Energy Generation from the CO Oxidation-Hydrogen Production Pathway in Rubrivivax gelatinosus

When incubated in the presence of CO gas, Rubrivivax gelatinosus CBS induces a CO oxidation-H₂ production pathway according to the stoichiometry CO + H₂O → CO₂ + H₂. Once induced, this pathway proceeds equally well in both light and darkness. When light is not present, CO can serve as the sole carbon source, supporting cell growth anaerobically with a cell doubling time of nearly 2 days. This observation suggests that the CO oxidation reaction yields energy. Indeed, new ATP synthesis was detected in darkness following CO additions to the gas phase of the culture, in contrast to the case for a control that received an inert gas such as argon. When the CO-to-H₂ activity was determined in the presence of the electron transport uncoupler carbonyl-cyanide m-chlorophenylhydrazone (CCCP), the rate of H₂ production from CO oxidation was enhanced nearly 40% compared to that of the control. Upon the addition of the ATP synthase inhibitor N,N′-dicyclohexylcarbodiimide (DCCD), we observed an inhibition of H₂ production from CO oxidation which could be reversed upon the addition of CCCP. Collectively, these data strongly suggest that the CO-to-H₂ reaction yields ATP driven by a transmembrane proton gradient, but the detailed mechanism of this reaction is not yet known. These findings encourage additional research aimed at long-term H₂ production from gas streams containing CO.     

Characterization of the oxygen tolerance of a hydrogenase linked to a carbon monoxide oxidation pathway in Rubrivivax gelatinosus

A hydrogenase linked to the carbon monoxide oxidation pathway in Rubrivivax gelatinosus displays tolerance to O2. When either whole-cell or membrane-free partially purified hydrogenase was stirred in full air (21% O2, 79% N2), its H2 evolution activity exhibited a half-life of 20 or 6 h, respectively, as determined by an anaerobic assay using reduced methyl viologen. When the partially purified hydrogenase was stirred in an atmosphere containing either 3.3 or 13% O2 for 15 min and evaluated by a hydrogen-deuterium (H-D) exchange assay, nearly 80 or 60% of its isotopic exchange rate was retained, respectively. When this enzyme suspension was subsequently returned to an anaerobic atmosphere, more than 90% of the H-D exchange activity was recovered, reflecting the reversibility of this hydrogenase toward O2 inactivation. Like most hydrogenases, the CO-linked hydrogenase was extremely sensitive to CO, with 50% inhibition occurring at 3.9 microM dissolved CO. Hydrogen production from the CO-linked hydrogenase was detected when ferredoxins of a prokaryotic source were the immediate electron mediator, provided they were photoreduced by spinach thylakoid membranes containing active water-splitting activity. Based on its appreciable tolerance to O2, potential applications of this hydrogenase are discussed.     

Characterization of the core complex of Rubrivivax gelatinosus in a mutant devoid of the LH2 antenna

The core complex of purple bacteria is a supramolecular assembly consisting of an array of light-harvesting LH1 antenna organized around the reaction center. It has been isolated and characterized in this work using a Rubrivivax gelatinosus mutant lacking the peripheral LH2 antenna. The purification did not modify the organization of the complex as shown by comparison with the intact membranes of the mutant. The protein components consisted exclusively of the reaction center, the associated tetraheme cyt c and the LH1 alphabeta subunits; no other protein which could play the role of pufX could be detected. The complex migrated as a single band in a sucrose gradient, and as a monomer in a native Blue gel electrophoresis. Comparison of its absorbance spectrum with those of the isolated RC and of the LH1 antenna as well as measurements of the bacteriochlorophyll/tetraheme cyt c ratio indicated that the mean number of LH1 subunits per RC-cyt c is near 16. The polypeptides of the LH1 antenna were shown to present several modifications. The alpha one was formylated at its N-terminal residue and the N-terminal methionine of beta was cleaved, as already observed for other Rubrivivax gelatinosus strains. Both modifications occurred possibly by post-translational processing. Furthermore the alpha polypeptides were heterogeneous, some of them having lost the 15 last residues of their C-terminus. This truncation of the hydrophobic C-terminal extension is similar to that observed previously for the alpha polypeptide of the Rubrivivax gelatinosus LH2 antenna and is probably due to proteolysis or to instability of this extension.     

