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Iron is an essential element for the hyperthermophilic archaeon Pyrococcus furiosus , and many of its iron-containing enzymes have been characterized. How iron assimilation is regulated, however, is unknown. The genome sequence contains genes encoding two putative iron-responsive transcription factors, DtxR and Fur. Global transcriptional profiles of the dtxR deletion mutant (ΔDTXR) and the parent strain under iron-sufficient and iron-limited conditions indicated that DtxR represses the expression of the genes encoding two putative iron transporters, Ftr1 and FeoAB, under iron-sufficient conditions. Under iron limitation, DtxR represses expression of the gene encoding the iron-containing enzyme aldehyde ferredoxin oxidoreductase and a putative ABC-type transporter. Analysis of the dtxR gene sequence indicated an incorrectly predicted translation start site, and the corrected full-length DtxR protein, in contrast to the truncated version, specifically bound to the promoters of ftr1 and feoAB , confirming its role as a transcription regulator. Expression of the gene encoding Ftr1 was dramatically upregulated by iron limitation, but no phenotype was observed for the ΔFTR1 deletion mutant under iron-limited conditions. The intracellular iron concentrations of ΔFTR1 and the parent strain were similar, suggesting that under the conditions tested, Ftr1 is not an essential iron transporter despite its response to iron. In contrast to DtxR, the Fur protein appears not to be a functional regulator in P. furiosus , since it did not bind to the promoters of any of the iron-regulated genes and the deletion mutant (ΔFUR) revealed no transcriptional responses to iron availability. DtxR is therefore the key iron-responsive transcriptional regulator in P. furiosus .

Inductively Coupled Plasma Mass Spectrometry (ICP-MS) is a highly sensitive elemental analysis technique that has been widely applied in many fields. Here we describe applications using a broad-scale approach to examine metal usage in biology. These protocols address questions such as: Which elements from the surrounding environment are taken up into the cells of a given organism? How does this vary between different organisms? Which metals are “bound” and which are “free”? With which type(s) of proteins are the “bound” metals associated? This allows for investigations into several branches of bioinorganic chemistry including uptake, toxicity, detoxification, bioremediation, and the discovery of new uses for elements. In the protocols presented here, there is an emphasis on metals, and, more narrowly, on transition metals because these comprise the majority of tightly protein-bound, low-abundance elements. Nonmetals, metalloids, main-group metals, and f-block metals are also analyzed and investigated. The sample preparation procedure requires acid lability for detection, which likely eliminates certain nonmetals, such as selenium. However, one advantage of the protocols described is that they are readily adapted to measure any element of interest. Curr. Protoc. Chem. Biol. 4:249-274 © 2012 by John Wiley & Sons, Inc. Keywords: metalloproteome; metallome; ICP-MS; metalloprotein; metalloenzyme; bioinorganic chemistry; metallobiochemistry

The vital nature of metal uptake and balance in biology is evident in the highly evolved strategies to facilitate metal homeostasis in all three domains of life. Several decades of study on metals and metalloproteins have revealed numerous essential bio-metal functions. Recent advances in mass spectrometry, X-ray scattering/absorption, and proteomics have exposed a much broader usage of metals in biology than expected. Even elements such as uranium, arsenic, and lead are implicated in biological processes as part of an emerging and expansive view of bio-metals. Here we discuss opportunities and challenges for established and newer approaches to study metalloproteins with a focus on technologies that promise to rapidly expand our knowledge of metalloproteins and metal functions in biology.

