Photoinactivation of the detoxifying enzyme nitrophenol reductase from Rhodobacter capsulatus

The phototrophic bacterium Rhodobacter capsulatus E1F1 detoxifies 2,4-dinitrophenol by inducing an NAD(P)H-dependent iron flavoprotein that reduces this compound to the less toxic end product 2-amino-4-nitrophenol. This nitrophenol reductase was stable in crude extracts containing carotenes, but it became rapidly inactivated when purified protein was exposed to intense white light or moderate blue light intensities, especially in the presence of exogenous flavins. Red light irradiation had no effect on nitrophenol reductase activity. Photoinactivation of the enzyme was irreversible and increased under anoxic conditions. This photoinactivation was prevented by reductants such as NAD(P)H and EDTA and by the excited flavin quencher iodide. Addition of superoxide dismutase, catalase, tryptophan or histidine did not affect photoinactivation of nitrophenol reductase, thus excluding these reactive dioxygen species as the inactivating agent. Substantial protection by 2,4-dinitrophenol also took place when the enzyme was irradiated at a wavelength coinciding with one of the absorption peaks of this compound (365nm). These results suggest that the lability of nitrophenol reductase was due to the absorption of blue light by the flavin prosthetic group, thus producing an excited flavin that might irreversibly oxidize some functional group(s) necessary for enzyme catalysis. Nitrophenol reductase may be preserved in vivo from blue light photoinactivation by the high content of carotenes and excess of reducing equivalents in phototrophic growing cells.Abbreviations 2,4-DNP 2,4-dinitrophenol - ANP 2-amino-4-nitrophenol - EDTA ethylenediamine tetraacetic acid - MES 2-(N-Morpholino) ethanesulfonic acid - NPR nitrophenol reductase     

Binding protein dependent transport of C4-dicarboxylates in Rhodobacter capsulatus

The characteristics of malate transport into aerobically grown cells of the purple photosynthetic bacterium Rhodobacter capsulatus were determined. A single transport system was distinguished kinetically which displayed a K_t value of 2.9 ± 1.2 μM and V_max of 43 ± 6 nmol · min^-1 · mg^-1 protein. Competition experiments indicated that the metabolically related C4-dicarboxylates succinate and fumarate are also transported by this system. Malate uptake was sensitive to osmotic shock and evidence from the binding of radiolabelled malate and succinate to periplasmic protein fractions indicated that transport is mediated by a dicarboxylate binding protein. The activity of the transport system was studied as a function of external and internal pH and it was found that a marked activation of uptake occurred at intracellular pH values greater than 7. The use of a high affinity binding protein dependent system to transport a major carbon and energy source suggests that Rhodobacter capsulatus would be capable of obtaining growth sustaining quantities of C4-dicarboxylates even if these were present at very low concentrations in the environment.     

Nitrate reductase activity in spheroplasts from Rhodobacter capsulatus E1F1 requires a periplasmic protein

Spheroplasts from Rhodobacter capsulatus E1F1 cells grown in nitrate maintained nitrate uptake and nitrate reductase activity only when they were illuminated under anaerobiosis in the presence of the periplasmic fraction and nitrate. The effects on nitrate uptake and nitrate reductase activity of spheroplasts were observed at low concentrations of periplasmic protein (about 50 x ml^-1). Periplasm from nitrate-grown cells was also required for nitrate reductase activity in spheroplasts isolated from ammonia-grown or diazotrophic cells which initially lacked this enzymatic activity. Both the maintenance of nitrate reductase in spheroplasts from nitrate-grown cells and the appearance of the activity in spheroplasts from diazotrophic cells were dependent on de novo protein synthesis. A periplasmic, 45-kDa protein which maintained the activity of nitrate reductase in spheroplasts was partially purified by gel filtration chromatography of periplasm obtained from nitrate-grown cells.Abbreviations NR nitrate reductase - CCCP carbonyl cyanide m-chlorophenylhydrazone - CAM chloramphenicol     

