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Kiseok Yoon - One of the best experts on this subject based on the ideXlab platform.
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Spectroscopic and functional properties of novel 2[4Fe-4S] cluster-containing ferredoxins from the green sulfur bacterium Chlorobium tepidum.
The Journal of biological chemistry, 2001Co-Authors: Kiseok Yoon, Russ Hille, Craig Hemann, Cedric E. Bobst, F. Robert TabitaAbstract:Abstract Two distinct ferredoxins, Fd I and Fd II, were isolated and purified to homogeneity from photoautotrophically grown Chlorobium tepidum, a moderately thermophilic green sulfur bacterium that assimilates carbon dioxide by the reductive tricarboxylic acid cycle. Both ferredoxins serve a crucial role as electron donors for reductive carboxylation, catalyzed by a key enzyme of this pathway, Pyruvate Synthase/Pyruvate ferredoxin oxidoreductase. The reduction potentials of Fd I and Fd II were determined by cyclic voltammetry to be −514 and −584 mV, respectively, which are more electronegative than any previously studied Fds in which two [4Fe-4S] clusters display a single transition. Further spectroscopic studies indicated that the CD spectrum of oxidized Fd I closely resembled that of Fd II; however, both spectra appeared to be unique relative to ferredoxins studied previously. Double integration of the EPR signal of the two Fds yielded approximately ∼2.0 spins per molecule, compatible with the idea thatC. tepidum Fd I and Fd II accept 2 electrons upon reduction. These results suggest that the C. tepidum Fd I and Fd II polypeptides each contain two bound [4Fe-4S] clusters.C. tepidum Fd I and Fd II are novel 2[4Fe-4S] Fds, which were shown previously to function as biological electron donors or acceptors for C. tepidum Pyruvate Synthase/Pyruvate ferredoxin oxidoreductase (Yoon, K.-S., Hille, R., Hemann, C. F., and Tabita, F. R. (1999) J. Biol. Chem. 274, 29772–29778). Kinetic measurements indicated that Fd I had ∼2.3-fold higher affinity than Fd II. The results of amino acid sequence alignments, molecular modeling, oxidation-reduction potentials, and spectral properties strongly indicate that the C. tepidumFds are chimeras of both clostridial-type and chromatium-type Fds, suggesting that the two Fds are likely intermediates in the evolutional development of 2[4Fe-4S] clusters compared with the well described clostridial and chromatium types.
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rubredoxin from the green sulfur bacterium chlorobium tepidum functions as an electron acceptor for Pyruvate ferredoxin oxidoreductase
Journal of Biological Chemistry, 1999Co-Authors: Kiseok Yoon, Russ Hille, Craig Hemann, Robert F TabitaAbstract:Abstract Rubredoxin (Rd) from the moderately thermophilic green sulfur bacterium Chlorobium tepidum was found to function as an electron acceptor for Pyruvate ferredoxin oxidoreductase (PFOR). This enzyme, which catalyzes the conversion of Pyruvate to acetyl-CoA and CO2, exhibited an absolute dependence upon the presence of Rd. However, Rd was incapable of participating in the Pyruvate Synthase or CO2 fixation reaction of C. tepidum PFOR, for which two different reduced ferredoxins are employed as electron donors. These results suggest a specific functional role for Rd in Pyruvate oxidation and provide the initial indication that the two important physiological reactions catalyzed by PFOR/Pyruvate Synthase are dependent on different electron carriers in the cell. The UV-visible spectrum of oxidized Rd, with a monomer molecular weight of 6500, gave a molar absorption coefficient at 492 nm of 6.89 mm −1 cm−1 with anA 492/A 280 ratio of 0.343 and contained one iron atom/molecule. Further spectroscopic studies indicated that the CD spectrum of oxidized C. tepidum Rd exhibited a unique absorption maximum at 385 nm and a shoulder at 420 nm. The EPR spectrum of oxidized Rd also exhibited unusual anisotropic resonances at g = 9.675 and g = 4.322, which is composed of a narrow central feature with broader shoulders to high and low field. The midpoint reduction potential of C. tepidum Rd was determined to be −87 mV, which is the most electronegative value reported for Rd from any source.
