular Calcium-Binding RTX Protein Homologue.

Bacteriophage-like gene transfer agents (GTAs) have been discovered in both of the prokaryotic branches of the three-domain phylogenetic tree of life. The production of a GTA (RcGTA) by the phototrophic alphaproteobacterium Rhodobacter capsulatus is regulated by quorum sensing and a phosphorelay homologous to systems in other species that control essential functions such as the initiation of chromosome replication and cell division. In wild-type strains, RcGTA is produced in <3% of cells in laboratory cultures. Mutants of R. capsulatus that exhibit greatly elevated production of RcGTA were created decades ago by chemical mutagenesis, but the nature and molecular consequences of the mutation were unknown. We show that the number of cells in a population that go on to express RcGTA genes is controlled by a stochastic process, in contrast to a genetic process. We used transposon mutagenesis along with a fluorescent protein reporter system and genome sequence data to identify a gene, rcc00280, that encodes an RTX family calcium-binding protein homologue. The Rc280 protein acts as an extracellular repressor of RcGTA gene expression by decreasing the percentage of cells that induce the production of RcGTA.IMPORTANCE GTAs catalyze horizontal gene transfer (HGT), which is important for genomic evolution because the majority of genes found in bacterial genomes have undergone HGT at some point in their evolution. Therefore, it is important to determine how the production of GTAs is regulated to understand the factors that modulate the frequency of gene transfer and thereby specify the tempo of evolution. This work describes a new type of genetic regulation in which an extracellular calcium-binding protein homologue represses the induction of the Rhodobacter capsulatus GTA, RcGTA.

Transcriptome analysis of Rhodobacter capsulatus grown on different nitrogen sources.

This study investigated the effect of different nitrogen sources, namely, ammonium chloride and glutamate, on photoheterotrophic metabolism of Rhodobacter capsulatus grown on acetate as the carbon source. Genes that were significantly differentially expressed according to Affymetrix microarray data were categorized into Clusters of Orthologous Groups functional categories and those in acetate assimilation, hydrogen production, and photosynthetic electron transport pathways were analyzed in detail. Genes related to hydrogen production metabolism were significantly downregulated in cultures grown on ammonium chloride when compared to those grown on glutamate. In contrast, photosynthetic electron transport and acetate assimilation pathway genes were upregulated. In detail, aceA encoding isocitrate lyase, a unique enzyme of the glyoxylate cycle and ccrA encoding the rate limiting crotonyl-CoA carboxylase/reductase enzyme of ethylmalonyl-coA pathway were significantly upregulated. Our findings indicate for the first time that R. capsulatus can operate both glyoxylate and ethylmalonyl-coA cycles for acetate assimilation.

Tuning purple bacteria salt-tolerance for photobioelectrochemical systems in saline environments.

The development of photobioelectrochemical systems is an exciting field requiring a combination of electrochemical, biological and material science knowledge. One of the main advantages of applying anoxygenic photosynthetic microorganisms versus non-photosynthetic bacteria is the possibility to utilize sunlight as the energy source, while removing organic contaminants from a solution. Since bacterial cells utilize energy to maintain the intracellular osmolarity, bacterial species that do not rely on organic species as an energy source have an advantage over species requiring them for their sustainment. Herein, we discuss the possible use of Rhodobacter capsulatus, an extremely versatile photosynthetic purple bacteria, for application in environments within a range of low to moderately high salinity (0-25 g L-1 NaCl). Bacterial cells' capability to adapt to changing salinity, and effects on bioelectrochemical performance will be presented, as well as major drawbacks and research needs to drive future efforts and discussions.

Functional flexibility of electron flow between quinol oxidation Qo site of cytochrome bc1 and cytochrome c revealed by combinatory effects of mutations in cytochrome b, iron-sulfur protein and cytochrome c1.