Assembly of genes into operons is generally viewed as an important process during the continual adaptation of microbes to changing environmental challenges. However, the genome reorganization events that drive this process are also the roots of instability for existing operons. We have determined that there exists a statistically significant trend that correlates the proportion of genes encoded in operons in archaea to their phylogenetic lineage. We have further characterized how microbes deal with operon instability by mapping and comparing transcriptome architectures of four phylogenetically diverse extremophiles that span the range of operon stabilities observed across archaeal lineages: a photoheterotrophic halophile (Halobacterium salinarum NRC-1), a hydrogenotrophic methanogen (Methanococcus maripaludis S2), an acidophilic and aerobic thermophile (Sulfolobus solfataricus P2), and an anaerobic hyperthermophile (Pyrococcus furiosus DSM 3638). We demonstrate how the evolution of transcriptional elements (promoters and terminators) generates new operons, restores the coordinated regulation of translocated, inverted, and newly acquired genes, and introduces completely novel regulation for even some of the most conserved operonic genes such as those encoding subunits of the ribosome. The inverse correlation (r = –0.92) between the proportion of operons with such internally located transcriptional elements and the fraction of conserved operons in each of the four archaea reveals an unprecedented view into varying stages of operon evolution. Importantly, our integrated analysis has revealed that organisms adapted to higher growth temperatures have lower tolerance for genome reorganization events that disrupt operon structures.

Virtually all cellular processes are carried out by dynamic molecular assemblies or multiprotein complexes, the compositions of which are largely undefined. They cannot be predicted solely from bioinformatics analyses nor are there well defined techniques currently available to unequivocally identify protein complexes (PCs). To address this issue, we attempted to directly determine the identity of PCs from native microbial biomass using Pyrococcus furiosus, a hyperthermophilic archaeon that grows optimally at 100 degrees C, as the model organism. Novel PCs were identified by large scale fractionation of the native proteome using non-denaturing, sequential column chromatography under anaerobic, reducing conditions. A total of 967 distinct P. furiosus proteins were identified by mass spectrometry (nano LC-ESI-MS/MS), representing approximately 80% of the cytoplasmic proteins. Based on the co-fractionation of proteins that are encoded by adjacent genes on the chromosome, 106 potential heteromeric PCs containing 243 proteins were identified, only 20 of which were known or expected. In addition to those of unknown function, novel and uncharacterized PCs were identified that are proposed to be involved in the metabolism of amino acids (10), carbohydrates (four), lipids (two), vitamins and metals (three), and DNA and RNA (nine). A further 30 potential PCs were classified as tentative, and the remaining potential PCs (13) were classified as weakly interacting. Some major advantages of native biomass fractionation for PC identification are that it provides a road map for the (partial) purification of native forms of novel and uncharacterized PCs, and the results can be utilized for the recombinant production of low abundance PCs to provide enough material for detailed structural and biochemical analyses.

We have performed a comprehensive characterization of global molecular changes for a model organism Pyrococcus furiosus using transcriptomic (DNA microarray), proteomic, and metabolomic analysis as it undergoes a cold adaptation response from its optimal 95 to 72 degrees C. Metabolic profiling on the same set of samples shows the down-regulation of many metabolites. However, some metabolites are found to be strongly up-regulated. An approach using accurate mass, isotopic pattern, database searching, and retention time is used to putatively identify several metabolites of interest. Many of the up-regulated metabolites are part of an alternative polyamine biosynthesis pathway previously established in a thermophilic bacterium Thermus thermophilus. Arginine, agmatine, spermidine, and branched polyamines N4-aminopropylspermidine and N4-( N-acetylaminopropyl)spermidine were unambiguously identified based on their accurate mass, isotopic pattern, and matching of MS/MS data acquired under identical conditions for the natural metabolite and a high purity standard. Both DNA microarray and semiquantitative proteomic analysis using a label-free spectral counting approach indicate the down-regulation of a large majority of genes with diverse predicted functions related to growth such as transcription, amino acid biosynthesis, and translation. Some genes are, however, found to be up-regulated through the measurement of their relative mRNA and protein levels. The complimentary information obtained by the various "omics" techniques is used to catalogue and correlate the overall molecular changes.