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Reductive TCA cycle in an aerobic bacterium, Hydrogenobacter thermophilus strain TK-6
Studies in Surface Science and Catalysis, 1998Co-Authors: Masaharu Ishii, Kiseok Yoon, Yasufumi Ueda, Toshihiro Ochiai, Nare Yun, Seiichi Takishita, Tohru Kodama, Yasuo IgarashiAbstract:Publisher Summary Hydrogenobacter thermophilus strain TK-6 is an aerobic thermophilic hydrogen-oxidizing bacterium isolated from hot spring in Izu, Japan. As for CO 2 fixation pathway of the strain, 14 CO 2 labeling experiment, in vitro enzyme assays, and purification of ATP:citrate lyase in the group supported the idea that the reductive TCA cycle is operative in strain TK-6. However, identification of a strong reductant that is needed for the Pyruvate Synthase and 2-oxoglutarate Synthase and clarification of an electron transport system were definitely needed. H. thermophilus was shown to belong to a very early branching order, the Aquificales . Also, strain TK-6 was isolated from hot spring, which implies that the strain may have its original ecological niche underground where little or no oxygen exists. Taking these things into consideration, it is of great interest to examine if the strain has its ability to grow anaerobically.
Ivan A. Berg - One of the best experts on this subject based on the ideXlab platform.
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Study of the distribution of autotrophic CO2 fixation cycles in Crenarchaeota.
Microbiology, 2010Co-Authors: Ivan A. Berg, W. Hugo Ramos-vera, Anna Petri, Harald Huber, Georg FuchsAbstract:Two new autotrophic carbon fixation cycles have been recently described in Crenarchaeota. The 3-hydroxypropionate/4-hydroxybutyrate cycle using acetyl-coenzyme A (CoA)/propionyl-CoA carboxylase as the carboxylating enzyme has been identified for (micro)aerobic members of the Sulfolobales. The dicarboxylate/4-hydroxybutyrate cycle using oxygen-sensitive Pyruvate Synthase and phosphoenolPyruvate carboxylase as carboxylating enzymes has been found in members of the anaerobic Desulfurococcales and Thermoproteales. However, Sulfolobales include anaerobic and Desulfurococcales aerobic autotrophic representatives, raising the question of which of the two cycles they use. We studied the mechanisms of autotrophic CO2 fixation in the strictly anaerobic Stygiolobus azoricus (Sulfolobales) and in the facultatively aerobic Pyrolobus fumarii (Desulfurococcales). The activities of all enzymes of the 3-hydroxypropionate/4-hydroxybutyrate cycle were found in the anaerobic S. azoricus. In contrast, the aerobic or denitrifying P. fumarii possesses all enzyme activities of the dicarboxylate/4-hydroxybutyrate cycle. We conclude that autotrophic Crenarchaeota use one of the two cycles, and that their distribution correlates with the 16S rRNA-based phylogeny of this group, rather than with the aerobic or anaerobic lifestyle.
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Autotrophic Carbon Dioxide Assimilation in Thermoproteales Revisited
Journal of bacteriology, 2009Co-Authors: W. Hugo Ramos-vera, Ivan A. Berg, Georg FuchsAbstract:For Crenarchaea, two new autotrophic carbon fixation cycles were recently described. Sulfolobales use the 3-hydroxypropionate/4-hydroxybutyrate cycle, with acetyl-coenzyme A (CoA)/propionyl-CoA carboxylase as the carboxylating enzyme. Ignicoccus hospitalis (Desulfurococcales) uses the dicarboxylate/4-hydroxybutyrate cycle, with Pyruvate Synthase and phosphoenolPyruvate carboxylase being responsible for CO2 fixation. In the two cycles, acetyl-CoA and two inorganic carbons are transformed to succinyl-CoA by different routes, whereas the regeneration of acetyl-CoA from succinyl-CoA proceeds via the same route. Thermoproteales would be an exception to this unifying concept, since for Thermoproteus neutrophilus, the reductive citric acid cycle was proposed as a carbon fixation mechanism. Here, evidence is presented for the operation of the dicarboxylate/4-hydroxybutyrate cycle in this archaeon. All required enzyme activities were detected in large amounts. The key enzymes of the cycle were strongly upregulated under autotrophic growth conditions, indicating their involvement in autotrophic CO2 fixation. The corresponding genes were identified in the genome. 14C-labeled 4-hydroxybutyrate was incorporated into the central building blocks in accordance with the key position of this compound in the cycle. Moreover, the results of previous 13C-labeling studies, which could be reconciled with a reductive citric acid cycle only when some assumptions were made, were perfectly in line with the new proposal. We conclude that the dicarboxylate/4-hydroxybutyrate cycle is operating in CO2 fixation in the strict anaerobic Thermoproteales as well as in Desulfurococcales.
