The light-harvesting core antenna (LH1) and the reaction centre (RC) of purple photosynthetic bacteria form a supramolecular complex (LH1-RC) to use sunlight energy in a highly efficient manner. Here we report the first near-atomic structure, to our knowledge, of a LH1-RC complex, namely that of a Ca(2+)-bound complex from Thermochromatium tepidum, which reveals detailed information on the arrangement and interactions of the protein subunits and the cofactors. The RC is surrounded by 16 heterodimers of the LH1 αβ-subunit that form a completely closed structure. The Ca(2+) ions are located at the periplasmic side of LH1. Thirty-two bacteriochlorophyll and 16 spirilloxanthin molecules in the LH1 ring form an elliptical assembly. The geometries of the pigment assembly involved in the absorption characteristics of the bacteriochlorophyll in LH1 and excitation energy transfer among the pigments are reported. In addition, possible ubiquinone channels in the closed LH1 complex are proposed based on the atomic structure.
High-potential iron-sulfur protein (HiPIP) is a soluble electron carrier protein of photosynthetic bacteria with an Fe4S4 cluster. Although structural changes accompanying the electron transfer are important for understanding of the functional mechanism, the changes have not been clarified in sufficient detail. We previously reported the high-resolution crystal structures of HiPIP from a thermophilic purple bacterium Thermochromatium tepidum in the reduced state. In order to perform a detailed comparison between the structures in different redox states, the oxidized structure should also be revealed at high resolution. Therefore, in the present study we performed a crystallographic analysis of oxidized HiPIP and a structural comparison with the reduced form at a high resolution of 0.8 Å. The comparison highlighted small but significant contraction in the iron-sulfur cluster. The changes in Fe-S bond lengths were similar to that predicted by theoretical calculation, although some discrepancies were also found. Almost distances between the sulfur atoms of the iron-sulfur cluster and the protein environment are elongated upon the oxidation. Positional changes of hydrogen atoms in the protein environment, such as on the amide-hydrogen of Cys75 in the proximity of the iron-sulfur cluster, were also observed in the accurate analyses. None of the water molecules exhibited significant changes in position or anisotropy of atomic displacement parameter between the two states, while the orientations of some water molecules were different.
Chitinase from T. kodakarensis (TkChiA) catalyzes the hydrolysis of chitin. The enzyme consists of two catalytic and three binding domains (ChBD1, ChBD2 and ChBD3). ChBD2 and ChBD3 can bind to not only chitin but also cellulose. In both domains, the intervals of the side chains of the three tryptophan residues, which are located on the molecular surface, correspond to twice the length of the lattice of the chitin. A binding model with crystalline chitin implies that the tryptophan residues and a glutamate residue interact with the hexose ring by CH-p interactions and the amide group by a hydrogen bond, respectively.
Heme A is an essential cofactor for respiratory terminal oxidases and vital for respiration in aerobic organisms. The final step of heme A biosynthesis is formylation of the C-8 methyl group of heme molecule by heme A synthase (HAS). HAS is a heme-containing integral membrane protein, and its structure and reaction mechanisms have remained unknown. Thus, little is known about HAS despite of its importance. Here we report the crystal structure of HAS from Bacillus subtilis at 2.2-Å resolution. The N- and C-terminal halves of HAS consist of four-helix bundles and they align in a pseudo twofold symmetry manner. Each bundle contains a pair of histidine residues and forms a heme-binding domain. The C-half domain binds a cofactor-heme molecule, while the N-half domain is vacant. Many water molecules are found in the transmembrane region and around the substrate-binding site, and some of them interact with the main chain of transmembrane helix. Comparison of these two domain structures enables us to construct a substrate-heme binding state structure. This structure implies that a completely conserved glutamate, Glu57 in B. subtilis, is the catalytic residue for the formylation reaction. These results provide valuable suggestions of the substrate-heme binding mechanism. Our results present significant insight into the heme A biosynthesis.
The lipidic cubic phase method is an effective approach for membrane protein crystallography. The in meso grown crystals are usually cryocooled directly without removing the host matrix from the harvested crystal surface. However, the host matrix often causes the appearance of scattering rings and an increase in background scattering during the data collection. Moreover, the frozen host matrix sometimes becomes opaque and it can hinder conventional crystal centering. In this study, several oils were examined for their ability to clean the host matrix and to provide cryoprotection for crystals grown in the lipidic cubic phase. Several of the tested oils appeared to be useful in terms of their effect on crystal stability and background scattering. This method should be of value for the collection of highly accurate data sets.
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