This chapter explores the metal chemistry of different types of marine hydrothermal environments, the metal requirements of hyperthermophiles, the nature and constraints of their interactions with metals, and the biogeochemical implications of hyperthermophile-metal interactions. Metalloenzymes form a major part of metalloproteomes and reflect the bioavailability of metals within a given environment that are (or were at some point during their evolution) the most accessible. Dissimilatory iron reduction has been studied extensively in just four groups of hyperthermophilic archaea and one hyperthermophilic bacterium. The reduction potential and pH of an environment also appear to affect the growth rates of hyperthermophiles on iron. It is clear that there is extensive interaction between metals and hyperthermophilic microbes from hydrothermal environments, and yet the study of the nature of these interactions is in its infancy. Dissimilatory metal reduction may be limited by the organism’s ability to transfer electrons from its cytoplasm to the typically insoluble metal acceptor. Therefore, it is necessary to determine the respiratory mechanisms and how they lead to energy conservation, especially because the physiological mechanisms for metal reduction in hyperthermophiles differ from those found in mesophilic bacteria based on the lack of polyheme c-type cytochromes, a periplasm, and an outer cell wall membrane. Further study of hyperthermophilemetal interactions in hydrothermal environments will create a new understanding of the basic principles that govern a broad array of metabolic processes and a significant portion of the Earth’s biosphere.

Although as many as half of all proteins are thought to require a metal cofactor, the metalloproteomes of microorganisms remain relatively unexplored. Microorganisms from different environments are likely to vary greatly in the metals that they assimilate, not just among the metals with well-characterized roles but also those lacking any known function. Herein we investigated the metal utilization of two microorganisms that were isolated from very similar environments and are of interest because of potential roles in the immobilization of heavy metals, such as uranium and chromium. The metals assimilated and their concentrations in the cytoplasm of Desulfovibrio vulgaris strain Hildenborough (DvH) and Enterobacter cloacae strain Hanford (EcH) varied dramatically, with a larger number of metals present in Enterobacter. For example, a total of 9 and 19 metals were assimilated into their cytoplasmic fractions, respectively, and DvH did not assimilate significant amounts of zinc or copper whereas EcH assimilated both. However, bioinformatic analysis of their genome sequences revealed a comparable number of predicted metalloproteins, 813 in DvH and 953 in EcH. These allowed some rationalization of the types of metal assimilated in some cases (Fe, Cu, Mo, W, V) but not in others (Zn, Nd, Ce, Pr, Dy, Hf and Th). It was also shown that U binds an unknown soluble protein in EcH but this incorporation was the result of extracellular U binding to cytoplasmic components after cell lysis.

The nucleotide sequence was obtained for the hypE gene in the cluster of structural and accessory genes required for the assembly and functioning of the membrane-bound, dimeric, (NiFe)hydrogenase in Azotobacter vinelandii. The hypE gene encodes a polypeptide of 341 amino acid residues which is rich in alanine, glycine, valine and proline and appears to be involved in maturation of the enzyme because chromosomal mutations in hypE block O2-dependent H2-oxidation and affect the amount, processing and localization of the (NiFe) hydrogenase alpha-subunit. The complete nucleotide sequence for the hydrogenase gene cluster in A. vinelandii has now been assembled into a contiguous sequence of 13,914 bp containing 16 potential genes which appear to be transcribed undirectionally. They are arranged in the order hoxK, hoxG, hoxZ, hoxM, hoxL, hoxO, hoxQ, hoxR, hoxT, hoxV, hypA, hypB, hypF, hypC, hypD and hypE. This cluster closely resembles those described for comparable (NiFe) hydrogenases in other bacteria.

