title: Molecular structure of flavocytochrome b2 at 2.4 A resolution.
abstract: The crystal structure of flavocytochrome b2 has been solved at 3.0 A resolution by the method of multiple isomorphous replacement with anomalous scattering. Area detector data from native and two heavy-atom derivative crystals were used. The phases were refined by the B.C. Wang phase-filtering procedure utilizing the 67% (v/v) solvent content of the crystals. A molecular model was built first on a minimap and then on computer graphics from a combination of maps both averaged and not averaged about the molecular symmetry axis. The structure was extended to 2.4 A resolution using film data recorded at a synchrotron and refined by the Hendrickson-Konnert procedure. The molecule, a tetramer of Mr 230,000, is located on a crystallographic 2-fold axis and possesses local 4-fold symmetry. Each subunit is composed of two domains, one binding a heme and the other an FMN prosthetic group. In subunit 1, both the cystochrome and the flavin-binding domain are visible in the electron density map. In subunit 2 the cytochrome domain is disordered. However, in the latter, a molecule of pyruvate, the product of the enzymatic reaction, is bound at the active site. The cytochrome domain consists of residues 1 to 99 and is folded in a fashion similar to the homologous soluble fragment of cytochrome b5. The flavin binding domain contains a parallel beta 8 alpha 8 barrel structure and is composed of residues 100 to 486. The remaining 25 residues form a tail that wraps around the molecular 4-fold axis and is in contact with each remaining subunit. The FMN moiety, which is located at the C-terminal end of the central beta-barrel, is mostly sequestered from solvent; it forms hydrogen bond interactions with main- and side-chain atoms from six of the eight beta-strands. The interaction of Lys349 with atoms N-1 and O-2 of the flavin ring is probably responsible for stabilization of the anionic form of the flavin semiquinone and hydroquinone and enhancing the reactivity of atom N-5 toward sulfite. The binding of pyruvate at the active site in subunit 2 is stabilized by interaction of its carboxylate group with the side-chain atoms of Arg376 and Tyr143. Residues His373 and Tyr254 interact with the keto-oxygen atom and are involved in catalysis. In contrast, four water molecules occupy the substrate-binding site in subunit 1 and Tyr143 forms a hydrogen bond to the ordered heme propionate group. Otherwise the two flavin-binding domains are identical within experimental error.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Department of Cell Biology and Physiology, Washington University School of Medicine, St Louis, MO 63110.
authors.forename: Z X
authors.initials: ZX
authors.lastname: Xia
authors.forename: F S
authors.initials: FS
authors.lastname: Mathews
mesh_terms: D000595:Amino Acid Sequence; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D003196:Computer Graphics; D004579:Electron Transport; D005415:Flavins / Q000378:metabolism; D005418:Flavodoxin / Q000378:metabolism; D006418:Heme; D006639:Histidine; D007501:Iron; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D042844:L-Lactate Dehydrogenase (Cytochrome); D008969:Molecular Sequence Data; D015394:Molecular Structure; D011487:Protein Conformation; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000378:metabolism; D014961:X-Ray Diffraction
chemical_list: D005415:Flavins; D005418:Flavodoxin; D013438:Sulfhydryl Compounds; D006418:Heme; D006639:Histidine; D007501:Iron; D007770:L-Lactate Dehydrogenase; D042844:L-Lactate Dehydrogenase (Cytochrome)