Transfer of electron from quinol to cytochrome c is an integral part of catalytic cycle of cytochrome bc1. It is a multi-step reaction involving: i) electron transfer from quinol bound at the catalytic Qo site to the Rieske iron-sulfur ([2Fe-2S]) cluster, ii) large-scale movement of a domain containing [2Fe-2S] cluster (ISP-HD) towards cytochrome c1, iii) reduction of cytochrome c1 by reduced [2Fe-2S] cluster, iv) reduction of cytochrome c by cytochrome c1. In this work, to examine this multi-step reaction we introduced various types of barriers for electron transfer within the chain of [2Fe-2S] cluster, cytochrome c1 and cytochrome c. The barriers included: impediment in the motion of ISP-HD, uphill electron transfer from [2Fe-2S] cluster to heme c1 of cytochrome c1, and impediment in the catalytic quinol oxidation. The barriers were introduced separately or in various combinations and their effects on enzymatic activity of cytochrome bc1 were compared. This analysis revealed significant degree of functional flexibility allowing the cofactor chains to accommodate certain structural and/or redox potential changes without losing overall electron and proton transfers capabilities. In some cases inhibitory effects compensated one another to improve/restore the function. The results support an equilibrium model in which a random oscillation of ISP-HD between the Qo site and cytochrome c1 helps maintaining redox equilibrium between all cofactors of the chain. We propose a new concept in which independence of the dynamics of the Qo site substrate and the motion of ISP-HD is one of the elements supporting this equilibrium and also is a potential factor limiting the overall catalytic rate.

Inhibition of bacteriochlorophyll biosynthesis in the purple phototrophic bacteria Rhodospirillumrubrum and Rhodobacter capsulatus grown in the presence of a toxic concentration of selenite.

Background In many works, the chemical composition of bacterially-produced elemental selenium nanoparticles (Se0-nanoparticles) was investigated using electron dispersive X-ray analysis. The results suggest that these particles should be associated with organic compounds. However, a complete analysis of their chemical composition is still missing. Aiming at identifying organic compounds associated with the Se0-nanoparticles produced by the purple phototrophic bacteria Rhodospirillum rubrum and Rhodobacter capsulatus (α group of the proteobacteria), we used MALDI-TOF spectrometry.Results This technic revealed that numerous signals obtained from particles produced by both species of bacteria were from metabolites of the photosynthetic system. Furthermore, not only bacteriochlorophyll a, bacteriopheophytin a, and bacteriopheophorbide a, which are known to accumulate in stationary phase cultures of these bacteria grown phototrophically in the absence of selenite, were identified. The particles were also associated with intermediary metabolites of the bacteriochlorophyll a biosynthesis pathway such as protoporphyrin IX, protoporphyrin IX monomethyl ester, bacteriochlorophyllide a and, most likely, Mg-protoporphyrin IX-monomethyl ester, as well as with oxidation products of the substrates of protochlorophyllide reductase and chlorin reductase.Conclusion Accumulation of intermediary metabolites of the bacteriochlorophyll biosynthesis pathway in these purple phototrophic bacteria was attributed to inhibition of oxygen-sensitive enzymes involved in this pathway. Consistent with this interpretation it has been reported that these bacteria reduce selenite intracellularly, that they contain high levels of glutathione and that the reduction of selenite with glutathione is a very fast reaction accompanied by the production of reactive oxygen species. As many enzymes involved in the biosynthesis of bacteriochlorophyll contain [Fe-S] clusters in their active site, which are known to be degraded in the presence of reactive oxygen species as well as in the presence of molecular oxygen, we concluded that the substrates of these enzymes accumulate in cells during selenite reduction.Association of metabolites of bacteriochlorophyll biosynthesis and degradation with the Se0-nanoparticles produced by Rhodospirillum rubrum and Rhodobacter capsulatus is proposed to result from coating of the nanoparticles with the intracytoplasmic membrane of these bacteria, where the photochemical apparatus is concentrated.

Bacterial PerO Permeases Transport Sulfate and Related Oxyanions.