H2 oxidation in Azotobacter vinelandii is catalyzed by a membrane-bound, alpha beta dimeric [NiFe] hydrogenase. Maturation of the enzyme involves cleavage of a putative N-terminal signal sequence in the beta subunit and removal of 15 amino acids from the C terminus of the alpha subunit. Cells limited for nickel exhibited low hydrogenase activities and contained an apparently large form of the alpha subunit. Addition of nickel to such cells increased hydrogenase activities fivefold over 2 h. The increase in the first hour did not require transcription and translation and correlated with processing of the large form of the alpha subunit (pre-alpha) to the small form (alpha) resembling the alpha subunit from the purified enzyme. In vivo, pre-alpha appeared soluble whereas the majority of alpha was membrane bound. Processing of pre-alpha to alpha was reproduced in vitro in membrane-depleted extracts of nickel-limited cells. Processing specifically required the addition of Ni2+, whereas Co2+, Cu2+, Ca2+, Fe2+, Mn2+, and Zn2+ were ineffective. However, Zn2+, Co2+, and Cu2+ inhibited nickel-dependent processing. Mg-ATP and Mg-GTP stimulated processing, whereas anaerobic conditions and/or the addition of dithiothreitol and sodium dithionite was unnecessary. Processing was not inhibited by the protease inhibitors phenylmethylsulfonyl fluoride, E64, and pepstatin.

Metal ion cofactors afford proteins virtually unlimited catalytic potential, enable electron transfer reactions and have a great impact on protein stability. Consequently, metalloproteins have key roles in most biological processes, including respiration (iron and copper), photosynthesis (manganese) and drug metabolism (iron). Yet, predicting from genome sequence the numbers and types of metal an organism assimilates from its environment or uses in its metalloproteome is currently impossible because metal coordination sites are diverse and poorly recognized. We present here a robust, metal-based approach to determine all metals an organism assimilates and identify its metalloproteins on a genome-wide scale. This shifts the focus from classical protein-based purification to metal-based identification and purification by liquid chromatography, high-throughput tandem mass spectrometry (HT-MS/MS) and inductively coupled plasma mass spectrometry (ICP-MS) to characterize cytoplasmic metalloproteins from an exemplary microorganism (Pyrococcus furiosus). Of 343 metal peaks in chromatography fractions, 158 did not match any predicted metalloprotein. Unassigned peaks included metals known to be used (cobalt, iron, nickel, tungsten and zinc; 83 peaks) plus metals the organism was not thought to assimilate (lead, manganese, molybdenum, uranium and vanadium; 75 peaks). Purification of eight of 158 unexpected metal peaks yielded four novel nickel- and molybdenum-containing proteins, whereas four purified proteins contained sub-stoichiometric amounts of misincorporated lead and uranium. Analyses of two additional microorganisms (Escherichia coli and Sulfolobus solfataricus) revealed species-specific assimilation of yet more unexpected metals. Metalloproteomes are therefore much more extensive and diverse than previously recognized, and promise to provide key insights for cell biology, microbial growth and toxicity mechanisms.

Azotobacter vinelandii contains a heterodimeric, membrane-bound [NiFe]hydrogenase capable of catalyzing the reversible oxidation of H2. The beta and alpha subunits of the enzyme are encoded by the structural genes hoxK and hoxG, respectively, which appear to form part of an operon that contains at least one further potential gene (open reading frame 3 [ORF3]). In this study, determination of the nucleotide sequence of a region of 2,344 bp downstream of ORF3 revealed four additional closely spaced or overlapping ORFs. These ORFs, ORF4 through ORF7, potentially encode polypeptides with predicted masses of 22.8, 11.4, 16.3, and 31 kDa, respectively. Mutagenesis of the chromosome of A. vinelandii in the area sequenced was carried out by introduction of antibiotic resistance gene cassettes. Disruption of hoxK and hoxG by a kanamycin resistance gene abolished whole-cell hydrogenase activity coupled to O2 and led to loss of the hydrogenase alpha subunit. Insertional mutagenesis of ORF3 through ORF7 with a promoterless lacZ-Kmr cassette established that the region is transcriptionally active and involved in H2 oxidation. We propose to call ORF3 through ORF7 hoxZ, hoxM, hoxL, hoxO, and hoxQ, respectively. The predicted hox gene products resemble those encoded by genes from hydrogenase-related operons in other bacteria, including Escherichia coli and Alcaligenes eutrophus.