Inhibition of aconitase and fumarase by nitrogen compounds in Rhodobacter capsulatus

High levels of aconitase and fumarase activities were found in Rhodobacter capsulatus E1F1 cells cultured with nitrate as the sole nitrogen source either under light-anaerobic or dark-aerobic conditions. Both activities were strongly and reversibly inhibited in vitro by nitrite or nitric oxide, whereas nitrate or hydroxylamine showed a lower effect. Other enzymes of the tricarboxylic acids cycle such as malate dehydrogenase or isocitrate dehydrogenase were not affected by these nitrogen compounds. When growing on nitrate in the dark R. capsulatus E1F1 cells accumulated nitrite intracellularly, so that an in vivo inhibition of aconitase and fumarase could account for the strong inhibition of growth observed in the presence of nitrite under dark-aerobic conditions.Abbreviations ACO aconitase - FUM fumarase - MDH malate dehydrogenase - ICDH isocitrate dehydrogenase - TCA tricarboxylic acid     

Tellurite uptake by cells of the facultative phototroph Rhodobacter capsulatus is a Delta pH-dependent process

The uptake by light-grown cells of Rhodobacter capsulatus of the highly toxic metalloid oxyanion tellurite (TeO(3)(2-)) was examined. We show that tellurite is rapidly taken up by illuminated cells in a process which is inhibited by the protonophore carbonyl cyanide-p-trifluoromethoxyphenyl-hydrazone (FCCP) and by the K(+)/H(+) exchanger nigericin. Notably, the light-driven membrane potential (Delta psi) is enhanced by K(2)TeO(3)> or =200 microM. Further, tellurite uptake is largely insensitive to valinomycin, strongly repressed by the sulfhydryl reagent N-ethylethylmaleimide (NEM) and competitively inhibited by phosphate. We conclude that tellurite is transported into cells by a Delta pH-dependent, non-electrogenic process which is likely to involve the phosphate transporter (PiT family).     

Characterization of the interaction of Rhodobacter capsulatus cytochrome c peroxidase with charge reversal mutants of cytochrome c(2)

Steady-state kinetics for the reaction of Rhodobacter capsulatus bacterial cytochrome c peroxidase (BCCP) with its substrate cytochrome c(2) were investigated. The Rb. capsulatus BCCP is dependent on calcium for activation as previously shown for the Pseudomonas aeruginosa BCCP and Paracoccus denitrificans enzymes. Furthermore, the activity shows a bell-shaped pH dependence with optimum at pH 7.0. Enzyme activity is greatest at low ionic strength and drops off steeply as ionic strength increases, resulting in an apparent interaction domain charge product of -13. All cytochromes c(2) show an asymmetric distribution of surface charge, with a concentration of 14 positive charges near the exposed heme edge of Rb. capsulatus c(2) which potentially may interact with approximately 6 negative charges, localized near the edge of the high-potential heme of the Rb. capsulatus BCCP. To test this proposal, we constructed charge reversal mutants of the 14 positively charged residues located on the front face of Rb. capsulatus cytochrome c(2) and examined their effect on steady-state kinetics with BCCP. Mutated residues in Rb. capsulatus cytochrome c(2) that showed the greatest effects on binding and enzyme activity are K12E, K14E, K54E, K84E, K93E, and K99E, which is consistent with the site of electron transfer being located at the heme edge. We conclude that a combination of long-range, nonspecific electrostatic interactions as well as localized salt bridges between, e.g., cytochrome c(2) K12, K14, K54, and K99 with BCCP D194, D241, and D6, account for the observed kinetics.     

