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An upgraded version of the sample changer ;CATS' (Cryogenic Automated Transfer System) that was developed on the FIP-BM30A beamline at the ESRF is presented. At present, CATS is installed at SLS (three systems), BESSY (one system), DLS (two systems) and APS (four systems for the LSCAT beamline). It consists mainly of an automated Dewar with an assortment of specific grippers designed to obtain a fast and reliable mounting/dismounting rate without jeopardizing the flexibility of the system. The upgraded system has the ability to manage any sample standard stored in any kind of puck.

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In anaerobic organisms, the decarboxylation of pyruvate, a crucial component of intermediary metabolism, is catalyzed by the metalloenzyme pyruvate: ferredoxin oxidoreductase (PFOR) resulting in the generation of low potential electrons and the subsequent acetylation of coenzyme A (CoA). PFOR is the only enzyme for which a stable acetyl thiamine diphosphate (ThDP)-based free radical reaction intermediate has been identified. The 1.87 A-resolution structure of the radical form of PFOR from Desulfovibrio africanus shows that, despite currently accepted ideas, the thiazole ring of the ThDP cofactor is markedly bent, indicating a drastic reduction of its aromaticity. In addition, the bond connecting the acetyl group to ThDP is unusually long, probably of the one-electron type already described for several cation radicals but not yet found in a biological system. Taken together, our data, along with evidence from the literature, suggest that acetyl-CoA synthesis by PFOR proceeds via a condensation mechanism involving acetyl (PFOR-based) and thiyl (CoA-based) radicals.

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Fe-only hydrogenases, as well as their NiFe counterparts, display unusual intrinsic high-frequency IR bands that have been assigned to CO and CN(-) ligation to iron in their active sites. FTIR experiments performed on the Fe-only hydrogenase from Desulfovibrio desulfuricans indicate that upon reduction of the active oxidized form, there is a major shift of one of these bands that is provoked, most likely, by the change of a CO ligand from a bridging position to a terminal one. Indeed, the crystal structure of the reduced active site of this enzyme shows that the previously bridging CO is now terminally bound to the iron ion that most likely corresponds to the primary hydrogen binding site (Fe2). The CO binding change may result from changes in the coordination sphere of Fe2 or its reduction. Superposition of this reduced active site with the equivalent region of a NiFe hydrogenase shows a remarkable coincidence between the coordination of Fe2 and that of the Fe ion in the NiFe cluster. Both stereochemical and mechanistic considerations suggest that the small organic molecule found at the Fe-only hydrogenase active site and previously modeled as 1,3-propanedithiolate may, in fact, be di-(thiomethyl)-amine.

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R-phycoerythrin, a light-harvesting component from the red algae Gracilaria chilensis, was crystallized by vapour diffusion using ammonium sulfate as precipitant agent. Red crystals grew after one week at 293 K and diffracted to 2.70 A resolution. Three serial macroseeding assays were necessary to grow a second larger crystal to dimensions of 0.68 x 0.16 x 0.16 mm. This crystal diffracted to 2.24 A resolution using synchrotron radiation at beamline BM14 of the European Synchrotron Radiation Facility (ESRF) at Grenoble, France and was used for structure determination. Data were collected at 100 K to a completeness of 98.6%. The crystal was trigonal, space group R3, with unit-cell parameters a = b = 187.3, c = 59.1 A, alpha = beta = 90, gamma = 120 degrees. Data treatment using the CCP4 suite of programs indicated that the crystal was twinned ((I(2))/(I)(2) = 1.41). Molecular replacement was performed with AMoRe using the R-phycoerythrin from Polysiphonia urceolata [Chang et al. (1996), J. Mol. Biol. 249, 424-440] as a search model. In order to overcome the twinning problem, SHELX97 was used for the crystallographic refinement. The twin fraction was 0.48, indicating a nearly perfect hemihedrally twinned crystal. The final R(work) and R(free) factors are 0.16 and 0.25, respectively. All the residues and chromophores of the alpha- and beta-chains are well defined in the electron-density maps. Some residues belonging to the gamma-linker are also recognizable.