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A dicarboxylate/4-hydroxybutyrate autotrophic carbon assimilation cycle in the hyperthermophilic Archaeum Ignicoccus hospitalis
Proceedings of the National Academy of Sciences of the United States of America, 2008Co-Authors: Harald Huber, Ivan A. Berg, Martin Gallenberger, Ulrike Jahn, Eva Eylert, Daniel Kockelkorn, Wolfgang Eisenreich, Georg FuchsAbstract:Ignicoccus hospitalis is an anaerobic, autotrophic, hyperthermophilic Archaeum that serves as a host for the symbiotic/parasitic Archaeum Nanoarchaeum equitans. It uses a yet unsolved autotrophic CO2 fixation pathway that starts from acetyl-CoA (CoA), which is reductively carboxylated to Pyruvate. Pyruvate is converted to phosphoenol-Pyruvate (PEP), from which glucogenesis as well as oxaloacetate formation branch off. Here, we present the complete metabolic cycle by which the primary CO2 acceptor molecule acetyl-CoA is regenerated. Oxaloacetate is reduced to succinyl-CoA by an incomplete reductive citric acid cycle lacking 2-oxoglutarate dehydrogenase or Synthase. Succinyl-CoA is reduced to 4-hydroxybutyrate, which is then activated to the CoA thioester. By using the radical enzyme 4-hydroxybutyryl-CoA dehydratase, 4-hydroxybutyryl-CoA is dehydrated to crotonyl-CoA. Finally, β-oxidation of crotonyl-CoA leads to two molecules of acetyl-CoA. Thus, the cyclic pathway forms an extra molecule of acetyl-CoA, with Pyruvate Synthase and PEP carboxylase as the carboxylating enzymes. The proposal is based on in vitro transformation of 4-hydroxybutyrate, detection of all enzyme activities, and in vivo-labeling experiments using [1-14C]4-hydroxybutyrate, [1,4-13C2], [U-13C4]succinate, or [1-13C]Pyruvate as tracers. The pathway is termed the dicarboxylate/4-hydroxybutyrate cycle. It combines anaerobic metabolic modules to a straightforward and efficient CO2 fixation mechanism.
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A proposed citramalate cycle for acetate assimilation in the purple non‐sulfur bacterium Rhodospirillum rubrum
FEMS Microbiology Letters, 2006Co-Authors: R. N. Ivanovsky, E. N. Krasil’nikova, Ivan A. BergAbstract:During phototrophic growth on acetate and CO2Rhodospirillum rubrum 2R contained malate Synthase but lacked isocitrate lyase. Acetate assimilation by R. rubrum cells was stimulated by Pyruvate, propionate glyoxylate, CO2 and H2. Acetate photoassimilation by R. rubrum cells in the presence of bicarbonate was accompanied by glyoxylate secretion, which increased after addition of fluoroacetate and decreased after addition of malonate. When acetyl-CoA was incubated with Pyruvate in cell-free extracts, citramalate was formed. Citramalate was also formed from propionyl-CoA and glyoxylate. The existence in R. rubrum of a CO2-dependent cyclic pathway of acetate oxidation to glyoxylate with citramalate as an intermediate is proposed. Inhibitor analysis of acetate and bicarbonate assimilation indicated that Pyruvate Synthase is not involved in acetate assimilation in R. rubrum. The possible anaplerotic sequences employed by R. rubrum during phototrophic growth on acetate are discussed.
