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, data analysis, and couples structural analysis with automated archiving. We subjected 50 representative proteins, mostly from Pyrococcus furiosus, to this pipeline, revealing 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.

Superoxide reductase from the hyperthermophilic anaerobe Pyrococcus furiosus uses electrons from reduced nicotinamide adenine dinucleotide phosphate, by way of rubredoxin and an oxidoreductase, to reduce superoxide to hydrogen peroxide, which is then reduced to water by peroxidases. Unlike superoxide dismutase, the enzyme that protects aerobes from the toxic effects of oxygen, SOR does not catalyze the production of oxygen from superoxide and therefore confers a selective advantage on anaerobes. Superoxide reductase and associated proteins are catalytically active 80 degrees C below the optimum growth temperature (100 degrees C) of P. furiosus, conditions under which the organism is likely to be exposed to oxygen.

Superoxide reductase (SOR) is a blue non-heme iron protein that functions in anaerobic microbes as a defense mechanism against reactive oxygen species by catalyzing the reduction of superoxide to hydrogen peroxide [Jenney, F. E., Jr., Verhagen, M. F. J. M., Cui, X. , and Adams, M. W. W. (1999) Science 286, 306-309]. Crystal structures of SOR from the hyperthermophilic archaeon Pyrococcus furiosus have been determined in the oxidized and reduced forms to resolutions of 1.7 and 2.0 A, respectively. SOR forms a homotetramer, with each subunit adopting an immunoglobulin-like beta-barrel fold that coordinates a mononuclear, non-heme iron center. The protein fold and metal center are similar to those observed previously for the homologous protein desulfoferrodoxin from Desulfovibrio desulfuricans [Coelho, A. V., Matias, P., Fülöp, V., Thompson, A., Gonzalez, A., and Carrondo, M. A. (1997) J. Bioinorg. Chem. 2, 680-689]. Each iron is coordinated to imidazole nitrogens of four histidines in a planar arrangement, with a cysteine ligand occupying an axial position normal to this plane. In two of the subunits of the oxidized structure, a glutamate carboxylate serves as the sixth ligand to form an overall six-coordinate, octahedral coordinate environment. In the remaining two subunits, the sixth coordination site is either vacant or occupied by solvent molecules. The iron centers in all four subunits of the reduced structure exhibit pentacoordination. The structures of the oxidized and reduced forms of SOR suggest a mechanism by which superoxide accessibility may be controlled and define a possible binding site for rubredoxin, the likely physiological electron donor to SOR.

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.

In attempts to develop a method of introducing DNA into Pyrococcus furiosus, we discovered a variant within the wild-type population that is naturally and efficiently competent for DNA uptake. A pyrF gene deletion mutant was constructed in the genome, and the combined transformation and recombination frequencies of this strain allowed marker replacement by direct selection using linear DNA. We have demonstrated the use of this strain, designated COM1, for genetic manipulation. Using genetic selections and counterselections based on uracil biosynthesis, we generated single-and double-deletion mutants of the two gene clusters that encode the two cytoplasmic hydrogenases. The COM1 strain will provide the basis for the development of more sophisticated genetic tools allowing the study and metabolic engineering of this important hyperthermophile.It would be difficult to overestimate the contribution of genetic manipulation to the study of any biological system, and it is an essential tool for the metabolic engineering of biosynthetic and substrate utilization pathways. This is particularly true for the archaea since, in spite of their environmental and industrial importance, coupled with their unique molecular features, much remains to be learned about their biology (2). The marine hyperthermophilic anaerobe Pyrococcus furiosus is of special interest not only for its ability to grow optimally at 100°C and the implications of this trait for its biology but also for industrial applications of its enzymes, as well as its capacity to produce hydrogen efficiently (4, 13, 44). The ability to apply genetic analyses of P. furiosus to underpin existing biochemical and molecular studies will contribute greatly to the establishment of P. furiosus as a model organism, particularly for biological hydrogen production.The development of genetic systems in the archaea, in general, presents many unique challenges given the extreme growth requirements of many of these organisms. To date, genetic systems of various levels of sophistication have been developed for representatives of all major groups of archaea, including halophiles, methanogens, thermoacidophiles, and hyperthermophiles (2,6,30,40,43,46). A variety of transformation methods are being used, including electroporation, heat shock with or without CaCl 2 treatment, phage-mediated transduction, spheroplast transformation, liposomes, and, very recently, even conjugation with Escherichia coli (2, 12). Transformation via natural competence has been reported in three archaeal species, in comparison to over 60 bacterial species that are known to exhibit this trait (16,36). Two of them are the methanogens Methanococcus voltae PS (7, 27) and Methanobacterium thermoautotrophicum Marburg (47); however, transformation frequencies were low, and there have been no follow-up studies regarding natural competence. The other is the hyperthermophile Thermococcus kodakarensis, which has an optimal growth temperature of 85°C. Its natural competence has enabled the development of genetic tools fo...