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A crystal structure of the C-terminal domain of Escherichia coli UvrB (UvrB') has been solved to 3.0 A resolution. The domain adopts a helix-loop-helix fold which is stabilised by the packing of hydrophobic side-chains between helices. From the UvrB' fold, a model for a domain of UvrC (UvrC') that has high sequence homology with UvrB' has been made. In the crystal, a dimerisation of UvrB domains is seen involving specific hydrophobic and salt bridge interactions between residues in and close to the loop region of the domain. It is proposed that a homologous mode of interaction may occur between UvrB and UvrC. This interaction is likely to be flexible, potentially spanning > 50 A.

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BACKGROUND: [NiFeSe] hydrogenases are metalloenzymes that catalyze the reaction H2<-->2H+ + 2e-. They are generally heterodimeric, contain three iron-sulfur clusters in their small subunit and a nickel-iron-containing active site in their large subunit that includes a selenocysteine (SeCys) ligand. RESULTS: We report here the X-ray structure at 2.15 A resolution of the periplasmic [NiFeSe] hydrogenase from Desulfomicrobium baculatum in its reduced, active form. A comparison of active sites of the oxidized, as-prepared, Desulfovibrio gigas and the reduced D. baculatum hydrogenases shows that in the reduced enzyme the nickel-iron distance is 0.4 A shorter than in the oxidized enzyme. In addition, the putative oxo ligand, detected in the as-prepared D. gigas enzyme, is absent from the D. baculatum hydrogenase. We also observe higher-than-average temperature factors for both the active site nickel-selenocysteine ligand and the neighboring Glu18 residue, suggesting that both these moieties are involved in proton transfer between the active site and the molecular surface. Other differences between [NiFeSe] and [NiFe] hydrogenases are the presence of a third [4Fe4S] cluster replacing the [3Fe4S] cluster found in the D. gigas enzyme, and a putative iron center that substitutes the magnesium ion that has already been described at the C terminus of the large subunit of two [NiFe] hydrogenases. CONCLUSIONS: The heterolytic cleavage of molecular hydrogen seems to be mediated by the nickel center and the selenocysteine residue. Beside modifying the catalytic properties of the enzyme, the selenium ligand might protect the nickel atom from oxidation. We conclude that the putative oxo ligand is a signature of inactive 'unready' [NiFe] hydrogenases.

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The 2.54 A resolution structure of Ni-Fe hydrogenase has revealed the existence of hydrophobic channels connecting the molecular surface to the active site. A crystallographic analysis of xenon binding together with molecular dynamics simulations of xenon and H2 diffusion in the enzyme interior suggest that these channels serve as pathways for gas access to the active site.

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Hydrogenases are proteins which metabolize the most simple of chemical compounds, molecular hydrogen, according to the reaction H2<-->2H+ + 2e-. These enzymes are found in many microorganisms of great biotechnological interest such as methanogenic, acetogenic, nitrogen fixing, photosynthetic or sulfate-reducing bacteria. The X-ray structure of a dimeric [NiFe] hydrogenase together with a wealth of biophysical, biochemical and genetic studies have revealed that the large subunit contains the bimetallic [Ni-Fe] active site, with biologically uncommon CO and CN ligands to the iron, whereas the small subunit contains three iron-sulfur cluster. During catalysis, the nickel atom is most likely responsible for a base-assisted heterolytic cleavage of the hydrogen molecule whereas the iron atom could be redox active. Specific channels are probably required for the transfer of the chemical reaction partners (H2, H+ and e-) between the active site, deeply buried inside the protein, and the molecular surface. The generation of a functional enzyme, including the assembly of the complex catalytic center, requires maturation and involves a large number of auxiliary proteins which have been partly characterized by molecular biology.

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