Protein dynamics: imidazole and 2-mercaptoethanol binding to the Rhodobacter capsulatus cytochrome c(2) mutant,glycine 95 proline

The Class I c-type cytochromes can bind exogenous ligands in the oxidized state, with the kinetics of ligand binding providing information on naturally occurring intramolecular dynamics. Typically, nitrogenous bases are used as ligands; however, it is less well known that 2-mercaptoethanol (BME), a commonly used cytochrome reducing agent, can form a complex with the heme. To better understand the cytochrome-mercaptan interaction, we have investigated the kinetics of binding of BME to wild type and mutants of Rhodobacter capsulatus cytochrome c(2) and to horse cytochrome c. Complex formation with the G95P mutant is apparent from the formation of a green color and a shift in the Soret peak to 418 nm from 410 nm upon addition of BME. Unlike horse cytochrome c and wild-type R. capsulatus cytochrome c(2), G95P permits the kinetics of formation of the BME-G95P complex to be measured since complex formation and reduction kinetics can be resolved. The affinity constant for the binding of BME to mutant G95P was strong ( approximately 1.5 x 10(5)M(-1)) and the kinetics of formation of the BME-G95P complex were found to undergo a change in rate-limiting step consistent with a concentration-independent protein rearrangement (68s(-1)) followed by second-order binding of BME ( approximately approximately 1.3 x 10(5)M(-1)s(-1)). The most remarkable characteristic of mutant G95P is the relatively large amount of high-spin species in equilibrium with the low- spin form, which can be estimated to be approximately 3% at pH 7. The BME binding kinetics, coupled with the kinetics of imidazole binding to G95P, allow us, for the first time, to specify all four rate constants describing the ligand binding reaction. Moreover, we can use the kinetic results to estimate the rate constants for ligand binding with the wild-type cytochrome c(2). This has also allowed us to quantify and more fully interpret cytochrome dynamics.     

Rhodobacter capsulatus strain BK5 possesses a membrane bound respiratory nitrate reductase rather than the periplasmic enzyme found in other strains

Rhodobacter capsulatus strain BK5 possesses a membrane bound respiratory nitrate reductase rather than the periplasmic enzyme found in other strains. The enzyme in strain BK5 is shown to be both functionally and structurally related to the nitrate reductase of Paracoccus denitrificans and Escherichia coli.Abbreviation TMAO trimethylamine-N-oxide     

Pore forming activity of the major outer membrane protein of Rhodobacter capsulatus in lipid bilayer membranes

Porin of the outer membrane of Rhodobacter capsulatus St. Louis (ATCC 23782) was isolated and reconstituted into lipid bilayer membranes. The porin was obtained either by the sodium dodecyl sulfate treatment of cell envelopes (SDS-porin) or by saline extraction of whole cells (NaCl-porin). Nanomolar concentrations of both porin preparations resulted in a strong conductance increase of the lipid bilayer membranes by many orders of magnitude. At small protein concentrations the conductance increased in a stepwise fashion, the average single channel conductance being about 0.35 nS in 0.1 M KCl for SDS-porin and NaCl-porin as well. The single channel conductance was a linear function of the specific conductance of the aqueous phase. The results were consistent with the assumption that the porin formed large water-filled transmembrane channels in the membrane. From the average value of the single channel conductance in 0.1 M KCl an effective channel diameter of about 1.5 nm was estimated for both types of porins.Abbreviations EDTA ethylenediamine tetraacetic acid - SDS sodium dodecyl sulfate     

Activity and expression of nitrogenase in Rhodobacter capsulatus under aerobiosis in the dark and in the light

Rhodobacter capsulatus was grown chemotrophically in the dark in oxygen-regulated chemostat culture and in the presence of limiting amounts of fixed N. When the oxygen partial pressure was varied, in situ nitrogen fixation occurred only at 1% of air saturation of the medium. By contrast, nitrogenase proteins and their activity measured in the absence of oxygen could be detected up to 30% of air saturation. This revealed that expression of nitrogenase is much less sensitive toward oxygen than the in situ function of the enzyme. At oxygen partial pressures > 1% of air saturation, the degree of modification of the Fe protein of nitrogenase was increased. Light was of no stimulatory effect on both the activity and the expression of nitrogenase. This holds true for growth at 1% or 5% of air saturation. At 5% of air saturation, however, high illumination enhanced the inhibitory effect of oxygen on nitrogenase formation.     