Phototrophic growth of Rubrivivax gelatinosus in poultry slaughterhouse wastewater

Rubrivivax gelatinosus was grown in Pfennig's synthetic medium (PM) and in treated wastewater from poultry slaughterhouse (TW) to assess growth profiles for biomass production. Cultures inoculated at 1% (v/v) were grown under anaerobiosis at 30+/-2 degrees C and 1400+/-200 lux for 12 days. Regular absorbance curves for R. gelatinosus were found both on PM and TW. On PM, the highest dry weight of biomass, 0.39 gL(-1), was achieved in the 216-h culture and the highest specific growth rate of 0.2960 h(-1) occurred in the 24-h culture. On TW, the highest biomass of 0.57 gL(-1) was also obtained in the 216-h culture and the highest specific growth rate, 0.1970 h(-1), was achieved in the 48-h culture. For productivity and chemical oxygen demand investigations, the cultivation was accomplished in the TW under anaerobiosis at 32+/-2 degrees C and 4000+/-500 lux, for 10 days. Productivity was 0.085 g biomass (d.w.) L(-1) day(-1), with a COD decrease of 91%.     

Purification and Characterization of Alkaline Serine Proteinase from Photosynthetic Bacterium,Rubrivivax gelatinosus KDDS1

In order to reduce the protein content of wastewater, photosynthetic bacteria producing proteinases were screened from wastewater of various sources and stocked in culture. An isolated strain, KDDS1, was identified as Rubrivivax gelatinosus, a purple nonsulfur bacterium that secretes proteinase under micro-aerobic conditions under light at 35°C. Molecular weight of the purified enzyme was estimated to be 32.5 kDa. The enzyme showed the highest activity at 45°C and pH 9.6, and the activity was completely inhibited by phenylmethyl sulfonyl fluoride (PMSF), but not by EDTA. The amino-terminal 24 amino acid sequence of the enzyme showed about 50% identity to those of serine proteinases from Pseudoalteromonas piscicida strain O-7 and Burkholderia pseudomallei. Thus, the enzyme from Rvi. gelatinosus KDDS1 was thought to be a serine-type proteinase. This was the first serine proteinase characterized from photosynthetic bacteria.     

Energy generation from the CO oxidation-hydrogen production pathway in Rubrivivax gelatinosus

When incubated in the presence of CO gas, Rubrivivax gelatinosus CBS induces a CO oxidation-H2 production pathway according to the stoichiometry CO + H2O --> CO2 + H2. Once induced, this pathway proceeds equally well in both light and darkness. When light is not present, CO can serve as the sole carbon source, supporting cell growth anaerobically with a cell doubling time of nearly 2 days. This observation suggests that the CO oxidation reaction yields energy. Indeed, new ATP synthesis was detected in darkness following CO additions to the gas phase of the culture, in contrast to the case for a control that received an inert gas such as argon. When the CO-to-H2 activity was determined in the presence of the electron transport uncoupler carbonyl-cyanide m-chlorophenylhydrazone (CCCP), the rate of H2 production from CO oxidation was enhanced nearly 40% compared to that of the control. Upon the addition of the ATP synthase inhibitor N,N'-dicyclohexylcarbodiimide (DCCD), we observed an inhibition of H2 production from CO oxidation which could be reversed upon the addition of CCCP. Collectively, these data strongly suggest that the CO-to-H2 reaction yields ATP driven by a transmembrane proton gradient, but the detailed mechanism of this reaction is not yet known. These findings encourage additional research aimed at long-term H2 production from gas streams containing CO.     

Kinetic variations determine the product pattern of phytoene desaturase from Rubrivivax gelatinosus

In bacteria and fungi, the degree of carotenoid desaturation is determined by a single enzyme, the CrtI-type phytoene desaturase. In different organisms, this enzyme can carry out either three, four or even five desaturation steps. The purple bacterium Rubrivivax gelatinosus is the only known species in which reaction products of a 3-step and a 4-step desaturation (i.e. neurosporene and lycopene derivatives) accumulate simultaneously. The properties of this phytoene desaturation to catalyze neurosporene or lycopene were analyzed by heterologous complementations in Escherichia coli and by in vitro studies. They demonstrated that high enzyme concentrations or low phytoene supply favor the formation of lycopene. Under these conditions, CrtI from Rhodobacter spheroides can be forced in vitro to lycopene formation although this carotene is not synthesized in this species. All results can be explained by a model based on the competition between phytoene and neurosporene for the substrate binding site of phytoene desaturase. Mutations in CrtI from Rvi. gelatinosus have been generated resulting in increased lycopene formation in Escherichia coli. This modification in catalysis is due to increased amounts of CrtI protein.     

