FhuA, the receptor for ferrichrome-iron in Escherichia coli , is a member of a family of integral outer membrane proteins, which, together with the energy-transducing protein TonB, mediate the active transport of ferric siderophores across the outer membrane of Gram-negative bacteria. The three-dimensional structure of FhuA is presented here in two conformations: with and without ferrichrome-iron at resolutions of 2.7 and 2.5 angstroms, respectively. FhuA is a β barrel composed of 22 antiparallel β strands. In contrast to the typical trimeric arrangement found in porins, FhuA is monomeric. Located within the β barrel is a structurally distinct domain, the “cork,” which mainly consists of a four-stranded β sheet and four short α helices. A single lipopolysaccharide molecule is noncovalently associated with the membrane-embedded region of the protein. Upon binding of ferrichrome-iron, conformational changes are transduced to the periplasmic pocket of FhuA, signaling the ligand-loaded status of the receptor. Sequence homologies and mutagenesis data are used to propose a structural mechanism for TonB-dependent siderophore-mediated transport across the outer membrane.

Peridinin-chlorophyll-protein, a water-soluble light-harvesting complex that has a bluegreen absorbing carotenoid as its main pigment, is present in most photosynthetic dinoflagellates. Its high-resolution (2.0 angstrom) x-ray structure reveals a noncrystallographic trimer in which each polypeptide contains an unusual jellyroll fold of the a-helical amino-and carboxyl-terminal domains. These domains constitute a scaffold with pseudo-twofold symmetry surrounding a hydrophobic cavity filled by two lipid, eight peridinin, and two chlorophyll a molecules. The structural basis for efficient excitonic energy transfer from peridinin to chlorophyll is found in the clustering of peridinins around the chlorophylls at van der Waals distances. Table 1. Crystallographic data. Data collection: Native1 and derivative data (5 IllM K 2 PtCI 4 , 1-day soak) were collected on a rotating anode source (CuKa, 40 kV, 100 mA) with a STOE (Darmstadt, Germany) imaging plate detector. Native2 was collected at the BW7B wiggler bealllline at DESY on a 30-cm MARresearch (Hamburg, Germany) image plate. All data were processed with XDS (26). Phasing: Six heavy-atom binding sites were found using SHELXS (27) in Patterson search mode, and four additional sites were found by inspection of difference Fourier maps. Refinement of heavy-atom parameters was done with DAREFI (28). The phases could be improved further by inclusion of the anomalous signal and the use of solvent flattening and noncrystallographic symmetry averaging of the trimer in the asymmetric unit (29). Model building and refinement: The resulting 2.9 Aelectron density map was readily interpret-able. An initial model of PCP was built with 0 (23). The full sequence could be included, and 10 pigments were modeled. The initial model was refined with X-PLOR (24), including simulated annealing, positional refinement, and manual rebuilding against Native1 and later Native2. Strong noncrystallographic symmetry restraints were applied throughout the refinement. ARP (30) was used to obtain unbiased atomic positions for well-connected density in the 2.0 Adifference electron-density map. The arrangement of these atoms, together with possible hydrogen-bonding patterns, was consistent with an interpretation as DGDG molecules. After refinement, the DGDG head groups obey good stereochemistry and show no difference density. tuolecules. Within each cluster, the efficiency of singlet energy transfer fr01n peridinin to chlorophyll is close to unity (7). Models of chromophore interaction within and atuong the clusters have previously been based on spectroscopic investigations (7,8). Structural infornlation, such as that available for 1uenl-brane-bound LHCs fron1 higher plants (3) and bacteria (9), has greatly enhanced our understanding of antenna systen1s having chlorophyll as the n1ain pignlent. The highresolution structure of PCP gives insight into the highly organized structural basis of lightharvesting by carotenoids and its efficient transfer to chlorophyll and should be of considerable value for re...

H2 turnover at the [FeFe]-hydrogenase cofactor (H-cluster) is assumed to follow a reversible heterolytic mechanism, first yielding a proton and a hydrido-species which again is double-oxidized to release another proton. Three of the four presumed catalytic intermediates (Hox, Hred/Hred and Hsred) were characterized, using various spectroscopic techniques. However, in catalytically active enzyme, the state containing the hydrido-species, which is eponymous for the proposed heterolytic mechanism, has yet only been speculated about. We use different strategies to trap and spectroscopically characterize this transient hydride state (Hhyd) for three wild-type [FeFe]-hydrogenases. Applying a novel set-up for real-time attenuated total-reflection Fourier-transform infrared spectroscopy, we monitor compositional changes in the state-specific infrared signatures of [FeFe]-hydrogenases, varying buffer pH and gas composition. We selectively enrich the equilibrium concentration of Hhyd, applying Le Chatelier’s principle by simultaneously increasing substrate and product concentrations (H2/H+). Site-directed manipulation, targeting either the proton-transfer pathway or the adt ligand, significantly enhances Hhyd accumulation independent of pH.

Crystal structures of semisynthetic [FeFe]-hydrogenases with variations in the [2Fe] cluster show little structural differences despite strong effects on activity.