Molecular structure of flavocytochrome b2 at 2.4 A resolution

J Mol Biol. 1990 Apr 20;212(4):837-63. doi: 10.1016/0022-2836(90)90240-M.

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)

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Chemical Phenomena
  • Chemistry, Physical
  • Computer Graphics
  • Electron Transport
  • Flavins / metabolism
  • Flavodoxin / metabolism
  • Heme
  • Histidine
  • Iron
  • L-Lactate Dehydrogenase (Cytochrome)
  • L-Lactate Dehydrogenase* / metabolism
  • Molecular Sequence Data
  • Molecular Structure
  • Protein Conformation
  • Substrate Specificity
  • Sulfhydryl Compounds / metabolism
  • X-Ray Diffraction

Substances

  • Flavins
  • Flavodoxin
  • Sulfhydryl Compounds
  • Heme
  • Histidine
  • Iron
  • L-Lactate Dehydrogenase
  • L-Lactate Dehydrogenase (Cytochrome)