Cytoplasmic and membrane-bound hydrogenases from Pyrococcus furiosus

2018 ◽  
pp. 153-168
Author(s):  
Chang-Hao Wu ◽  
Dominik K. Haja ◽  
Michael W.W. Adams
Keyword(s):  
Pyrococcus Furiosus ◽  
Membrane Bound

scholarly journals pH Homeostasis and Sodium Ion Pumping by Multiple Resistance and pH Antiporters in Pyrococcus furiosus

2021 ◽  
Vol 12 ◽  
Author(s):  
Dominik K. Haja ◽  
Michael W. W. Adams
Keyword(s):  
Pyrococcus Furiosus ◽  
Gas Production ◽  
Hydrogen Gas ◽  
Sodium Ion ◽  
Redox Activity ◽  
Deletion Strain ◽  
Multiple Resistance ◽  
Ph Homeostasis ◽  
Membrane Bound ◽  
Ion Pumping

Multiple Resistance and pH (Mrp) antiporters are seven-subunit complexes that couple transport of ions across the membrane in response to a proton motive force (PMF) and have various physiological roles, including sodium ion sensing and pH homeostasis. The hyperthermophilic archaeon Pyrococcus furiosus contains three copies of Mrp encoding genes in its genome. Two are found as integral components of two respiratory complexes, membrane bound hydrogenase (MBH) and the membrane bound sulfane sulfur reductase (MBS) that couple redox activity to sodium translocation, while the third copy is a stand-alone Mrp. Sequence alignments show that this Mrp does not contain an energy-input (PMF) module but contains all other predicted functional Mrp domains. The P. furiosus Mrp deletion strain exhibits no significant changes in optimal pH or sodium ion concentration for growth but is more sensitive to medium acidification during growth. Cell suspension hydrogen gas production assays using the deletion strain show that this Mrp uses sodium as the coupling ion. Mrp likely maintains cytoplasmic pH by exchanging protons inside the cell for extracellular sodium ions. Deletion of the MBH sodium-translocating module demonstrates that hydrogen gas production is uncoupled from ion pumping and provides insights into the evolution of this Mrp-containing respiratory complex.

scholarly journals Key Role for Sulfur in Peptide Metabolism and in Regulation of Three Hydrogenases in the Hyperthermophilic ArchaeonPyrococcus furiosus

2001 ◽  
Vol 183 (2) ◽  
pp. 716-724 ◽  
Author(s):  
Michael W. W. Adams ◽  
James F. Holden ◽  
Angeli Lal Menon ◽  
Gerrit J. Schut ◽  
Amy M. Grunden ◽  
...  
Keyword(s):  
Growth Rates ◽  
Elemental Sulfur ◽  
Pyrococcus Furiosus ◽  
Growth Conditions ◽  
Growth Media ◽  
Hyperthermophilic Archaeon ◽  
Order Of Magnitude ◽  
Membrane Bound ◽  
Peptide Metabolism ◽  
Specific Activities

ABSTRACT The hyperthermophilic archaeon Pyrococcus furiosusgrows optimally at 100°C by the fermentation of peptides and carbohydrates. Growth of the organism was examined in media containing either maltose, peptides (hydrolyzed casein), or both as the carbon source(s), each with and without elemental sulfur (S0). Growth rates were highest on media containing peptides and S0, with or without maltose. Growth did not occur on the peptide medium without S0. S0 had no effect on growth rates in the maltose medium in the absence of peptides. Phenylacetate production rates (from phenylalanine fermentation) from cells grown in the peptide medium containing S0 with or without maltose were the same, suggesting that S0 is required for peptide utilization. The activities of 14 of 21 enzymes involved in or related to the fermentation pathways of P. furiosus were shown to be regulated under the five different growth conditions studied. The presence of S0 in the growth media resulted in decreases in specific activities of two cytoplasmic hydrogenases (I and II) and of a membrane-bound hydrogenase, each by an order of magnitude. The primary S0-reducing enzyme in this organism and the mechanism of the S0 dependence of peptide metabolism are not known. This study provides the first evidence for a highly regulated fermentation-based metabolism in P. furiosus and a significant regulatory role for elemental sulfur or its metabolites.

scholarly journals Insights into the Metabolism of Elemental Sulfur by the Hyperthermophilic Archaeon Pyrococcus furiosus: Characterization of a Coenzyme A- Dependent NAD(P)H Sulfur Oxidoreductase

2007 ◽  
Vol 189 (12) ◽  
pp. 4431-4441 ◽  
Author(s):  
Gerrit J. Schut ◽  
Stephanie L. Bridger ◽  
Michael W. W. Adams
Keyword(s):  
Gene Cluster ◽  
Elemental Sulfur ◽  
Coenzyme A ◽  
Specific Activity ◽  
Pyrococcus Furiosus ◽  
Side Reaction ◽  
Primary Response ◽  
Secondary Response ◽  
Hyperthermophilic Archaeon ◽  
Membrane Bound

