Rapid-reaction kinetics of the bifurcating NAD+-dependent NADPH:ferredoxin oxidoreductase NfnI from Pyrococcus furiosus
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SUBMITTER: Ortiz S
PROVIDER: S-EPMC10724689 | biostudies-literature | 2023 Oct
REPOSITORIES: biostudies-literature
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Project description:We have investigated the equilibrium properties and rapid-reaction kinetics of the isolated butyryl-CoA dehydrogenase (bcd) component of the electron-bifurcating crotonyl-CoA-dependent NADH:ferredoxin oxidoreductase (EtfAB-bcd) from Megasphaera elsdenii. We find that a neutral FADH• semiquinone accumulates transiently during both reduction with sodium dithionite and with NADH in the presence of catalytic concentrations of EtfAB. In both cases full reduction of bcd to the hydroquinone is eventually observed, but the accumulation of FADH• indicates that a substantial portion of reduction occurs in sequential one-electron processes rather than a single two-electron event. In rapid-reaction experiments following the reaction of reduced bcd with crotonyl-CoA and oxidized bcd with butyryl-CoA, long-wavelength-absorbing intermediates are observed that are assigned to bcdred:crotonyl-CoA and bcdox:butyryl-CoA charge-transfer complexes, demonstrating their kinetic competence in the course of the reaction. In the presence of crotonyl-CoA there is an accumulation of semiquinone that is unequivocally the anionic FAD•- rather than the neutral FADH• seen in the absence of substrate, indicating that binding of substrate/product results in ionization of the bcd semiquinone. In addition to fully characterizing the rapid-reaction kinetics of both the oxidative and reductive half-reactions, our results demonstrate that one-electron processes play an important role in the reduction of bcd in EtfAB-bcd.
| S-EPMC10320503 | biostudies-literature
Project description:Formaldehyde ferredoxin oxidoreductase from Pyrococcus furiosus is a homotetrameric protein with one tungstodipterin and one [4Fe-4S] cubane per 69-kDa subunit. The enzyme kinetics have been studied under steady-state conditions at 80 degrees C and pre-steady state conditions at 50 degrees C, in the latter case via monitoring of the relatively weak (epsilon approximately 2 mM(-1) cm(-1)) optical spectrum of the tungsten cofactor. The steady-state data are consistent with a substrate substituted-enzyme mechanism for three substrates (formaldehyde plus two ferredoxin molecules). The KM value for free formaldehyde (21 microM) with ferredoxin as an electron acceptor is approximately 3 times lower than the value measured when benzyl viologen is used as an acceptor. The KM of ferredoxin (14 microM) is an order of magnitude less than previously reported values. An explanation for this discrepancy may be the fact that high concentrations of substrate are inhibitory and denaturing to the enzyme. Pre-steady-state difference spectra reveal peak shifts and a lack of isosbestic points, an indication that several processes happen in the first seconds of the reaction. Two fast processes (kobs1 = 4.7 s(-1), kobs2 = 1.9 s(-1)) are interpreted as oxidation of the substrate followed by rearrangement of the active site. Alternatively, these processes could be the entry/binding of the substrate followed by its oxidation. The release of the product and the electron shuffling over the tungsten and iron-sulfur center in the absence of an external electron acceptor are slower (kobs3 = 6.10 x 10(-2 )s(-1), kobs4 = 2.18 x 10(-2 )s(-1)). On the basis of these results in combination with results from previous electron paramagnetic resonance studies an activation route plus catalytic redox cycle is proposed.
| S-EPMC2099461 | biostudies-other
Project description:The hyperthermophilic archaea Pyrococcus furiosus and Thermococcus litoralis contain the tungstoenzymes aldehyde ferredoxin oxidoreductase, a homodimer, and formaldehyde ferredoxin oxidoreductase, a homotetramer. herein we report the cloning and sequencing of the P. furiosus gene aor (605 residues; M(r), 66,630) and the T. litoralis gene for (621 residues; M(r), 68,941).
| S-EPMC177251 | biostudies-other
Project description:The hyperthermophilic archaeon Pyrococcus furiosus expresses five aldehyde oxidoreductase (AOR) enzymes, all containing a tungsto-bispterin cofactor. The growth of this organism is fully dependent on the presence of tungsten in the growth medium. Previous studies have suggested that molybdenum is not incorporated in the active site of these enzymes. Application of the radioisotope (99)Mo in metal isotope native radioautography in gel electrophoresis (MIRAGE) technology to P. furiosus shows that molybdenum can in fact be incorporated in all five AOR enzymes. Mo(V) signals characteristic for molybdopterin were observed in formaldehyde oxidoreductase (FOR) in electron paramagnetic resonance (EPR)-monitored redox titrations. Our finding that the aldehyde oxidation activity of FOR and WOR5 (W-containing oxidoreductase 5) correlates only with the residual tungsten content suggests that the Mo-containing AORs are most likely inactive. An observed W/Mo antagonism is indicative of tungstate-dependent negative feedback of the expression of the tungstate/molybdate ABC transporter. An intracellular selection mechanism for tungstate and molybdate processing has to be present, since tungsten was found to be preferentially incorporated into the AORs even under conditions with comparable intracellular concentrations of tungstate and molybdate. Under the employed growth conditions of starch as the main carbon source in a rich medium, no tungsten- and/or molybdenum-associated proteins are detected in P. furiosus other than the high-affinity transporter, the proteins of the metallopterin insertion machinery, and the five W-AORs.