We present an efficient pipeline enabling high-throughput analysis of protein structure in solution with small angle X-ray scattering (SAXS). Our SAXS pipeline combines automated sample handling of microliter volumes, temperature and anaerobic control, rapid data collection and data analysis, and couples structural analysis with automated archiving. We subjected 50 representative proteins, mostly from Pyrococcus furiosus, to this pipeline and found that 30 were multimeric structures in solution. SAXS analysis allowed us to distinguish aggregated and unfolded proteins, define global structural parameters and oligomeric states for most samples, identify shapes and similar structures for 25 unknown structures, and determine envelopes for 41 proteins. We believe that high-throughput SAXS is an enabling technology that may change the way that structural genomics research is done.

The structure of prolidase from the hyperthermophilic archaeon Pyrococcus furiosus (Pfprol) has been solved and refined at 2.0 A resolution. This is the first structure of a prolidase, i.e., a peptidase specific for dipeptides having proline as the second residue. The asymmetric unit of the crystals contains a homodimer of the enzyme. Each of the two protein subunits has two domains. The C-terminal domain includes the catalytic site, which is centered on a dinuclear metal cluster. In the as-isolated form of Pfprol, the active-site metal atoms are Co(II) [Ghosh, M., et al. (1998) J. Bacteriol. 180, 4781-9]. An unexpected finding is that in the crystalline enzyme the active-site metal atoms are Zn(II), presumably as a result of metal exchange during crystallization. Both of the Zn(II) atoms are five-coordinate. The ligands include a bridging water molecule or hydroxide ion, which is likely to act as a nucleophile in the catalytic reaction. The two-domain polypeptide fold of Pfprol is similar to the folds of two functionally related enzymes, aminopeptidase P (APPro) and creatinase. In addition, the catalytic C-terminal domain of Pfprol has a polypeptide fold resembling that of the sole domain of a fourth enzyme, methionine aminopeptidase (MetAP). The active sites of APPro and MetAP, like that of Pfprol, include a dinuclear metal center. The metal ligands in the three enzymes are homologous. Comparisons with the molecular structures of APPro and MetAP suggest how Pfprol discriminates against oligopeptides and in favor of Xaa-Pro substrates. The crystal structure of Pfprol was solved by multiple-wavelength anomalous dispersion. The crystals yielded diffraction data of relatively high quality and resolution, despite the fact that one of the two protein subunits in the asymmetric unit was found to be significantly disordered. The final R and R(free) values are 0.24 and 0.28, respectively.

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.

Publisher Summary In most aerobic organisms the oxidative decarboxylation of pyruvate to acetyl-CoA is catalyzed by a large NAD-dependent pyruvate dehydrogenase complex. However, the same reaction in anaerobic organisms is catalyzed by a reversible, ferredoxin-dependent, pyruvate oxidoreductase (POR). PORs have been purified from archaea, bacteria, and anaerobic eukaryotic protists. A number of organisms, mostly archaea, are known to contain at least three 2-keto acid oxidoreductases (KORs) in addition to POR, and these are able to utilize substrates other than pyruvate. The enzymes are indolepyruvate ferredoxin oxidoreductase (IOR), 2-ketoisovalerate ferredoxin oxidoreductase (VOR), and 2-ketoglutarate ferredoxin oxidoreductase (KGOR). This article describes the purification of POR, VOR, and IOR from P. furiosus and KGOR from T. litoralis and some of the molecular and catalytic properties of this enzyme family.

The hyperthermophilic archaeon Pyrococcus furiosus grows 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 (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.

Publisher Summary An early study with Pyrococcus furiosus showed that growth of this hyperthermophilic archaeon is stimulated by the addition of tungsten to the medium. Subsequently, three distinct tungsten-containing enzymes were purified from this and related organisms. They are aldehyde ferredoxin oxidoreductase (AOR), which has been purifed from P. furiosus , Pyrococcus strain ES-4, and Thermococcus strain ES-1, formaldehyde ferredoxin oxidoreductase (FOR), which has been purified from Thermococcus litoralis and P. furiosus, 6 and glyceraldehyde-3-phosphate ferredoxin oxidoreductase (GAPOR), which has been obtained so far only from P. furiosus . All three enzymes catalyze the oxidation of various types of aldehyde using ferredoxin (Fd) as the physiological electron acceptor. This chapter describes the purification of AOR, FOR, and GAPOR from P. furiosus and summarizes some of their properties.