ABSTRACT The hyperthermophilic archaeon Pyrococcus furiosus uses carbohydrates as a carbon source and produces acetate, CO2, and H2 as end products. When S0 is added to a growing culture, within 10 min the rate of H2 production rapidly decreases and H2S is detected. After 1 hour cells contain high NADPH- and coenzyme A-dependent S0 reduction activity (0.7 units/mg, 85°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 S0 addition. This primary response to S0 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 S0 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 S0. A secondary response to S0 is observed 30 min after S0 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 S0-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 S0-reducing system in autotrophic archaea and bacteria.

scholarly journals Reinvestigation of the Steady-State Kinetics and Physiological Function of the Soluble NiFe-Hydrogenase I of Pyrococcus furiosus

2007 ◽  
Vol 190 (5) ◽  
pp. 1584-1587 ◽  
Author(s):  
Daan J. van Haaster ◽  
Pedro J. Silva ◽  
Peter-Leon Hagedoorn ◽  
Jaap A. Jongejan ◽  
Wilfred R. Hagen
Keyword(s):  
Steady State ◽  
Hydrogen Production ◽  
Physiological Function ◽  
Pyrococcus Furiosus ◽  
High Hydrogen ◽  
Hydrogen Consumption ◽  
Steady State Kinetics ◽  
Production Activity ◽  
Membrane Bound ◽  
Consumption Activity

ABSTRACT Pyrococcus furiosus has two types of NiFe-hydrogenases: a heterotetrameric soluble hydrogenase and a multimeric transmembrane hydrogenase. Originally, the soluble hydrogenase was proposed to be a new type of H2 evolution hydrogenase, because, in contrast to all of the then known NiFe-hydrogenases, the hydrogen production activity at 80°C was found to be higher than the hydrogen consumption activity and CO inhibition appeared to be absent. NADPH was proposed to be the electron donor. Later, it was found that the membrane-bound hydrogenase exhibits very high hydrogen production activity sufficient to explain cellular H2 production levels, and this seems to eliminate the need for a soluble hydrogen production activity and therefore leave the soluble hydrogenase without a physiological function. Therefore, the steady-state kinetics of the soluble hydrogenase were reinvestigated. In contrast to previous reports, a low K m for H2 (∼20 μM) was found, which suggests a relatively high affinity for hydrogen. Also, the hydrogen consumption activity was 1 order of magnitude higher than the hydrogen production activity, and CO inhibition was significant (50% inhibition with 20 μM dissolved CO). Since the K m for NADP+ is ∼37 μM, we concluded that the soluble hydrogenase from P. furiosus is likely to function in the regeneration of NADPH and thus reuses the hydrogen produced by the membrane-bound hydrogenase in proton respiration.

scholarly journals Characterization of membrane-bound sulfane reductase: A missing link in the evolution of modern day respiratory complexes

2018 ◽  
Vol 293 (43) ◽  
pp. 16687-16696 ◽  
Author(s):  
Chang-Hao Wu ◽  
Gerrit J. Schut ◽  
Farris L. Poole ◽  
Dominik K. Haja ◽  
Michael W. W. Adams
Keyword(s):  
Complex I ◽  
Pyrococcus Furiosus ◽  
Atp Synthesis ◽  
Genetically Engineered ◽  
Hydrogen Gas ◽  
Hyperthermophilic Archaea ◽  
Cysteine Residues ◽  
Sulfane Sulfur ◽  
Respiratory Complexes ◽  
Membrane Bound

Hyperthermophilic archaea contain a hydrogen gas–evolving,respiratory membrane–bound NiFe-hydrogenase (MBH) that is very closely related to the aerobic respiratory complex I. During growth on elemental sulfur (S°), these microorganisms also produce a homologous membrane-bound complex (MBX), which generates H2S. MBX evolutionarily links MBH to complex I, but its catalytic function is unknown. Herein, we show that MBX reduces the sulfane sulfur of polysulfides by using ferredoxin (Fd) as the electron donor, and we rename it membrane-bound sulfane reductase (MBS). Two forms of affinity-tagged MBS were purified from genetically engineered Pyrococcus furiosus (a hyperthermophilic archaea species): the 13-subunit holoenzyme (S-MBS) and a cytoplasmic 4-subunit catalytic subcomplex (C-MBS). S-MBS and C-MBS reduced dimethyl trisulfide (DMTS) with comparable Km (∼490 μm) and Vmax values (12 μmol/min/mg). The MBS catalytic subunit (MbsL), but not that of complex I (NuoD), retains two of four NiFe-coordinating cysteine residues of MBH. However, these cysteine residues were not involved in MBS catalysis because a mutant P. furiosus strain (MbsLC85A/C385A) grew normally with S°. The products of the DMTS reduction and properties of polysulfides indicated that in the physiological reaction, MBS uses Fd (Eo′ = −480 mV) to reduce sulfane sulfur (Eo′ −260 mV) and cleave organic (RSnR, n ≥ 3) and anionic polysulfides (Sn2−, n ≥ 4) but that it does not produce H2S. Based on homology to MBH, MBS also creates an ion gradient for ATP synthesis. This work establishes the electrochemical reaction catalyzed by MBS that is intermediate in the evolution from proton- to quinone-reducing respiratory complexes.

