Marc F. J. M. Verhagen

Key Role for Sulfur in Peptide Metabolism and in Regulation of Three Hydrogenases in the Hyperthermophilic Archaeon Pyrococcus furiosus

The hyperthermophilic archaeon Pyrococcus furiosus grows optimally at 100 degrees 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 (S(0)). Growth rates were highest on media containing peptides and S(0), with or without maltose. Growth did not occur on the peptide medium without S(0). S(0) 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 S(0) with or without maltose were the same, suggesting that S(0) 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 S(0) 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 S(0)-reducing enzyme in this organism and the mechanism of the S(0) 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.

Purification and Characterization of a Membrane-Bound Hydrogenase from the Hyperthermophilic Archaeon Pyrococcus furiosus

Highly washed membrane preparations from cells of the hyperthermophilic archaeon Pyrococcus furiosus contain high hydrogenase activity (9.4 micromol of H(2) evolved/mg at 80 degrees C) using reduced methyl viologen as the electron donor. The enzyme was solubilized with n-dodecyl-beta-D-maltoside and purified by multistep chromatography in the presence of Triton X-100. The purified preparation contained two major proteins (alpha and beta) in an approximate 1:1 ratio with a minimum molecular mass near 65 kDa and contained approximately 1 Ni and 4 Fe atoms/mol. The reduced enzyme gave rise to an electron paramagnetic resonance signal typical of the so-called Ni-C center of mesophilic NiFe-hydrogenases. Neither highly washed membranes nor the purified enzyme used NAD(P)(H) or P. furiosus ferredoxin as an electron carrier, nor did either catalyze the reduction of elemental sulfur with H(2) as the electron donor. Using N-terminal amino acid sequence information, the genes proposed to encode the alpha and beta subunits were located in the genome database within a putative 14-gene operon (termed mbh). The deduced sequences of the two subunits (Mbh 11 and 12) were distinctly different from those of the four subunits that comprise each of the two cytoplasmic NiFe-hydrogenases of P. furiosus and show that the alpha subunit contains the NiFe-catalytic site. Six of the open reading frames (ORFs) in the operon, including those encoding the alpha and beta subunits, show high sequence similarity (>30% identity) with proteins associated with the membrane-bound NiFe-hydrogenase complexes from Methanosarcina barkeri, Escherichia coli, and Rhodospirillum rubrum. The remaining eight ORFs encode small (<19-kDa) hypothetical proteins. These data suggest that P. furiosus, which was thought to be solely a fermentative organism, may contain a previously unrecognized respiratory system in which H(2) metabolism is coupled to energy conservation.

Cellobiose Dehydrogenase from the Fungi Phanerochaete chrysosporium and Humicola insolens A FLAVOHEMOPROTEIN FROM HUMICOLA INSOLENS CONTAINS 6-HYDROXY-FAD AS THE DOMINANT ACTIVE COFACTOR

Cellobiose dehydrogenases (CDH) were purified from cellulose-grown cultures of the fungi Phanerochaete chrysosporium and Humicola insolens. The pH optimum of the cellobiose-cytochrome c oxidoreductase activity ofP. chrysosporium CDH was acidic, whereas that of H. insolens CDH was neutral. The absorption spectra of the two CDHs showed them to be typical hemoproteins, but there was a small difference in the visible region. Limited proteolysis between the heme and flavin domains was performed to investigate the cofactors. There was no difference in absorption spectrum between the heme domains ofP. chrysosporium and H. insolens CDHs. The midpoint potentials of heme at pH 7.0 were almost identical, and no difference in pH dependence was observed over the range of pH 3–9. The pH dependence of cellobiose oxidation by the flavin domains was similar to that of the native CDHs, indicating that the difference in the pH dependence of the catalytic activity between the two CDHs is because of the flavin domains. The absorption spectrum of the flavin domain fromH. insolens CDH has absorbance maxima at 343 and 426 and a broad absorption peak at 660 nm, whereas that of P. chrysosporium CDH showed a normal flavoprotein spectrum. Flavin cofactors were extracted from the flavin domains and analyzed by high-performance liquid chromatography. The flavin cofactor fromH. insolens was found to be a mixture of 60% 6-hydroxy-FAD and 40% FAD, whereas that from P. chrysosporium CDH was normal FAD. After reconstitution of the deflavo-proteins it was found that flavin domains containing 6-hydroxy-FAD were clearly active but their cellobiose oxidation rates were lower than those of flavin domains containing normal FAD. Reconstitution of flavin cofactor had no effect on the optimum pH. From these results, it is concluded that the pH dependence is not because of the flavin cofactor but is because of the protein molecule.