Pyruvate ferredoxin oxidoreductase (POR) from the hyperthermophilic archaeon Pyrococcus furiosus (Pf) catalyzes the final oxidative step in carbohydrate fermentation in which pyruvate is oxidized to acetyl-CoA and CO2, coupled to the reduction of ferredoxin (Fd). POR is composed of two 'catalytic units' of molecular mass approximately 120 kDa. Each unit consists of four subunits, alpha beta gamma delta, with masses of approximately 44, 36, 20, and 12 kDa, respectively, and contains at least two [4Fe-4S] clusters. The precise mechanism of catalysis and the role of the individual subunits are not known. The gene encoding the delta-subunit of Pf POR has been expressed in E. coli, and the protein was purified after reconstitution with iron and sulfide. The reconstituted delta-subunit (recPOR-delta) is monomeric with a mass of 11 879 +/- 1.2 Da as determined by mass spectrometry, in agreement with that predicted from the gene sequence. Purified recPOR-delta contains 8 Fe mol/mol and remained intact when incubated at 85 degreesC for 2 h, as judged by its visible absorption properties. The reduced form of the protein exhibited an EPR spectrum characteristic of two, spin-spin interacting [4Fe-4S]1+ clusters. When compared with the EPR properties of the reduced holoenzyme, the latter was shown to contain a third [4Fe-4S]1+ cluster in addition to the two within the delta-subunit. The reduction potential of the two 4Fe clusters in isolated recPOR-delta (-403 +/- 8 mV at pH 8.0 and 24 degreesC) decreased linearly with temperature (-1.55 mV/ degreesC) up to 82 degreesC. RecPOR-delta replaced Pf Fd as an in vitro electron carrier for two oxidoreductases from Pf, POR and Fd:NADP oxidoreductase, and the POR holoenzyme displayed a higher apparent affinity for its own subunit (apparent Km = 1.0 microM at 80 degreesC) than for Fd (apparent Km = 4.4 microM). The molecular and spectroscopic properties and amino acid sequence of the isolated delta-subunit suggest that it evolved from an 8Fe-type Fd by the addition of approximately 40 residues at the N-terminus, and that this extension enabled it to interact with additional subunits within POR.

High throughput solution structural analyses by small angle X-ray scattering efficiently enables the characterization of shape and assembly for nearly any purified protein. Crystallography has provided a deep and broad survey of macromolecular structure. Shape and assembly from SAXS in combination with available structures is often enough to answer critical mechanistic questions both enhancing the value of a structure and obviating larger crystallographic projects. Moreover, SAXS is a solution based technique, sample requirement are modest and compatible with many other biophysical methods. Here we present our high throughput SAXS data collection and analysis pipeline as applied to structural genomics targets, and metabolic pathways. Our goals of metabolic engineering and understanding protein mediated reactions rely on knowing the shape and assembly state of reactive complexes under an array of conditions. Given the number of gene products involved in metabolic networks, SAXS will play an important role in characterizing the structure of each individually, in complex with partners, and in various contexts. SAXS is well positioned to bridge the rapid output of bioinformatics and the relatively slow output of high resolution structural techniques.

The Azotobacter vinelandii [NiFe]hydrogenase-encoding structural genes were isolated from an A. vinelandii genomic cosmid library. Nucleotide (nt) sequence analysis showed that the two genes, hoxK and hoxG, which encode the small and large subunits of the enzyme, respectively, form part of an operon that contains at least one other gene. The hoxK gene encodes a polypeptide of 358 amino acids (aa) (39,209 Da). The deduced aa sequence encodes a possible 45-aa N-terminus extension, not present in the purified A. vinelandii hydrogenase small subunit, which could be a cellular targeting sequence. The hoxG gene is downstream form, and overlaps hoxK by 4 nt and encodes a 602-aa polypeptide of 66,803 Da. The hoxK and hoxG gene products display homology to aa sequences of hydrogenase small and large subunits, respectively, from other organisms. The hoxG gene lies 16 nt upstream from a third open reading frame which could encode a 27,729-Da (240-aa) hydrophobic polypeptide containing 53% nonpolar and 11% aromatic aa. The significance of this possible third gene is not known at present.

