The 2.6‐Å refined structure of the Escherichia coli recombinant Saccharomyces cerevisiae flavocytochrome b₂‐sulfite complex

Abstract

Flavocytochrome b2 from Saccharomyces cerevisiae catalyzes the oxidation of L-lactate to pyruvate and the electron transfer to cytochrome c in the mitochondrial intermembrane space. It is a homotetramer with a molecular weight of 4 x 58 kDa, each monomer of which is composed of 2 distinct domains, the one carrying FMN and the other, a "bS-like" heme. The native structure has been described at a resolution of 2.4 A (Xia ZX, Mathews FS, 1990, JMol Bio/212:837-863). The heme domains protrude from the central body of the tetramer consisting of the 4 FMN binding domains. Because only 2 heme domains are visible in the electron density map, the other 2 are probably disordered. We crystallized the Escherichia coli recombinant flavocytochrome b2 from S. cerevisiae inhibited by sulfite. Although the crystals were obtained under very different conditions from those of the pyruvate-containing native enzyme, they were found to be isostructural (P 3, 2 1, a = b = 164.5 A , c = 114.0 A). The 2.6-A X-ray structure was extensively refined with X-PLOR (R = 17.3%), which made it possible to describe in detail the recombinant flavocytochrome b2 molecular structure. There exist few differences between the native and recombinant structures, in line with the fact that they show similar kinetic behavior, and they further confirm the intrinsic mobility of the heme domain (Labeyrie F, Beloil JC, Thomas MA, 1988, Biochim Biophys Acta 953:134-141). This structure will be used as a starting model in the structural resolution of flavocytochrome b2 point mutants.

The Roles of Two Amino Acid Residues in the Active Site of l-Lactate Monooxygenase

“…DISCUSSION An active site model of LMO (Fig. 2) based on the known structures of flavocytochrome b 2 (11,12) and glycolate oxidase (4) features two amino acids previously unstudied in this enzyme that are close to the substrate binding site. Arginine 187 in LMO is conserved throughout the FMN-dependent family of enzymes that catalyze the oxidation of L-␣-hydroxy acids.…”

“…The peptide sequence shows considerable homology with other members of this enzyme family (7). On the basis of the known structures of flavocytochrome b 2 (11,12) and glycolate oxidase (4), which show a strong similarity in the folding pattern around the flavin (13), a model of the active site of LMO has been made (Fig. 2).…”

“…The known structures of recombinant flavocytochrome b 2 (12) and glycolate oxidase in the presence (22) and absence (4) of bound active site inhibitors show that the equivalent residues in these enzymes (arginine 289 and arginine 164, respectively) are close to the active site and may therefore be involved in catalysis. Mutation of arginine 289 to lysine in flavocytochrome b 2 (23) resulted in a 4-fold increase in K m toward L-lactate and a 12-fold decrease in k cat in steadystate assays.…”

QM/MM study ofl-lactate oxidation by flavocytochrome b2

“…3,6 In the crystal structure of the R289K variant in complex with sulfite, the water molecule has been displaced by the lysine side chain, which adopts more or less the orientation of R289 in the proximal position, and interacts with flavin-bound sulfite. 7 The homologue of R289 is in the proximal position in most structures of family members.…”

“…Several residues in that proteasesensitive loop are invisible in all structures. 3,[5][6][7] There is evidence that residues of this loop interact with the active site during catalysis, since the mere proteolysis of one specific peptide bond in the loop induces alterations of the kinetic parameters. 9 Despite the amount of experimental data collected over the past decades, two mechanistic hypotheses (see Figure 3) remain in debate.…”