Pyrococcus furiosus: large-scale cultivation and enzyme purification.

Publisher Summary Pyrococcus furiosus is currently one of the most studied of the hyperthermophilic microorganisms mainly because of the relative ease with which it can be cultivated and good growth yields. The organism was originally isolated from a geothermally heated shallow sea vent off the coast of Italy by Stetter and co-workers. It grows at temperatures ranging from 70° to 105° with an optimum at 100° using complex peptide mixtures or sugars, e.g., starch, glycogen, maltose, and cellobiose as carbon and energy sources. Unlike many other heterotrophic hyperthermophiles, significant growth of P. furiosus is not obligately dependent on elemental sulfur (S°), although if it is added to the growth medium it is reduced by the organism to H 2 S. This chapter presents the methods used to grow P. furiosus up to the 600-liter scale and to prepare cell-free extracts for the large-scale purification of proteins. In addition, a summary is provided of the elution patterns of different enzymes when the cell-free extract is applied to an anion-exchange chromatography column.

On the reduction potentials of Fe and Cu-Zn containing superoxide dismutases

The reduction potentials of bovine erythrocyte copper-zinc superoxide dismutase and Escherichia coli iron superoxide dismutase were determined in EPR-monitored redox titrations in homogeneous solution. The copper-zinc enzyme is reduced and reoxidized with a midpoint potential of +120 mV versus standard hydrogen electrode (SHE) at pH 7.5. The iron enzyme can be reduced with an apparent midpoint potential of -67 mV versus SHE at pH 7.5. However, reaction with ferricyanide affords only slow, partial re-oxidation. Cyclic voltammetry of the copper-zinc enzyme in the presence of 50 mM Sc3+ at pH 4.0 using a glassy carbon electrode results in asymmetric voltammograms. The midpoint potential of the enzyme at this pH value, calculated as the average of the anodic and cathodic peak potentials, is +400 mV versus SHE. The physiological relevance of this value is limited, since EPR experiments indicated that reduction of the copper-zinc enzyme at pH 4.0 is not reversible. Consequences of the irreversible behavior of the two dismutases for the previously reported studies on their redox properties are discussed.

Heterologous expression and properties of the γ-subunit of the Fe-only hydrogenase from Thermotoga maritima

Thermotoga maritima is a hyperthermophilic bacterium that contains a complex, heterotrimeric (alpha(beta)gamma) Fe-only hydrogenase. Sequence analysis indicates that the gene encoding the smallest subunit (gamma), hydC, contains a predicted iron-sulfur cluster binding motif. However, characterization of the native gamma-subunit has been hampered by interference from and the inability to separate intact gamma-subunit from the other two subunits (alpha and beta). To investigate the function and properties of the isolated gamma-subunit, the gene encoding HydG was expressed in Escherichia coli. Two forms of the recombinant protein were obtained with molecular masses of 10 and 18 kDa, respectively. Both contained a single [2Fe-2S] cluster based on metal analysis, EPR and UV-visible spectroscopy. NH2-terminal sequencing revealed that the 10 kDa protein is a truncated form of the intact gamma-subunit and lacks the first 65 amino acid residues. The midpoint potential of the 18 kDa form was -356 mV at pH 7.0 and 25 degrees C, as measured by direct electrochemistry, and was pH dependent with a pK(ox) of 7.5 and a pK(red) of 7.7. The oxidized, recombinant gamma-subunit was stable at 80 degrees C under anaerobic conditions with a half-life greater than 24 h, as judged by the UV-visible spectrum of the [2Fe-2S] cluster. In the presence of air the protein was less stable and denatured with a half-life of approx. 2.5 h. The recombinant gamma-subunit was electron transfer competent and was efficiently reduced by pyruvate ferredoxin oxidoreductase from Pyrococcus furiosus, with a Km of 5microM and a Vmax of 9 U/mg. In contrast, native T. maritima hydrogenase holoenzyme and its separated alpha-subunit were much less effective electron donors for the gamma-subunit, with a V(max) of 0.01 U/mg and 0.1 U/mg, respectively.