The combination of UV/visible/NIR absorption, CD and variable-temperature magnetic circular dichroism (VTMCD), EPR, and X-ray absorption (XAS) spectroscopies has been used to investigate the electronic and structural properties of the oxidized and reduced forms of Pyrococcus furiosus superoxide reductase (SOR) as a function of pH and exogenous ligand binding. XAS shows that the mononuclear ferric center in the oxidized enzyme is very susceptible to photoreduction in the X-ray beam. This observation facilitates interpretation of ground- and excited-state electronic properties and the EXAFS results for the oxidized enzyme in terms of the published X-ray crystallographic data (Yeh, A. P.; Hu, Y.; Jenney, F. E.; Adams, M. W. W.; Rees, D. C. Biochemistry 2000, 39, 2499-2508). In the oxidized state, the mononuclear ferric active site has octahedral coordination with four equatorial histidyl ligands and axial cysteinate and monodentate glutamate ligands. Fe EXAFS are best fit by one Fe-S at 2.36 A and five Fe-N/O at an average distance of 2.12 A. The EPR-determined spin Hamiltonian parameters for the high-spin (S = (5)/(2)) ferric site in the resting enzyme, D = -0.50 +/- 0.05 cm(-1) and E/D = 0.06, are consistent with tetragonally compressed octahedral coordination geometry. UV/visible absorption and VTMCD studies facilitate resolution and assignment of pi His --> Fe(3+)(t(2g)) and (Cys)S(p) --> Fe(3+)(t(2g)) charge-transfer transitions, and the polarizations deduced from MCD saturation magnetization studies indicate that the zero-field splitting (compression) axis corresponds to one of the axes with trans-histidyl ligands. EPR and VTMCD studies provide evidence of azide, ferrocyanide, hydroxide, and cyanide binding via displacement of the glutamate ligand. For azide, ferrocyanide, and hydroxide, ligand binding occurs with retention of the high-spin (S = 5/2) ground state (E/D = 0.27 and D < 0 for azide and ferrocyanide; E/D = 0.25 and D = +1.1 +/- 0.2 cm(-1) for hydroxide), whereas cyanide binding results in a low-spin (S = 1/2) species (g = 2.29, 2.25, 1.94). The ground-state and charge-transfer/ligand-field excited-state properties of the low-spin cyanide-bound derivative are shown to be consistent with a tetragonally elongated octahedral coordination with the elongation axis corresponding to an axis with trans-histidyl ligands. In the reduced state, the ferrous site of SOR is shown to have square-pyramidal coordination geometry in frozen solution with four equatorial histidines and one axial cysteine on the basis of XAS and UV and NIR VTMCD studies. Fe EXAFS are best fit by one Fe-S at 2.37 A and four Fe-N/O at an average distance of 2.15 A. VTMCD reveals a high-spin (S = 2) ferrous site with (Cys)S(p) --> Fe(2+) charge-transfer transitions in the UV region and (5)T(2g) --> (5)E(g) ligand-field transitions in the NIR region at 12400 and <5000 cm(-1). The ligand-field bands indicate square-pyramidal coordination geometry with 10Dq < 8700 cm(-1) and a large excited-state splitting, Delta (5)E(g) > 7400 cm(-1). Analys...

A persistent puzzle in the field of biological electron transfer is the conserved iron-sulfur cluster motif in both high potential iron-sulfur protein (HiPIP) and ferredoxin (Fd) active sites. Despite this structural similarity, HiPIPs react oxidatively at physiological potentials, whereas Fds are reduced. Sulfur K-edge x-ray absorption spectroscopy uncovers the substantial influence of hydration on this variation in reactivity. Fe-S covalency is much lower in natively hydrated Fd active sites than in HiPIPs but increases upon water removal; similarly, HiPIP covalency decreases when unfolding exposes an otherwise hydrophobically shielded active site to water. Studies on model compounds and accompanying density functional theory calculations support a correlation of Fe-S covalency with ease of oxidation and therefore suggest that hydration accounts for most of the difference between Fd and HiPIP reduction potentials.

Rubredoxin from the hyperthermophile Pyrococcus furiosus is the most thermostable protein characterized to date with an estimated global unfolding rate of 10 ؊6 s ؊1 at 100°C. In marked contrast to these slow global dynamics, hydrogen exchange experiments here demonstrate that conformational opening for solvent access occurs in the Ϸmillisecond time frame or faster at 28°C for all amide positions. Under these conditions all backbone amides with exchange protection factors between 10 4 and 10 6 , for which EX2 exchange kinetics were directly verified, have exchange activation energy values within 2-3 kcal͞mol of that observed for unstructured peptides. The conformational flexibility of this protein is thus sufficient for water and base catalyst access to the exchanging amide with quite limited structural disruption. The common hypothesis that enhanced conformational rigidity in the folded native state underlies the increased thermal stability of hyperthermophile proteins is not supported by these data.