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Carbon Metabolism of Filamentous Anoxygenic Phototrophic Bacteria of the Family Oscillochloridaceae
Microbiology, 2005Co-Authors: Ivan A. Berg, N. V. Ugol’kova, E. N. Krasil’nikova, O. I. Keppen, R. N. IvanovskyAbstract:The carbon metabolism of representatives of the family Oscillochloridaceae (Oscillochloris trichoides DG6 and the recent isolates Oscillochloris sp. R, KR, and BM) has been studied. Based on data from an inhibitory analysis of autotrophic CO2 assimilation and measurements of the activities of the enzymes involved in this process, it is concluded that, in all Oscillochloris strains, CO2 fixation occurs via the operation of the Calvin cycle. PhosphoenolPyruvate (PEP), which is formed in this cycle, can be involved in the metabolism via the following reaction sequence: PEP (+ CO2) --> oxalacetate --> malate --> fumarate --> succinate --> succinyl-CoA (+ CO2) --> 2-oxoglutarate (+ CO2) --> isocitrate. Acetate, utilized as and additional carbon source, can be carboxylated to Pyruvate by Pyruvate Synthase and further involved in the metabolism via the above reaction sequence. Propionyl-CoA Synthase and malonyl-CoA reductase, the key enzymes of the 3-hydroxypropionate cycle, have not been detected in Oscillochloris representatives.
Yasuo Igarashi - One of the best experts on this subject based on the ideXlab platform.
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Enzymatic and electron paramagnetic resonance studies of anabolic Pyruvate synthesis by Pyruvate: ferredoxin oxidoreductase from Hydrogenobacter thermophilus.
The FEBS journal, 2009Co-Authors: Takeshi Ikeda, Masaharu Ishii, Masahiro Yamamoto, Hiroyuki Arai, Daijiro Ohmori, Yasuo IgarashiAbstract:Pyruvate: ferredoxin oxidoreductase (POR; EC 1.2.7.1) catalyzes the thiamine pyrophosphate-dependent oxidative decarboxylation of Pyruvate to form acetyl-CoA and CO(2). The thermophilic, obligate chemolithoautotrophic hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus TK-6, assimilates CO(2) via the reductive tricarboxylic acid cycle. In this cycle, POR acts as Pyruvate Synthase catalyzing the reverse reaction (i.e. reductive carboxylation of acetyl-CoA) to form Pyruvate. The Pyruvate synthesis reaction catalyzed by POR is an energetically unfavorable reaction and requires a strong reductant. Moreover, the reducing equivalents must be supplied via its physiological electron mediator, a small iron-sulfur protein ferredoxin. Therefore, the reaction is difficult to demonstrate in vitro and the reaction mechanism has been poorly understood. In the present study, we coupled the decarboxylation of 2-oxoglutarate catalyzed by 2-oxoglutarate: ferredoxin oxidoreductase (EC 1.2.7.3), which generates sufficiently low-potential electrons to reduce ferredoxin, to drive the energy-demanding Pyruvate synthesis by POR. We demonstrate that H. thermophilus POR catalyzes Pyruvate synthesis from acetyl-CoA and CO(2), confirming the operation of the reductive tricarboxylic acid cycle in this bacterium. We also measured the electron paramagnetic resonance spectra of the POR intermediates in both the forward and reverse reactions, and demonstrate the intermediacy of a 2-(1-hydroxyethyl)- or 2-(1-hydroxyethylidene)-thiamine pyrophosphate radical in both reactions. The reaction mechanism of the reductive carboxylation of acetyl-CoA is also discussed.
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Reductive TCA cycle in an aerobic bacterium, Hydrogenobacter thermophilus strain TK-6
Studies in Surface Science and Catalysis, 1998Co-Authors: Masaharu Ishii, Kiseok Yoon, Yasufumi Ueda, Toshihiro Ochiai, Nare Yun, Seiichi Takishita, Tohru Kodama, Yasuo IgarashiAbstract:Publisher Summary Hydrogenobacter thermophilus strain TK-6 is an aerobic thermophilic hydrogen-oxidizing bacterium isolated from hot spring in Izu, Japan. As for CO 2 fixation pathway of the strain, 14 CO 2 labeling experiment, in vitro enzyme assays, and purification of ATP:citrate lyase in the group supported the idea that the reductive TCA cycle is operative in strain TK-6. However, identification of a strong reductant that is needed for the Pyruvate Synthase and 2-oxoglutarate Synthase and clarification of an electron transport system were definitely needed. H. thermophilus was shown to belong to a very early branching order, the Aquificales . Also, strain TK-6 was isolated from hot spring, which implies that the strain may have its original ecological niche underground where little or no oxygen exists. Taking these things into consideration, it is of great interest to examine if the strain has its ability to grow anaerobically.