The regulation of cytochrome c oxidase of Rhodobacter capsulatus by light and oxygen

In order to distinguish between the regulatory effects of oxygen tension and light intensity on cytochrome c oxidase protein and enzymatic activity cells of Rhodobacter capsulatus were shifted from phototrophic (anaerobic, light) growth to aerobic-light, aerobic-dark and to anaerobic-dark conditions, respectively. During shift-experiments the formation of oxidase protein and regulation of oxidase activity was followed by immunological and enzymatic means. The results support the idea, that the formation of oxidase protein is regulated by oxygen tension and light intensity changes, whereas the regulation of oxidase activity seems only to be correlated to the oxygen tension. A DNA sequence involved in the oxygen-dependent regulation of cytochrome oxidase could be identified in the regulation-deficient oxidase mutant H41 of R. capsulatus. Immunological investigations of cytochrome c₂ from mutant H41 demonstrated at the same time the participation of the c₂-polypeptide in the regulation of cytochrome c oxidase.Abbreviations Bchl bacteriochlorophyll - CIE crossed immuno-electrophoresis - DMSO dimethyl sulfoxide     

Control of nitrogenase in chemostat cultures of Rhodobacter capsulatus grown on ammonium at different illuminations

Rhodobacter capsulatus strain 37b4 was grown phototrophically in chemostat cultures with 2 mM of ammonium chloride and 30 mM of malate at a constant dilution rate of 0.075 h^-1. When illumination was raised from 3000 to 30000 lx, steady state biomass levels as well as malate uptake increased linearly with increasing illumination. Yet, in no case external ammonium could be detected in the culture fluid. Specific nitrogenase activity increased by a factor of ten between 3000 and 15000 lx and approached constancy above 15 000 lx. When samples were anaerobically withdrawn from the chemostat and subsequently grown in batch cultures under saturating light conditions, biomass increased to a constant level, independently of the illumination used in the previous chemostat culture. In fact, the specific nitrogen contents of cells were 0.195 and 0.154 (g of N per g of protein) with chemostat cultures adapted to 3000 and 30000 lx, respectively. With the former cultures, specific nitrogen contents decreased to 0.142 g of nitrogen per g of cell protein upon incubation in a batch system. This suggests the existence of free nitrogen compounds in cells of chemostat cultures, the concentrations of which decrease while protein levels increase with increasing energy supply. Intracellular amino acid pools revealed slightly elevated levels of major amino acids in low-light cultures as compared to high-light cultures. On the basis of intracellular levels of ammonium, however, no significant differences could be detected. Since, in addition, malate consumption increased linearly with increasing illumination, it is proposed that light controls nitrogenase in Rhodobacter capsulatus via the C/N ratio, as represented by malate and ammonium consumption, rather than directly.     

Capsule polysaccharide-protein-peptidoglycan complex in the cell envelope of Rhodobacter capsulatus

The capsule polysaccharide-protein-peptidoglycan complex (insoluble in boiling sodium dodecyl sulfate and hot phenol-water) from cell envelopes of Rhodobacter capsulatus St. Louis was characterized. Hydrofluoric, hydrochloric acid or alkaline hydrolysis solubilized the polysaccharide moiety, whereas the protein-peptidoglycan moiety remained insoluble. On treatment of the protein-peptidoglycan moiety with lysozyme, the protein with peptidoglycan-residues bound was solubilized. It showed a single, broad peptide band (M _r=about 17,000) on sodium dodecyl sulfate polyacrylamide gel-electrophoresis. The same protein was obtained by lysozyme digestion (without preceding hydrofluoric or hydrochloric acid treatment) of the protein-peptidoglycan complex of the phage-resistant mutant Rhodobacter capsulatus St. Louis RC1^-, in which the capsule polysaccharide is present in a free form. A protein-peptidoglycan complex was isolated also from the capsulefree Rhodobacter capsulatus 37b4. Covalent binding between the protein and peptidoglycan moieties is likely for all three strains as is the lipoprotein nature of the protein moiety. The polysaccharide moiety of the complete complex from the wild-type Rhodobacter capsulatus St. Louis was at least partly removable from the complex in the presence of high salt concentrations or ethylene diamine tetraacetate. A specific amino acid pattern (with Ser, Gly, Glu, and Ala dominating) remained constantly associated with the capsule polysaccharide moiety independent of the separation procedure.Abbreviations A₂pm diaminopimelic acid - Cetavlon cetyltrimethyl-ammonium bromide - EDTA ethylene-diaminetetraacetate, disodium salt - HF hydrofluoric acid - HPLC high-performance liquid chromatography - PAGL polyacrylamide gel-electrophoresis - SDS sodium dodecyl sulfate - TCA trichloroacetic acid     

