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The D14C variant of Pyrococcus furiosus ferredoxin provides an extraordinary framework to investigate a [3Fe–4S] cluster at two oxidation levels and compare the results to its physiologic [4Fe–4S] counterpart in the very same protein. Our spectroscopic and computational study reveals vibrational property changes related to the electronic and structural aspects of both Fe–S clusters. [ABSTRACT FROM AUTHOR]

The ferrodoxin (Fd) of the Pyrococcus furiosus is distinct from other Fds derived from hyperthermophilic bacteria since it exhibits a number of rare properties. The Fd of these archaeon strains were found to possess a highly-stable molecular heterogeneity, an Asp cluster ligand and a flexible disulfide bond. Although other hyperthermophilic archaea a 13-member alpha helix bond, that of Pyrococcus furiosus feature a number of extensions which lead to the formation of tertiary contacts.

Nuclear inelastic scattering of 57Fe labeled [NiFe] hydrogenase is shown to give information on different states of the enzyme. It was thus possible to detect and assign FeCO and FeCN bending and stretching vibrations of the active site outside the spectral range of the FeS cluster normal modes. [ABSTRACT FROM AUTHOR]

Blick ins Zentrum: Die Methode der inelastischen kernresonanten Streuung (NRVS) wurde genutzt, um 57Fe ‐ markierte [NiFe] ‐ Hydrogenase in unterschiedlichen Zuständen des Enzyms zu untersuchen. Damit gelang es, die Fe ‐ CO ‐ und Fe ‐ CN ‐ Beuge ‐ und ‐ Streckschwingung außerhalb des Spektralbereichs der Normalmoden der Fe ‐ S ‐ Cluster zu detektieren und zuzuordnen (siehe Spektrum). [ABSTRACT FROM AUTHOR]

Structural and functional characterization of the entire protein complement (the proteome) of an organism can provide an infrastructure upon which questions about biological pathways and systems biology can be framed. The technology necessary to perform this proteome-level structural and functional characterization is under development in numerous structural genomics and functional genomics initiatives. Given the ubiquity of metal active sites in a proteome, it seems appropriate to ask whether comprehensive local structural characterization of metal sites within a proteome (metalloproteomics) is either a valid or obtainable goal. With a proteome-wide knowledge of the active-site structures of all metalloproteins, one could start to ask how metal insertion, cluster assembly and metalloprotein expression are affected by growth conditions or developmental status etc. High-throughput X-ray absorption spectroscopy (HTXAS) is being developed as a technology for investigating the metalloproteome. In creating a pipeline from genome to metalloproteome, several bottlenecks to high-throughput determination of metal-site structures must be overcome. For example, automation of arraying small samples for XAS examination must be invented, automation of rapid data collection of multiple low-volume low-concentration samples must be developed, automation of data reduction and analysis must be perfected. Discussed here are the promises and the pitfalls of HTXAS development, including the results of initial feasibility experiments. [ABSTRACT FROM AUTHOR]

Presents a study which concluded that cytochrome cy of Rhodobacter capsulatus is attached to the cytoplasmis membrane by an uncleaved signal sequence-like anchor. Research methodology; Statistical information gathered from the study; Analysis of results obtained.

Rubredoxin from the hyperthermophilic archaeon Pyrococcus furiosus maintains its native structure at high temperatures (373 K). In order to investigate the role of hydrogen bonding, hydration and chain dynamics in this thermostability, wavelength-resolved Laue neutron diffraction data have been collected from the W3Y single mutant (Trp3→Tyr3) on the spallation neutron protein crystallography station (PCS) at Los Alamos Neutron Science Center. Data were measured at room temperature from nine crystal settings, each of approximately 12 h duration. The total data-measurement period was less than 5 d from a single crystal that had undergone H[sub2]/D[sub2]O exchange. The nominal resolution of the data is 2.1 Å. [ABSTRACT FROM AUTHOR]

The active site of [NiFe] hydrogenase is an excellent catalyst for hydrogen conversion with an intriguing ability to recover from exposure to dioxygen. In their Communication on page 724 ff., W. Lubitz, S. P. Cramer, and co‐workers use the synchrotron technique of nuclear resonance vibrational spectroscopy to identify normal modes associated with active‐site FeCN and FeCO motion. [ABSTRACT FROM AUTHOR]

Das aktive Zentrum der [NiFe] ‐ Hydrogenase ist ein exzellenter Katalysator der Wasserstoffumsetzung mit der faszinierenden Fähigkeit, sich nach Sauerstoffexposition zurückzubilden. W. Lubitz, S. P. Cramer et al. nutzen in ihrer Zuschrift auf S. 752 ff. die Synchrotrontechnik der kernresonanten inelastischen Streuung, um Normalmoden der Fe ‐ CN ‐ und Fe ‐ CO ‐ Bewegung im aktiven Zentrum zu identifizieren. [ABSTRACT FROM AUTHOR]

The D14C variant of Pyrococcus furiosus ferredoxin provides an extraordinary framework to investigate a [3Fe-4S] cluster at two oxidation levels and compare the results to its physiologic [4Fe-4S] counterpart in the very same protein. Our spectroscopic and computational study reveals vibrational property changes related to the electronic and structural aspects of both Fe-S clusters.

