Hydrogenase of the hyperthermophile Pyrococcus furiosus is an elemental sulfur reductase or sulfhydrogenase: evidence for a sulfur-reducing hydrogenase ancestor.

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.

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Antimicrobial Activity of the Iron-Sulfur Nitroso Compound Roussin's Black Salt [Fe4S3(NO)7] on the Hyperthermophilic Archaeon Pyrococcus furiosus

Applied and Environmental Microbiology ◽

10.1128/aem.02562-08 ◽

2009 ◽

Vol 75 (7) ◽

pp. 1820-1825 ◽

Cited By ~ 6

Author(s):

Scott D. Hamilton-Brehm ◽

Gerrit J. Schut ◽

Michael W. W. Adams

Keyword(s):

Nitric Oxide ◽

Antimicrobial Agent ◽

Pyrococcus Furiosus ◽

Cell Lysis ◽

Nitroso Compound ◽

Hyperthermophilic Archaeon ◽

New Class ◽

Iron Sulfur ◽

Sulfur Reductase ◽

Potential Use

ABSTRACT The iron-sulfur nitroso compound [Fe4S3(NO)7]− is a broad-spectrum antimicrobial agent that has been used for more than 100 years to combat pathogenic anaerobes. Known as Roussin's black salt (RBS), it contains seven moles of nitric oxide, the release of which was always assumed to mediate its cytotoxicity. Using the hyperthermophilic archaeon Pyrococcus furiosus, it is demonstrated through growth studies, membrane analyses, and scanning electron microscopy that nitric oxide does not play a role in RBS toxicity; rather, the mechanism involves membrane disruption leading to cell lysis. Moreover, insoluble elemental sulfur (S0), which is reduced by P. furiosus to hydrogen sulfide, prevents cell lysis by RBS. It is proposed that S0 also directly interacts with the membranes of P. furiosus during its transfer into the cell, ultimately for reduction by a cytosolic NADPH sulfur reductase. RBS is proposed to be a new class of inorganic antimicrobial agent that also has potential use as an inert cell-lysing agent.

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Characterization of Hydrogenase II from the Hyperthermophilic Archaeon Pyrococcus furiosus and Assessment of Its Role in Sulfur Reduction

Journal of Bacteriology ◽

10.1128/jb.182.7.1864-1871.2000 ◽

2000 ◽

Vol 182 (7) ◽

pp. 1864-1871 ◽

Cited By ~ 105

Author(s):

Kesen Ma ◽

Robert Weiss ◽

Michael W. W. Adams

Keyword(s):

Amino Acid ◽

Elemental Sulfur ◽

Pyrococcus Furiosus ◽

Electron Paramagnetic Resonance Spectroscopy ◽

Amino Acid Sequences ◽

Electron Donors ◽

Resonance Spectroscopy ◽

Paramagnetic Resonance ◽

Nucleotide Binding Sites ◽

Γ Subunit

ABSTRACT The fermentative hyperthermophile Pyrococcus furiosuscontains an NADPH-utilizing, heterotetrameric (αβγδ), cytoplasmic hydrogenase (hydrogenase I) that catalyzes both H2 production and the reduction of elemental sulfur to H2S. Herein is described the purification of a second enzyme of this type, hydrogenase II, from the same organism. Hydrogenase II has an M r of 320,000 ± 20,000 and contains four different subunits withM rs of 52,000 (α), 39,000 (β), 30,000 (γ), and 24,000 (δ). The heterotetramer contained Ni (0.9 ± 0.1 atom/mol), Fe (21 ± 1.6 atoms/mol), and flavin adenine dinucleotide (FAD) (0.83 ± 0.1 mol/mol). NADPH and NADH were equally efficient as electron donors for H2 production withKm values near 70 μM andk cat/Km values near 350 min−1 mM−1. In contrast to hydrogenase I, hydrogenase II catalyzed the H2-dependent reduction of NAD (Km , 128 μM;k cat/Km , 770 min−1 mM−1). Ferredoxin from P. furiosus was not an efficient electron carrier for either enzyme. Both H2 and NADPH served as electron donors for the reduction of elemental sulfur (S0) and polysulfide by hydrogenase I and hydrogenase II, and both enzymes preferentially reduce polysulfide to sulfide rather than protons to H2using NADPH as the electron donor. At least two [4Fe-4S] and one [2Fe-2S] cluster were detected in hydrogenase II by electron paramagnetic resonance spectroscopy, but amino acid sequence analyses indicated a total of five [4Fe-4S] clusters (two in the β subunit and three in the δ subunit) and one [2Fe-2S] cluster (in the γ subunit), as well as two putative nucleotide-binding sites in the γ subunit which are thought to bind FAD and NAD(P)(H). The amino acid sequences of the four subunits of hydrogenase II showed between 55 and 63% similarity to those of hydrogenase I. The two enzymes are present in the cytoplasm at approximately the same concentration. Hydrogenase II may become physiologically relevant at low S0concentrations since it has a higher affinity than hydrogenase I for both S0 and polysulfide.