[19] Fe-only hydrogenase from Thermotoga maritirria

Publisher Summary Hydrogenases catalyze the reversible oxidation of hydrogen gas (H2). The Fe-hydrogenases contain iron as the only metal and this is present in the form of both conventional and novel iron-sulfur dusters. This type of hydrogenase is found in anaerobic bacteria and in the hydrogenosomes of some unicellular, anaerobic eukaryotes. To date Fe-only hydrogenases have not been identified in archaea. This chapter focuses on the Fe-hydrogenase from the hyperthermophilic bacterium, Thermotoga maritima. This strictly anaerobic, hyperthermophilic bacterium grows optimally at 80 ° using sugars or peptides as a carbon and energy source. Herein are described the methods used to grow T. maritima and to purify and characterize its Fe-hydrogenase. So far, this is the only known hyperthermophilic example of this type of enzyme.

Differences between positive and negative ion stabilities of metal–sulfur cluster proteins: an electrospray ionization fourier transform ion cyclotron resonance study

Abstract The stability of iron–sulfur proteins during electrospray ionization in both positive ion and negative ion modes was investigated using Fourier transform ion cyclotron resonance mass spectrometry. Positive ion and negative ion mode mass spectra of iron and zinc rubredoxin from Clostridium pasteurianum and ferredoxins from Pyrococcus furiosus containing [3Fe–4S], [4Fe–4S], [3FeNi–4S], and [3FeMn–4S] clusters are compared. The results demonstrate that all clusters are stable as negative ions, whereas only the [4M–4S], (M = Fe, Mn, Ni) clusters are stable as positive ions. This is the first direct evidence of the existence of the [3FeMn–4S]-containing cluster from Pyrococcus furiosus. The formal oxidation state of each metal–sulfur cluster is determined by the mass-to-charge measurement, and is found to be the same for both positive and negative ions.

[3] Ferrodoxin from Pyrococcus furiosus

Publisher Summary Ferredoxins (Fd) are small proteins that contain iron-sulfur clusters as a redox active group. They are ubiquitous in biological systems and play integral roles in a wide variety of electron transfer processes, including respiration, photosynthesis, and fermentation. There are two main varieties: the 4Fe-type, which contain one and sometimes two cubane-type [4Fe-4S] clusters, and the 2Fe-type, which contain a [2Fe-2S] cluster (the S represents inorganic sulfide). Both types are covalently attached to their proteins via Fe-S bonds between the Fe atoms of the cluster and the sulfur atoms of four cysteine residues. Ferredoxins have been purified from a variety of microbial and eukaryotic sources and have been extensively studied. The first to be characterized from a hyperthermophile was from Pyrococcus furiosus ; in fact, this was one of the first proteins to be obtained from such an organism. The ease of purification and remarkable stability of P. furiosus ferredoxin, together with the availability of the recombinant protein, has enabled it to become one of the best studied of all ferredoxins, with numerous investigations into the properties of both the protein and of its single [4Fe-4S] cluster. This chapter describes the purification of ferredoxin from P. furiosus , and the purification of the recombinant protein from Escherichia coli , together with several mutant forms. Some of the methods that have been developed in characterizing this protein are also described in the chapter.