The Hydrogenase Activity of the Molybdenum/Copper-containing Carbon Monoxide Dehydrogenase of Oligotropha carboxidovorans

The reaction of the air-tolerant CO dehydrogenase from Oligotropha carboxidovorans with H₂ has been examined. Like the Ni-Fe CO dehydrogenase, the enzyme can be reduced by H₂ with a limiting rate constant of 5.3 s⁻¹ and a dissociation constant K_d of 525 μm; both k_red and k_red/K_d, reflecting the breakdown of the Michaelis complex and the reaction of free enzyme with free substrate in the low [S] regime, respectively, are largely pH-independent. During the reaction with H₂, a new EPR signal arising from the Mo/Cu-containing active site of the enzyme is observed which is distinct from the signal seen when the enzyme is reduced by CO, with greater g anisotropy and larger hyperfine coupling to the active site ^63,65Cu. The signal also exhibits hyperfine coupling to at least two solvent-exchangeable protons of bound substrate that are rapidly exchanged with solvent. Proton coupling is also evident in the EPR signal seen with the dithionite-reduced native enzyme, and this coupling is lost in the presence of bicarbonate. We attribute the coupled protons in the dithionite-reduced enzyme to coordinated water at the copper site in the native enzyme and conclude that bicarbonate is able to displace this water from the copper coordination sphere. On the basis of our results, a mechanism for H₂ oxidation is proposed which involves initial binding of H₂ to the copper of the binuclear center, displacing the bound water, followed by sequential deprotonation through a copper-hydride intermediate to reduce the binuclear center.     

Oxidation of Carbon Monoxide in Cell Extracts of Pseudomonas carboxydovorans

Extracts of aerobically, CO-autotrophically grown cells of Pseudomonas carboxydovorans were shown to catalyze the oxidation of CO to CO(2) in the presence of methylene blue, pyocyanine, thionine, phenazine methosulfate, or toluylene blue under strictly anaerobic conditions. Viologen dyes and NAD(P)(+) were ineffective as electron acceptors. The same extracts catalyzed the oxidation of formate and of hydrogen gas; the spectrum of electron acceptors was identical for the three substrates, CO, formate, and H(2). The CO- and the formate-oxidizing activities were found to be soluble enzymes, whereas hydrogenase was membrane bound exclusively. The rates of oxidation of CO, formate, and H(2) were measured spectrophotometrically following the reduction of methylene blue. The rate of carbon monoxide oxidation followed simple Michaelis-Menten kinetics; the apparent K(m) for CO was 45 muM. The reaction rate was maximal at pH 7.0, and the temperature dependence followed the Arrhenius equation with an activation energy (DeltaH(0)) of 35.9 kJ/mol (8.6 kcal/mol). Neither free formate nor hydrogen gas is an intermediate of the CO oxidation reaction. This conclusion is based on the differential sensitivity of the activities of formate dehydrogenase, hydrogenase, and CO dehydrogenase to heat, hypophosphite, chlorate, cyanide, azide, and fluoride as well as on the failure to trap free formate or hydrogen gas in coupled optical assays. These results support the following equation for CO oxidation in P. carboxydovorans: CO + H(2)O --> CO(2) + 2 H(+) + 2e(-) The CO-oxidizing activity of P. carboxydovorans differed from that of Clostridium pasteurianum by not reducing viologen dyes and by a pH optimum curve that did not show an inflection point.     

Two-dimensional structure of the native light-harvesting complex LH2 from Rubrivivax gelatinosus and of a truncated form.

The light-harvesting complex LH2 of Rubrivivax gelatinosus has an oligomeric structure built from alpha-beta heterodimers containing three bacteriochlorophylls and one carotenoid each. The alpha subunit (71 residues) presents a C-terminal hydrophobic extension (residues 51-71) which is prone to attack by an endogenous protease. This extension can also be cleaved by a mild thermolysin treatment, as demonstrated by electrophoresis and by matrix-assisted laser desorption-time of flight mass spectrometry. This cleavage does not affect the pigment binding sites as shown by absorption spectroscopy. Electron microscopy was used to investigate the structures of the native and thermolysin cleaved forms of the complexes. Two-dimensional crystals of the reconstituted complexes were examined after negative staining and cryomicroscopy. Projection maps at 10 A resolution were calculated, demonstrating the nonameric ring-like organization of alpha-beta subunits. The cleaved form presents the same structural features. We conclude that the LH2 complex is structurally homologous to the Rhodopseudomonas acidophila LH2. The hydrophobic C-terminal extension does not fold back in the membrane, but lays out on the periplasmic surface of the complex.     

Photooxidative stress stimulates illegitimate recombination and mutability in carotenoid-less mutants of Rubrivivax gelatinosus.