E. N. Krasil’nikova - One of the best experts on this subject based on the ideXlab platform.
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A proposed citramalate cycle for acetate assimilation in the purple non‐sulfur bacterium Rhodospirillum rubrum
FEMS Microbiology Letters, 2006Co-Authors: R. N. Ivanovsky, E. N. Krasil’nikova, Ivan A. BergAbstract:During phototrophic growth on acetate and CO2Rhodospirillum rubrum 2R contained malate Synthase but lacked isocitrate lyase. Acetate assimilation by R. rubrum cells was stimulated by Pyruvate, propionate glyoxylate, CO2 and H2. Acetate photoassimilation by R. rubrum cells in the presence of bicarbonate was accompanied by glyoxylate secretion, which increased after addition of fluoroacetate and decreased after addition of malonate. When acetyl-CoA was incubated with Pyruvate in cell-free extracts, citramalate was formed. Citramalate was also formed from propionyl-CoA and glyoxylate. The existence in R. rubrum of a CO2-dependent cyclic pathway of acetate oxidation to glyoxylate with citramalate as an intermediate is proposed. Inhibitor analysis of acetate and bicarbonate assimilation indicated that Pyruvate Synthase is not involved in acetate assimilation in R. rubrum. The possible anaplerotic sequences employed by R. rubrum during phototrophic growth on acetate are discussed.
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Carbon Metabolism of Filamentous Anoxygenic Phototrophic Bacteria of the Family Oscillochloridaceae
Microbiology, 2005Co-Authors: I. A. Berg, N. V. Ugol’kova, E. N. Krasil’nikova, O. I. Keppen, R. N. IvanovskyAbstract:The carbon metabolism of representatives of the family Oscillochloridaceae (Oscillochloris trichoides DG6 and the recent isolates Oscillochloris sp. R, KR, and BM) has been studied. Based on data from an inhibitory analysis of autotrophic CO_2 assimilation and measurements of the activities of the enzymes involved in this process, it is concluded that, in all Oscillochloris strains , CO_2 fixation occurs via the operation of the Calvin cycle. PhosphoenolPyruvate (PEP), which is formed in this cycle, can be involved in the metabolism via the following reaction sequence: PEP (+CO_2) å oxalacetate å malate å fumarate å succinate å succinyl-CoA (+CO_2) å 2-oxoglutarate. Acetate, utilized as an additional carbon source, can be carboxylated to Pyruvate by Pyruvate Synthase and further involved in the metabolism via the above reaction sequence. Propionyl-CoA Synthase and malonyl-CoA reductase, the key enzymes of the 3-hydroxypropionate cycle, have not been detected in Oscillochloris representatives.
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A Study of the Mechanism of Acetate Assimilation in Purple Nonsulfur Bacteria Lacking the Glyoxylate Shunt: Acetate Assimilation in Rhodobacter sphaeroides
Microbiology, 2005Co-Authors: L. V. Filatova, E. N. Krasil’nikova, I. A. Berg, A. A. Tsygankov, T. V. Laurinavichene, R.n. IvanobskyAbstract:The mechanism of acetate assimilation in the purple nonsulfur bacterium Rhodobacter sphaeroides , which lacks the glyoxylate shunt, has been studied. It has been found that the growth of this bacterium in batch and continuous cultures and the assimilation of acetate in cell suspensions are not stimulated by bicarbonate. The consumption of acetate is accompanied by the excretion of glyoxylate and Pyruvate into the medium, stimulated by glyoxylate and Pyruvate, and inhibited by citramalate. The respiration of cells in the presence of acetate is stimulated by glyoxylate, Pyruvate, citramalate, and mesaconate. These data suggest that the citramalate cycle may function in Rba. sphaeroides in the form of an anaplerotic pathway instead of the glyoxylate shunt. At the same time, the low ratio of fixation rates for bicarbonate and acetate exhibited by the Rba. sphaeroides cells (approximately 0.1), as well as the absence of the stimulatory effect of acetate on the fixation of bicarbonate in the presence of the Calvin cycle inhibitor iodoacetate, suggests that Pyruvate Synthase is not involved in acetate assimilation in the bacterium Rba. sphaeroides .