Recovery of the alternative oxidase dependent electron flow by fusion of membrane vesicles from Rhodobacter capsulatus mutant strains

Membranes from the facultative photosynthetic bacterium Rhodobacter capsulatus can be fused by sonication in vitro to form hybrid membranes, some of which are vesicles. The organization of the redox complexes involved in alternative-oxidase dependent electron transport of R. capsulatus can be studied by testing for in vitro complementation of this activity lacking in membranes from genetic mutants. The results obtained in complementation tests between well characterized mutants support current models of organization of electron transport components in R. capsulatus and confirm the in situ existence of independent functional entities such as NADH-dehydrogenase, reaction center, bacteriochlorophyll light-harvesting complexes and alternative-oxidase (cyt. o). The demonstration that artefactual mixing between different membrane entities may only be induced by treatments such as ultrasonic irradiation promoting intervesicular recombination has been taken as evidence that photorespiratory processes truly reflect an in vivo situation.     

Phosphotransfer in Rhodobacter sphaeroides chemotaxis

The two-component sensing system controlling bacterial chemotaxis is one of the best studied in biology. Rhodobacter sphaeroides has a complex chemosensory pathway comprising two histidine protein kinases (CheAs) and eight downstream response regulators (six CheYs and two CheBs) rather than the single copies of each as in Escherichia coli. We used in vitro analysis of phosphotransfer to start to determine why R.sphaeroides has these multiple homologues. CheA(1) and CheA(2) contain all the key motifs identified in the histidine protein kinase family, except for conservative substitutions (F-L and F-I) within the F box of CheA(2), and both are capable of ATP-dependent autophosphorylation. While the K(m) values for ATP of CheA(1) and CheA(2) were similar to that of E.coli, the k(cat) value was three times lower, but similar to that measured for the related Sinorhizobium meliloti CheA. However, the two CheAs differed both in their ability to phosphorylate the various response regulators and the rates of phosphotransfer. CheA(2) phosphorylated all of the CheYs and both CheBs, whilst CheA(1) did not phosphorylate either CheB and phosphorylated only the response regulators encoded within its own genetic locus (CheY(1), CheY(2), and CheY(5)) and CheY(3). The dephosphorylation rates of the R.sphaeroides CheBs were much slower than the E.coli CheB. The dephosphorylation rate of CheY(6), encoded by the third chemosensory locus, was ten times faster than that of the E.coli CheY. However, the dephosphorylation rates of the remaining R.sphaeroides CheYs were comparable to that of E.coli CheY.     

Two conserved non-canonical histidines are essential for activity of the cbb 3-type oxidase in Rhodobacter capsulatus