Carotenoids are essential to protection against photooxidative damage in photosynthetic and non-photosynthetic organisms. In a previous study, we reported the disruption of crtD and crtC carotenoid genes in the purple bacterium Rubrivivax gelatinosus, resulting in mutants that synthesized carotenoid intermediates. Here, carotenoid-less mutants have been constructed by disruption of the crtB gene. To study the biological role of carotenoids in photoprotection, the wild-type and the three carotenoid mutants were grown under different conditions. When exposed to photooxidative stress, only the carotenoid-less strains (crtB-) gave rise with a high frequency to four classes of mutants. In the first class, carotenoid biosynthesis was partially restored. The second class corresponded to photosynthetic-deficient mutants. The third class corresponded to mutants in which the LHI antenna level was decreased. In the fourth class, synthesis of the photosynthetic apparatus was inhibited only in aerobiosis. Molecular analyses indicated that the oxidative stress induced mutations and illegitimate recombination. Illegitimate recombination events produced either functional or non-functional chimeric genes. The R. gelatinosus crtB- strain could be very useful for studies of the SOS response and of illegitimate recombination induced by oxidants in bacteria.     

Structural and kinetics properties of a mutated phytoene desaturase from Rubrivivax gelatinosus with modified product specificity

The phytoene desaturase CrtI from Rubrivivax gelatinosus catalyzes simultaneously a three- and four-step desaturation producing both neurosporene and lycopene. These carotenes are intermediates for the synthesis of spheroidene and spirilloxanthin, respectively. Two different mutation libraries for the crtI gene from R. gelatinosus were constructed to screen for modified enzymes which synthesize almost exclusively either neurosporene or lycopene. The resulting mutants carried between one and four amino acid exchanges and at least one of them affected the secondary protein structure by shortening or extending one of the helices. A prominent amino acid which was exchanged in the neurosporene or lycopene-forming desaturase was leucine 208. Enzyme kinetic studies were carried out with the L208 modified desaturase and the specificities for phytoene and neurosporene as substrates determined. Higher and lower values correlate well with the higher or lower potential for the synthesis of lycopene from neurosporene. TopPred analysis of the mutations of L208 indicated that the location is in a highly hydrophobic membrane-integrated region which is a good candidate for the substrate-binding site of the desaturase.     

HiPIP in Rubrivivax gelatinosus is firmly associated to the membrane in a conformation efficient for electron transfer towards the photosynthetic reaction centre

High potential iron-sulfur protein (HiPIP), a small soluble redox protein, has been shown to serve in vivo as electron donor to the photosynthetic reaction centre (RC) in Rubrivivax gelatinosus [Biochemistry 34 (1995) 11736]. The results of time-resolved optical spectroscopy on membrane-fragments from this organism indicates that the photooxidized RC is re-reduced by HiPIP even in the absence of the soluble fraction. This implies that a significant fraction of HiPIP can firmly bind to the membrane in a conformation able to interact with the RCs. Salt treatment of the membrane-fragments abolishes these re-reduction kinetics, demonstrating the presence of HiPIP on the membrane due to association with the RC rather than due to simple trapping in hypothetical chromatophores. The existence of such a functional complex in membranes is confirmed and its structure further examined by electron paramagnetic resonance (EPR) performed on membrane-fragments. Orientation-dependent EPR spectra of HiPIP were recorded on partially ordered membranes, oxidized either chemically or photochemically. Whereas hardly any preferential orientation of the HiPIP was seen in the chemically oxidised sample, a subpopulation of HiPIP showing specific orientations could be photooxidised. This fraction arises from the electron transfer complex between HiPIP and the RC.     

Carbon Monoxide Abrogates Ischemic Insult to Neuronal Cells via the Soluble Guanylate Cyclase-cGMP Pathway

Purpose

Carbon monoxide (CO) is an accepted cytoprotective molecule. The extent and mechanisms of protection in neuronal systems have not been well studied. We hypothesized that delivery of CO via a novel releasing molecule (CORM) would impart neuroprotection in vivo against ischemia-reperfusion injury (IRI)-induced apoptosis of retinal ganglion cells (RGC) and in vitro of neuronal SH-SY5Y-cells via activation of soluble guanylate-cyclase (sGC).