| S-EPMC2916431 | biostudies-literature
Project description:Previous studies have shown that the hyperthermophilic archaeon Pyrococcus furiosus contains four distinct cytoplasmic 2-ketoacid oxidoreductases (ORs) which differ in their substrate specificities, while the hyperthermophilic bacterium Thermotoga maritima contains only one, pyruvate ferredoxin oxidoreductase (POR). These enzymes catalyze the synthesis of the acyl (or aryl) coenzyme A derivative in a thiamine PPi-dependent oxidative decarboxylation reaction with reduction of ferredoxin. We report here on the molecular analysis of the POR (por) and 2-ketoisovalerate ferredoxin oxidoreductase (vor) genes from P. furiosus and of the POR gene from T. maritima, all of which comprise four different subunits. The operon organization for P. furiosus POR and VOR was porG-vorDAB-porDAB, wherein the gamma subunit is shared by the two enzymes. The operon organization for T. maritima POR was porGDAB. The three enzymes were 46 to 53% identical at the amino acid level. Their delta subunits each contained two ferredoxin-type [4Fe-4S] cluster binding motifs (CXXCXXCXXXCP), while their beta subunits each contained four conserved cysteines in addition to a thiamine PPi-binding domain. Amino-terminal sequence comparisons show that POR, VOR, indolepyruvate OR, and 2-ketoglutarate OR of P. furiosus all belong to a phylogenetically homologous OR family. Moreover, the single-subunit pyruvate ORs from mesophilic and moderately thermophilic bacteria and from an amitochondriate eucaryote each contain four domains which are phylogenetically homologous to the four subunits of the hyperthermophilic ORs (27% sequence identity). Three of these subunits are also homologous to the dimeric POR from a mesophilic archaeon, Halobacterium halobium (21% identity). A model is proposed to account for the observed phenotypes based on genomic rearrangements of four ancestral OR subunits.
| S-EPMC177646 | biostudies-other
Project description:Moorella thermoacetica was long the only model organism used to study the biochemistry of acetogenesis from CO(2). Depending on the growth substrate, this Gram-positive bacterium can either form H(2) or consume it. Despite the importance of H(2) in its metabolism, a hydrogenase from the organism has not yet been characterized. We report here the purification and properties of an electron-bifurcating [FeFe]-hydrogenase from M. thermoacetica and show that the cytoplasmic enzyme efficiently catalyzes both H(2) formation and H(2) uptake. The purified heterotrimeric iron-sulfur flavoprotein (HydABC) catalyzed the coupled reduction of ferredoxin (Fd) and NAD(+) with H(2) at 55 °C at pH 7.5 at a specific rate of about 100 ?mol min(-1) mg protein(-1) and the reverse reaction, the coupled reduction of protons to H(2) with reduced ferredoxin and NADH, at a specific rate of about 10 ?mol min(-1) mg protein(-1) in the stoichiometry Fd(ox) + NAD(+) + 2H(2) Fd(red)(2-) + NADH + 3H(+). When ferredoxin from Clostridium pasteurianum, NAD(+), and the enzyme were incubated at pH 7.0 under 100% H(2) in the gas phase (E(0)' = -414 mV), more than 95% of the ferredoxin (E(0)' = -400 mV) was reduced, which indicated that ferredoxin reduction with H(2) is driven by the exergonic reduction of NAD(+) (E(0)' = -320 mV) with H(2). In the absence of NAD(+), ferredoxin was not reduced. We identified the genes encoding HydABC within the transcriptional unit hydCBAX and mapped the transcription start site.