Rhodobacter capsulatus synthesizes the high-affinity ABC transporters CysTWA and ModABC to specifically import the chemically related oxyanions sulfate and molybdate, respectively. In addition, R. capsulatus has the low-affinity permease PerO acting as a general oxyanion transporter, whose elimination increases tolerance to molybdate and tungstate. Although PerO-like permeases are widespread in bacteria, their function has not been examined in any other species to date. Here, we present evidence that PerO permeases from the alphaproteobacteria Agrobacterium tumefaciens, Dinoroseobacter shibae, Rhodobacter sphaeroides, and Sinorhizobium meliloti and the gammaproteobacterium Pseudomonas stutzeri functionally substitute for R. capsulatus PerO in sulfate uptake and sulfate-dependent growth, as shown by assimilation of radioactively labeled sulfate and heterologous complementation. Disruption of perO genes in A. tumefaciens, R. sphaeroides, and S. meliloti increased tolerance to tungstate and, in the case of R. sphaeroides, to molybdate, suggesting that heterometal oxyanions are common substrates of PerO permeases. This study supports the view that bacterial PerO permeases typically transport sulfate and related oxyanions and, hence, form a functionally conserved permease family.IMPORTANCE Despite the widespread distribution of PerO-like permeases in bacteria, our knowledge about PerO function until now was limited to one species, Rhodobacter capsulatus In this study, we showed that PerO proteins from diverse bacteria are functionally similar to the R. capsulatus prototype, suggesting that PerO permeases form a conserved family whose members transport sulfate and related oxyanions.

Complex Stability During the Transport Cycle of a Subclass I ECF Transporter.

The mechanism of energy-coupling factor (ECF) transporters, a special type of ATP-binding-cassette importers for micronutrients in prokaryotes, is a matter of controversial discussion. Among subclass II ECF transporters, a single ECF interacts with several substrate-binding integral membrane proteins (S units) for individual solutes. Release and catch of the S unit, previously observed experimentally for a subclass II system, was proposed as the mechanism of all ECF transporters. The BioM2NY biotin transporter is a prototype of subclass I systems, among which the S unit is dedicated to a specific ECF. Here we simulated the transport cycle using purified BioM2NY in detergent solution. BioM2NY complexes were stable during all steps. ATP binding was a prerequisite for biotin capture and ATP hydrolysis for subsequent biotin release. The data demonstrate that S units of subclass I ECF transporters do not have to dissociate from holotransporter complexes for high-affinity substrate binding, indicating mechanistic differences between the two subclasses.

Identifying involvement of Lys251/Asp252 pair in electron transfer and associated proton transfer at the quinone reduction site of Rhodobacter capsulatus cytochrome bc1.

Describing dynamics of proton transfers in proteins is challenging, but crucial for understanding processes which use them for biological functions. In cytochrome bc1, one of the key enzymes of respiration or photosynthesis, proton transfers engage in oxidation of quinol (QH2) and reduction of quinone (Q) taking place at two distinct catalytic sites. Here we evaluated by site-directed mutagenesis the contribution of Lys251/Asp252 pair (bacterial numbering) in electron transfers and associated with it proton uptake to the quinone reduction site (Qi site). We showed that the absence of protonable group at position 251 or 252 significantly changes the equilibrium levels of electronic reactions including the Qi-site mediated oxidation of heme bH, reverse reduction of heme bH by quinol and heme bH/Qi semiquinone equilibrium. This implicates the role of H-bonding network in binding of quinone/semiquinone and defining thermodynamic properties of Q/SQ/QH2 triad. The Lys251/Asp252 proton path is disabled only when both protonable groups are removed. With just one protonable residue from this pair, the entrance of protons to the catalytic site is sustained, albeit at lower rates, indicating that protons can travel through parallel routes, possibly involving water molecules. This shows that proton paths display engineering tolerance for change as long as all the elements available for functional cooperation secure efficient proton delivery to the catalytic site.