Two Distinct Types of Microfilaments in the Cytoplasm of Human Adenohypophysial Cells

1973 ◽  
Vol 31 ◽  
pp. 668-669
Author(s):  
E. Horvath ◽  
K. Kovacs ◽  
I. E. Stratmann ◽  
C. Ezrin
Keyword(s):  
Pituitary Tumors ◽  
Average Diameter ◽  
Anterior Lobe ◽  
Uranyl Acetate ◽  
Type I ◽  
Breast Cancer Patients ◽  
Human Pituitary ◽  
Severe Diabetes ◽  
Pituitary Glands ◽  
Membrane Bound

Surgically removed human pituitary glands as well as pituitary tumors fixed in glutaraldehyde, postfixed in osmium tetroxide, embedded in epon resin, stained with uranyl acetate and lead citrate have been investigated by electron microscopy in order to correlate ultrastructure with functional activity. In the course of this study two distinct types of microfilaments have been identified in the cytoplasm of adenohypophysiocytes.Type I microfilaments (Fig. 1) were found in the cytoplasm of anterior lobe cells of five female subjects with disseminated mammary cancer and two patients with severe diabetes mellitus. The breast cancer patients were treated pre-operatively for various periods of time with different doses of oxysteroids. The microfilaments had an average diameter of JO A, formed parallel bundles, were scattered irregularly in the cytoplasm and were frequently located in the perikaryon. They were not membrane-bound and failed to show any periodicity.

Intranuclear Crystals in Regenerating Canine Kidneys

1973 ◽  
Vol 31 ◽  
pp. 412-413
Author(s):  
D.G. Osborne ◽  
L.J. McCormack ◽  
M.O. Magnusson ◽  
W.S. Kiser
Keyword(s):  
Epithelial Cell ◽  
Epithelial Cells ◽  
Tubular Epithelial Cell ◽  
Tubular Epithelial Cells ◽  
Cell Nuclei ◽  
Crystalline Structures ◽  
Small Crystals ◽  
Membrane Bound

During a project in which regenerative changes were studied in autotransplanted canine kidneys, intranuclear crystals were seen in a small number of tubular epithelial cells. These crystalline structures were seen in the control specimens and also in regenerating specimens; the main differences being in size and number of them. The control specimens showed a few tubular epithelial cell nuclei almost completely occupied by large crystals that were not membrane bound. Subsequent follow-up biopsies of the same kidneys contained similar intranuclear crystals but of a much smaller size. Some of these nuclei contained several small crystals. The small crystals occurred at one week following transplantation and were seen even four weeks following transplantation. As time passed, the small crystals appeared to fuse to form larger crystals.

Membrane-bound vesicles associated with the microvilli in the midgut of the freshwater microcrustacean, Daphnia magna

1983 ◽  
Vol 41 ◽  
pp. 480-481
Author(s):  
Patricia L. Jansma
Keyword(s):  
Daphnia Magna ◽  
Intestinal Epithelial Cells ◽  
Uranyl Acetate ◽  
Intestinal Epithelial ◽  
Chlamydomonas Reinhardii ◽  
Thin Sections ◽  
Saturated Water ◽  
Cacodylate Buffer ◽  
Membrane Bound ◽  
Ethanol Series

The presence of the membrane bound vesicles or blebs on the intestinal epithelial cells has been demonstrated in a variety of vertebrates such as chicks, piglets, hamsters, and humans. The only invertebrates shown to have these microvillar blebs are two species of f1ies. While investigating the digestive processes of the freshwater microcrustacean, Daphnia magna, the presence of these microvillar blebs was noticed.Daphnia magna fed in a suspension of axenically grown green alga, Chlamydomonas reinhardii for one hour were narcotized with CO2 saturated water. The intestinal tracts were excised in 2% glutaraldehyde in 0.2 M cacodyl ate buffer and then placed in fresh 2% glutaraldehyde for one hour. After rinsing in 0.1 M cacodylate buffer, the sample was postfixed in 2% OsO4, dehydrated with a graded ethanol series, infiltrated and embedded with Epon-Araldite. Thin sections were stained with uranyl acetate and Reynolds lead citrate before viewing with the Philips EM 200.

Sign in / Sign up

Export Citation Format

Share Document