Cytochrome cbb ₃ oxidase, a member of the heme–copper oxidase superfamily, catalyses the reduction of oxygen to water and generates a proton gradient. Cytochrome c oxidases are characterized by a catalytic subunit (subunit I) containing two hemes and one copper ion ligated by six invariant histidine residues, which are diagnostic of heme–copper oxidases in all type of the heme–copper oxidase superfamily. Alignments of the amino acid sequences of subunit I (FixN or CcoN) of the cbb ₃-type oxidases show that catalytic subunit also contains six non-canonical histidine residues that are conserved in all CcoN subunits of the cbb ₃ oxidase, but not the catalytic subunits of other members of heme–copper oxidases superfamily. The function of these six CcoN-specific conserved histidines of cbb ₃-type oxidase in R. capsulatus is unknown. To analyze the contribution of the two invariant histidines of CcoN, H300 and H394, in activity and assembly of the Rhodobacter capsulatus cbb ₃-type oxidase, they were substituted for valine and alanine, respectively by site-directed mutagenesis. H300V and H394A mutations were analyzed with respect to their activity and assembly. It was found that H394A mutation led to a defect in the assembly of both CcoP and CcoO in the membrane, which results in almost complete loss of activity and that although the H300V mutant is normally assembled in the membrane and retain their stability, its catalytic activity is significantly reduced when compared with wild-type oxidase.     

The development of the photosynthetic apparatus and energy transduction in malate-limited phototrophic cultures of Rhodobacter capsulatus

The question was studied whether limited availability of the carbon source controls the development of the photosynthetic apparatus in Rhodobacter capsulatus. The organisms were grown phototrophically in a chemostat limited by malate as the sole source of reducing equivalents and carbon. The incident light-energy flux, representing the only energy source, was kept constant. Steady state levels of protein and dry weight of cells as well as molar growth yield coefficients (Y) decreased with increasing dilution rate (D, representing the growth rate, ) up to about D=0.14 h^-1. At higher D-values biomass levels as well as Y stayed largely constant. The specific rate of malate consumption leading to biomass production increased linearly while the rate representative of processes other than conversion of carbon into biomass increased almost exponentially with . Specific bacteriochlorophyll (Bchl) contents of cells as well as the specific rate of Bchl synthesis were rather low at low D-values. They increased as D was increased. Light energy fluxes required to half-maximally saturate proton extrusion by whole cells decreased when D was increased up to 0.1 h^-1; at higher D-values, however, they reached constancy. Maximal rates of proton extrusion as well as of photophosphorylation calculated on a Bchl basis decreased when D was increased up to 0.14 h^-1 and reached constancy at higher D-values. The results suggest that the availability of the growth limiting substrate controls the formation of the photosynthetic apparatus and, consequently, its functional properties including the efficiency of light-energy transduction. A relationship is assumed between malate conversion into biomass, i.e. Y-values, and the efficiency of light-energy transduction.Abbreviations ALA 5-aminoleyulinic acid - Bchl bacteriochlorophyll - D dilution rate [h^-1] - R Rhodobacter - Y molar growth yield coefficient - growth rate [h^-1]     

The relationship of biomass,polysaccharide and H2 formation in the wild-type and nifA/nifB mutants of Rhodobacter capsulatus

The production of biomass, polysaccharide storage material and H₂ from malate was studied in the wild-type and mutants RdcI, RdcII and RdcI/cII of Rhodobacter capsulatus. The mutants are defective in either copy I, copy II or both copies of the nitrogenase genes nifA and nifB. Stationary phase levels of biomass, polysaccharide and H₂ were determined in phototrophic batch cultures grown with 30 mM of d,l-malate and either 2, 5, or 8 mM of ammonium or 7 mM of glutamate. Calculation of the amounts of malate converted into the three products revealed that, at 8 mM of ammonium and 7 mM of glutamate, malate consumption and product formation were balanced. But with decreasing ammonium concentrations malate not converted into biomass was utilized with decreasing efficiency in polysaccharide and H₂ formation. This suggests formation of unknown products at the lower ammonium concentrations. Under conditions of optimal N supply, 80% of the malate not used for biomass production was converted by the wild-type and strain RdcII to H₂ and CO₂. Mutant RdcI exhibited slightly decreased H₂ production. The double mutant did not evolve H₂ but accumulated increased amounts of polysaccharide. However, the amounts of polysaccharide were lower than should be expected if all of the spare malate, not utilized by the double mutant for H₂ production, was converted into storage material. This and incomplete conversion of malate into known products at low ammonium supplies suggests that polysaccharide accumulation does not compete with the process of H₂ formation for malate.