This paper describes a method for the rapid isolation of phycobilisomes using a cationic detergent, CTAB (cetyltrimethylammonium bromide). The method has distinct advantages over those currently in use in that (i) release of intact phycobilisomes from cells in the presence of CTAB occurs in 40 s (as compared to 40–60 min of incubation required with Triton X-100), thereby reducing the chances of proteolysis of the component phycobiliproteins; and (ii) these phycobilisome preparations have reduced chlorophyll contamination in the initial stages. In addition this method also helps retain the structural and functional properties, as evidenced by spectroscopy and sodium dodecyl sulfate-polyacrylamide gel analysis.

Two electrophoretic forms of the large subunit of the soluble periplasmic [NiFe] hydrogenase from Desulfovibrio gigas have been detected by Western analysis. The faster moving form co-migrates with the large subunit from purified, active enzyme. Amino acid sequence and composition of the C-terminal tryptic peptide of the large subunit from purified hydrogenase revealed that it is 15 amino acids shorter than that predicted by the nucleotide sequence. Processing of the nascent large subunit occurs by C-terminal cleavage between His536 and Val537, residues which are highly conserved among [NiFe] hydrogenases. Mutagenesis of the analogous residues, His582 and Val583, in the E. coli hydrogenase-1 (HYD1) large subunit resulted in significant decrease in processing and HYD1 activity.

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.

Background Metal-containing proteins comprise a diverse and sizable category within the proteomes of organisms, ranging from proteins that use metals to catalyze reactions to proteins in which metals play key structural roles. Unfortunately, reliably predicting that a protein will contain a specific metal from its amino acid sequence is not currently possible. We recently developed a generally-applicable experimental technique for finding metalloproteins on a genome-wide scale. Applying this metal-directed protein purification approach (ICP-MS and MS/MS based) to the prototypical microbe Pyrococcus furiosus conclusively demonstrated the extent and diversity of the uncharacterized portion of microbial metalloproteomes since a majority of the observed metal peaks could not be assigned to known or predicted metalloproteins. However, even using this technique, it is not technically feasible to purify to homogeneity all metalloproteins in an organism. In order to address these limitations and complement the metal-directed protein purification, we developed a computational infrastructure and statistical methodology to aid in the pursuit and identification of novel metalloproteins. Results We demonstrate that our methodology enables predictions of metal-protein interactions using an experimental data set derived from a chromatography fractionation experiment in which 870 proteins and 10 metals were measured over 2,589 fractions. For each of the 10 metals, cobalt, iron, manganese, molybdenum, nickel, lead, tungsten, uranium, vanadium, and zinc, clusters of proteins frequently occurring in metal peaks (of a specific metal) within the fractionation space were defined. This resulted in predictions that there are from 5 undiscovered vanadium- to 13 undiscovered cobalt-containing proteins in Pyrococcus furiosus. Molybdenum and nickel were chosen for additional assessment producing lists of genes predicted to encode metalloproteins or metalloprotein subunits, 22 for nickel including seven from known nickel-proteins, and 20 for molybdenum including two from known molybdo-proteins. The uncharacterized proteins are prime candidates for metal-based purification or recombinant approaches to validate these predictions. Conclusions We conclude that the largely uncharacterized extent of native metalloproteomes can be revealed through analysis of the co-occurrence of metals and proteins across a fractionation space. This can significantly impact our understanding of metallobiochemistry, disease mechanisms, and metal toxicity, with implications for bioremediation, medicine and other fields.