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Carbon Metabolism of Filamentous Anoxygenic Phototrophic Bacteria of the Family Oscillochloridaceae
Microbiology, 2005Co-Authors: Ivan A. Berg, N. V. Ugol’kova, E. N. Krasil’nikova, O. I. Keppen, R. N. IvanovskyAbstract:The carbon metabolism of representatives of the family Oscillochloridaceae (Oscillochloris trichoides DG6 and the recent isolates Oscillochloris sp. R, KR, and BM) has been studied. Based on data from an inhibitory analysis of autotrophic CO2 assimilation and measurements of the activities of the enzymes involved in this process, it is concluded that, in all Oscillochloris strains, CO2 fixation occurs via the operation of the Calvin cycle. PhosphoenolPyruvate (PEP), which is formed in this cycle, can be involved in the metabolism via the following reaction sequence: PEP (+ CO2) --> oxalacetate --> malate --> fumarate --> succinate --> succinyl-CoA (+ CO2) --> 2-oxoglutarate (+ CO2) --> isocitrate. Acetate, utilized as and additional carbon source, can be carboxylated to Pyruvate by Pyruvate Synthase and further involved in the metabolism via the above reaction sequence. Propionyl-CoA Synthase and malonyl-CoA reductase, the key enzymes of the 3-hydroxypropionate cycle, have not been detected in Oscillochloris representatives.
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The mechanism of acetate assimilation in purple nonsulfur bacteria lacking the glyoxylate pathway: acetate assimilation in Rhodobacter sphaeroides cells
Mikrobiologiia, 2005Co-Authors: L. V. Filatova, E. N. Krasil’nikova, Ivan A. Berg, A. A. Tsygankov, T. V. LaurinavicheneAbstract:The mechanism of acetate assimilation in the purple nonsulfur bacterium Rhodobacter sphaeroides, which lacks the glyoxylate pathway, is studied. It is found that the growth of this bacterium in batch and continuous cultures and the assimilation of acetate in cell suspensions are not stimulated by bicarbonate. The consumption of acetate is accompanied by the excretion of glyoxylate and Pyruvate into the medium, stimulated by glyoxylate and Pyruvate, and inhibited by citramalate. The respiration of cells in the presence of acetate is stimulated by glyoxylate, Pyruvate, citramalate, and mesaconate. These data suggest that the citramalate cycle may function in Rba. sphaeroides in the form of an anaplerotic pathway instead of the glyoxylate pathway. At the same time, the low ratio of fixation rates for bicarbonate and acetate exhibited by the Rba. sphaeroides cells (approximately 0.1), as well as the absence of the stimulatory effect of acetate on the fixation of bicarbonate in the presence of the Calvin cycle inhibitor iodoacetate, suggests that Pyruvate Synthase is not involved in acetate assimilation in the bacterium Rba. sphaeroides.
Robert F Tabita - One of the best experts on this subject based on the ideXlab platform.
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rubredoxin from the green sulfur bacterium chlorobium tepidum functions as an electron acceptor for Pyruvate ferredoxin oxidoreductase
Journal of Biological Chemistry, 1999Co-Authors: Kiseok Yoon, Russ Hille, Craig Hemann, Robert F TabitaAbstract:Abstract Rubredoxin (Rd) from the moderately thermophilic green sulfur bacterium Chlorobium tepidum was found to function as an electron acceptor for Pyruvate ferredoxin oxidoreductase (PFOR). This enzyme, which catalyzes the conversion of Pyruvate to acetyl-CoA and CO2, exhibited an absolute dependence upon the presence of Rd. However, Rd was incapable of participating in the Pyruvate Synthase or CO2 fixation reaction of C. tepidum PFOR, for which two different reduced ferredoxins are employed as electron donors. These results suggest a specific functional role for Rd in Pyruvate oxidation and provide the initial indication that the two important physiological reactions catalyzed by PFOR/Pyruvate Synthase are dependent on different electron carriers in the cell. The UV-visible spectrum of oxidized Rd, with a monomer molecular weight of 6500, gave a molar absorption coefficient at 492 nm of 6.89 mm −1 cm−1 with anA 492/A 280 ratio of 0.343 and contained one iron atom/molecule. Further spectroscopic studies indicated that the CD spectrum of oxidized C. tepidum Rd exhibited a unique absorption maximum at 385 nm and a shoulder at 420 nm. The EPR spectrum of oxidized Rd also exhibited unusual anisotropic resonances at g = 9.675 and g = 4.322, which is composed of a narrow central feature with broader shoulders to high and low field. The midpoint reduction potential of C. tepidum Rd was determined to be −87 mV, which is the most electronegative value reported for Rd from any source.