Nuclear inelastic scattering of (57)Fe labeled [NiFe] hydrogenase is shown to give information on different states of the enzyme. It was thus possible to detect and assign Fe-CO and Fe-CN bending and stretching vibrations of the active site outside the spectral range of the Fe-S cluster normal modes., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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 advent of the complete genome sequences of various organisms in the mid-1990s raised the issue of how one could determine the function of hypothetical proteins. While insight might be obtained from a 3D structure, the chances of being able to predict such a structure is limited for the deduced amino acid sequence of any uncharacterized gene. A template for modeling is required, but there was only a low probability of finding a protein closely-related in sequence with an available structure. Thus, in the late 1990s, an international effort known as structural genomics (SG) was initiated, its primary goal to "fill sequence-structure space" by determining the 3D structures of representatives of all known protein families. This was to be achieved mainly by X-ray crystallography and it was estimated that at least 5,000 new structures would be required. While the proteins (genes) for SG have subsequently been derived from hundreds of different organisms, extremophiles and particularly thermophiles have been specifically targeted due to the increased stability and ease of handling of their proteins, relative to those from mesophiles. This review summarizes the significant impact that extremophiles and proteins derived from them have had on SG projects worldwide. To what extent SG has influenced the field of extremophile research is also discussed.

The hypothetical protein PF0899 is a 95-residue peptide from the hyperthermophilic archaeon Pyrococcus furiosus that represents a gene family with six members. P. furiosus ORF PF0899 has been cloned, expressed and crystallized and its structure has been determined by the Southeast Collaboratory for Structural Genomics (http://www.secsg.org). The structure was solved using the SCA2Structure pipeline from multiple data sets and has been refined to 1.85 A against the highest resolution data set collected (a presumed gold derivative), with a crystallographic R factor of 21.0% and R(free) of 24.0%. The refined structure shows some structural similarity to a wedge-shaped domain observed in the structure of the major capsid protein from bacteriophage HK97, suggesting that PF0899 may be a structural protein.

PF0610, a protein from the hyperthermophile Pyrococcus furiosus, has homologues only in other archaeal species and in three species of Fe(III)-reducing bacteria. It is thought to have a helix-turn-helix (HTH) domain at the N-terminus and possesses two CXXC motifs characteristic of metal binding proteins. We have determined the solution structure of the Zn-bound protein using NMR. PF0610 is a novel winged helix-turn-helix (wHTH) protein with a rubredoxin-like Zn ribbon as its W1 segment. In addition, it possesses a large number of basic residues on its surface. Clusters of basic residues can be found on both helix H3 and the metal-binding loops of W1, suggesting that it might be a DNA-binding protein. Accordingly, gel shift assays using both linear and circular DNA showed that PF0610 does bind DNA, at least in a sequence-independent fashion. Modeling the PF0610-DNA interaction based on other wHTH protein-DNA structures revealed that besides helix H3, basic residues around the second CXXC motif in the metal-binding loop could make extensive contacts with DNA. However, the bulkiness of the W1 region implies that the DNA conformation may be distorted upon PF0610 binding. PF0610 is the first protein known to have a Zn ribbon-embedded wHTH fold and, as such, has potential roles both as a metal-dependent transcription regulator and as a component of the chromosome packing system in P. furiosus. The discovery of this novel structure represents the addition of another branch to the winged HTH protein family and could contribute to our understanding of transcription regulatory processes in P. furiosus.

Superoxide reductase (SOR) and P450 enzymes contain similar [Fe(N)4(SCys)] active sites and, although they catalyze very different reactions, are proposed to involve analogous low-spin (hydro)peroxo-Fe(III) intermediates in their respective mechanisms that can be modeled by cyanide binding. The equatorial FeN4 ligation by four histidine ligands in CN-SOR and the heme in CN-P450cam is directly compared by 14N ENDOR, while the axial Fe-CN and Fe-S bonding is probed by 13C ENDOR of the cyanide ligand and 1Hbeta ENDOR measurements to determine the spin density delocalization onto the cysteine sulfur. There are small, but notable, differences in the bonding between Fe(III) and its ligands in the two enzymes. The ENDOR measurements are complemented by DFT computations that support the semiempirical equation used to compute spin densities on metal-coordinated cysteinyl and shed light on bonding changes as the Fe-C-N linkage bends. They further indicate that H bonds to the cysteinyl thiolate sulfur ligand reduce the spin density on the sulfur in both active sites to a degree that exceeds the difference induced by the alternative sets of "in-plane" nitrogen ligands.