| S-EPMC3591994 | biostudies-literature
Project description:Small RNA Sequencing from Pyrococcus furiosus Keywords: Small RNA Analysis
2009-12-28 | GSE16968 | GEO
Project description:Pyrococcus furiosus is a hyperthermophilic archaeon which grows optimally near 100 degreesC by fermenting peptides and sugars to produce organic acids, CO2, and H2. Its growth requires tungsten, and two different tungsten-containing enzymes, aldehyde ferredoxin oxidoreductase (AOR) and glyceraldehyde-3-phosphate ferredoxin oxidoreductase (GAPOR), have been previously purified from P. furiosus. These two enzymes are thought to function in the metabolism of peptides and carbohydrates, respectively. A third type of tungsten-containing enzyme, formaldehyde ferredoxin oxidoreductase (FOR), has now been characterized. FOR is a homotetramer with a mass of 280 kDa and contains approximately 1 W atom, 4 Fe atoms, and 1 Ca atom per subunit, together with a pterin cofactor. The low recovery of FOR activity during purification was attributed to loss of sulfide, since the purified enzyme was activated up to fivefold by treatment with sulfide (HS-) under reducing conditions. FOR uses P. furiosus ferredoxin as an electron acceptor (Km = 100 microM) and oxidizes a range of aldehydes. Formaldehyde (Km = 15 mM for the sulfide-activated enzyme) was used in routine assays, but the physiological substrate is thought to be an aliphatic C5 semi- or dialdehyde, e.g., glutaric dialdehyde (Km = 1 mM). Based on its amino-terminal sequence, the gene encoding FOR (for) was identified in the genomic database, together with those encoding AOR and GAPOR. The amino acid sequence of FOR corresponded to a mass of 68.7 kDa and is highly similar to those of the subunits of AOR (61% similarity and 40% identity) and GAPOR (50% similarity and 23% identity). The three genes are not linked on the P. furiosus chromosome. Two additional (and nonlinked) genes (termed wor4 and wor5) that encode putative tungstoenzymes with 57% (WOR4) and 56% (WOR5) sequence similarity to FOR were also identified. Based on sequence motif similarities with FOR, both WOR4 and WOR5 are also proposed to contain a tungstobispterin site and one [4Fe-4S] cluster per subunit.
| S-EPMC93494 | biostudies-literature
Project description:The hyperthermophilic archaeon Pyrococcus furiosus uses carbohydrates as a carbon source and produces acetate, CO2, and H2 as end products. When S(0) is added to a growing culture, within 10 min the rate of H2 production rapidly decreases and H(2)S is detected. After 1 hour cells contain high NADPH- and coenzyme A-dependent S(0) reduction activity (0.7 units/mg, 85 degrees C) located in the cytoplasm. The enzyme responsible for this activity was purified to electrophoretic homogeneity (specific activity, 100 units/mg) and is termed NAD(P)H elemental sulfur oxidoreductase (NSR). NSR is a homodimeric flavoprotein (M(r), 100,000) and is encoded by PF1186. This designation was previously assigned to the gene encoding an enzyme that reduces coenzyme A disulfide, which is a side reaction of NSR. Whole-genome DNA microarray and quantitative PCR analyses showed that the expression of NSR is up-regulated up to sevenfold within 10 min of S(0) addition. This primary response to S(0) also involves the up-regulation (>16-fold) of a 13-gene cluster encoding a membrane-bound oxidoreductase (MBX). The cluster encoding MBX is proposed to replace the homologous 14-gene cluster that encodes the ferredoxin-oxidizing, H2-evolving membrane-bound hydrogenase (MBH), which is down-regulated >12-fold within 10 min of S(0) addition. Although an activity for MBX could not be demonstrated, it is proposed to conserve energy by oxidizing ferredoxin and reducing NADP, which is used by NSR to reduce S(0). A secondary response to S(0) is observed 30 min after S(0) addition and includes the up-regulation of genes encoding proteins involved in amino acid biosynthesis and iron metabolism, as well as two so-called sulfur-induced proteins termed SipA and SipB. This novel S(0)-reducing system involving NSR and MBX has been found so far only in the heterotrophic Thermococcales and is in contrast to the cytochrome- and quinone-based S(0)-reducing system in autotrophic archaea and bacteria.
| S-EPMC1913366 | biostudies-literature
Project description:A scheme for the detoxification of superoxide in Pyrococcus furiosus has been previously proposed in which superoxide reductase (SOR) reduces (rather than dismutates) superoxide to hydrogen peroxide by using electrons from reduced rubredoxin (Rd). Rd is reduced with electrons from NAD(P)H by the enzyme NAD(P)H:rubredoxin oxidoreductase (NROR). The goal of the present work was to reconstitute this pathway in vitro using recombinant enzymes. While recombinant forms of SOR and Rd are available, the gene encoding P. furiosus NROR (PF1197) was found to be exceedingly toxic to Escherichia coli, and an active recombinant form (rNROR) was obtained via a fusion protein expression system, which produced an inactive form of NROR until cleavage. This allowed the complete pathway from NAD(P)H to the reduction of SOR via NROR and Rd to be reconstituted in vitro using recombinant proteins. rNROR is a 39.9-kDa protein whose sequence contains both flavin adenine dinucleotide (FAD)- and NAD(P)H-binding motifs, and it shares significant similarity with known and putative Rd-dependent oxidoreductases from several anaerobic bacteria, both mesophilic and hyperthermophilic. FAD was shown to be essential for activity in reconstitution assays and could not be replaced by flavin mononucleotide (FMN). The bound FAD has a midpoint potential of -173 mV at 23 degrees C (-193 mV at 80 degrees C). Like native NROR, the recombinant enzyme catalyzed the NADPH-dependent reduction of rubredoxin both at high (80 degrees C) and low (23 degrees C) temperatures, consistent with its proposed role in the superoxide reduction pathway. This is the first demonstration of in vitro superoxide reduction to hydrogen peroxide using NAD(P)H as the electron donor in an SOR-mediated pathway.
| S-EPMC1065123 | biostudies-literature