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

Journal of Bacteriology ◽

10.1128/jb.00031-07 ◽

2007 ◽

Vol 189 (12) ◽

pp. 4431-4441 ◽

Cited By ~ 109

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.

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The Elemental Sulfur-Responsive Protein (SipA) from the Hyperthermophilic Archaeon Pyrococcus furiosus Is Regulated by Sulfide in an Iron-Dependent Manner

Journal of Bacteriology ◽

10.1128/jb.00660-10 ◽

2010 ◽

Vol 192 (21) ◽

pp. 5841-5843 ◽

Cited By ~ 6

Author(s):

Sonya M. Clarkson ◽

Elizabeth C. Newcomer ◽

Everett G. Young ◽

Michael W. W. Adams

Keyword(s):

Elemental Sulfur ◽

Iron Sulfide ◽

Pyrococcus Furiosus ◽

Protein A ◽

Dependent Manner ◽

Intracellular Iron ◽

Hyperthermophilic Archaeon

ABSTRACT The gene (sipA) encoding the sulfur-induced protein A (PF2025) is highly upregulated during growth of Pyrococcus furiosus on elemental sulfur (S0). Expression of sipA is regulated by sulfide, the product of S0 reduction, but in an iron-dependent manner. SipA is proposed to play a role in intracellular iron sulfide detoxification.

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Minimal sulfur requirement for growth and sulfur-dependent metabolism of the hyperthermophilic archaeonStaphylothermus marinus

Archaea ◽

10.1155/2003/626017 ◽

2003 ◽

Vol 1 (3) ◽

pp. 191-197 ◽

Cited By ~ 14

Author(s):

Xiaolei Hao ◽

Kesen Ma

Keyword(s):

Elemental Sulfur ◽

Optimal Growth ◽

Physical Support ◽

Metabolic Functions ◽

Metabolic Transition ◽

Heterotrophic Metabolism ◽

Dependent Growth ◽

Sulfur Containing ◽

Sulfur Reductase ◽

Sulfur Containing Compounds

Staphylothermus marinusis an anaerobic hyperthermophilic archaeon that uses peptides as carbon and energy sources. Elemental sulfur (S°) is obligately required for its growth and is reduced to H2S. The metabolic functions and mechanisms of S° reduction were explored by examining S°-dependent growth and activities of key enzymes present in this organism. All three forms of S° tested—sublimed S°, colloidal S° and polysulfide—were used byS. marinus, and no other sulfur-containing compounds could replace S°. Elemental sulfur did not serve as physical support but appeared to function as an electron acceptor. The minimal S° concentration required for optimal growth was 0.05% (w/v). At this concentration, there appeared to be a metabolic transition from H2production to S° reduction. Some enzymatic activities related to S°-dependent metabolism, including sulfur reductase, hydrogenase, glutamate dehydrogenase and electron transfer activities, were detected in cell-free extracts ofS. marinus.These results indicate that S° plays an essential role in the heterotrophic metabolism ofS. marinus. Reducing equivalents generated by the oxidation of amino acids from peptidolysis may be transferred to sulfur reductase and hydrogenase, which then catalyze the production of H2S and H2, respectively.

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