Methods

To mimic ischemic respiratory arrest, SH-SY5Y-cells were incubated with rotenone (100 nmol/L, 4 h) ± CORM ALF186 (10–100 µmol/L) or inactivated ALF186 lacking the potential of releasing CO. Apoptosis and reactive oxygen species (ROS) production were analyzed using flow-cytometry (Annexin V, mitochondrial membrane potential, CM-H₂DCFDA) and Western blot (Caspase-3). The impact of ALF186± respiratory arrest on cell signaling was assessed by measuring expression of nitric oxide synthase (NOS) and soluble guanylate-cyclase (sGC) and by analyzing cellular cGMP levels. The effect of ALF186 (10 mg/kg iv) on retinal IRI in Sprague-Dawley rats was assessed by measuring densities of fluorogold-labeled RGC after IRI and by analysis of apoptosis-related genes in retinal tissue.

Results

ALF186 but not inactivated ALF186 inhibited rotenone-induced apoptosis (Annexin V positive cells: 25±2% rotenone vs. 14±1% ALF186+rotenone, p<0.001; relative mitochondrial membrane potential: 17±4% rotenone vs. 55±3% ALF186+rotenone, p<0.05). ALF186 increased cellular cGMP levels (33±5 nmol/L vs. 23±3 nmol/L; p<0.05) and sGC expression. sGC-inhibition attenuated ALF186-mediated protection (relative mitochondrial membrane potential: 55±3% ALF186+rotenone vs. 20±1% ODQ+ALF186+rotenone, p<0.05). ALF186 protected RGC in vivo (IRI 1255±327 RGC/mm² vs. ALF186+IRI 2036±83; p<0.05) while sGC inhibition abolished the protective effects of ALF186 (ALF186+IRI 2036±83 RGC/mm² vs. NS-2028+ALF186+IRI 1263±170, p<0.05).

Conclusions

The CORM ALF186 inhibits IRI-induced neuronal cell death via activation of sGC and may be a useful treatment option for acute ischemic insults to the retina and the brain.     

Interaction site for high-potential iron-sulfur protein on the tetraheme cytochrome subunit bound to the photosynthetic reaction center of Rubrivivax gelatinosus

We have recently demonstrated, using site-directed mutagenesis, that soluble cytochromes interact with the Rubrivivax gelatinosus photosynthetic reaction center (RC) in the vicinity of the low-potential heme 1 (c-551, Em = 70 mV) of the tetraheme cytochrome subunit, the fourth heme from the special pair of bacteriochlorophyll [Osyczka, A., et al. (1998) Biochemistry 37, 11732-11744]. Although the mutations generated in that study did not show clear effects on the electron transfer from high-potential iron-sulfur protein (HiPIP), which is the major physiological electron donor to the RC in this bacterium, we report here that other site-directed mutations near the solvent-exposed edge of the same low-potential heme 1, V67K (valine-67 substituted by lysine) and E79K/E85K/E93K (glutamates-79, -85, and -93, all replaced by lysines), considerably inhibit the electron transfer from HiPIP to the RC. Thus, it is concluded that HiPIP, like soluble cytochromes, binds to the RC in the vicinity of the exposed part of the low-potential heme 1 of the cytochrome subunit, although some differences in the configurations of the HiPIP-RC and cytochrome c-RC transient complexes may be postulated.     

Fluorescence life-times and aggregation states of the core light harvesting complex B875 from Rubrivivax gelatinosus

The core light-harvesting complex B875 isolated from the purple bacterium Rubrivivax gelatinosus and its different spectral forms B820 and B840, which are depleted of carotenoid, were investigated by steady-state and time-resolved fluorescence, and by electron microscopy. Images of B875 have been shown to contain cyclic oligomers with a diameter of 150–200 Å and with a central hole of 25 Å [Jirsakova V, Reiss-Husson F and Ranck JL (1996) Biochim Biophys Acta 1277: 150–160]. Dilute B820 samples contained heterogeneous, compact particles that tend to aggregate with increasing concentration of protein, forming clumps without any visible substructure. At the same time the absorption maximum of such aggregates shifted to 840 nm. Fluorescence emission and life times were analyzed by single photon counting. In B875 samples the major component emitted at 892 nm with a life time of 0.64 ns. B820 samples emitted at 830 nm with a life-time of 1 ns. An additional short life-time component of 0.3–0.4 ns was found in B820 and emitted at about 860 nm; its contribution increased with the B820 concentration. This latter component is attributed to the fluorescence quenching occuring within the non-native aggregates of B820 formed in the absence of carotenoid. When the B875 antenna was reconstituted from B820 subunit and hydroxyspheroidene, it presented an emission spectrum and a fluorescence decay identical to those observed in the native core complex, pointing to the structural role of the carotenoid for the proper architecture of this antenna.