Structural studies on recombinant and point mutants of flavocytochrome b2.

Flavocytochrome b2 from S cerevisiae is a homotetramer with a molecular mass of 4 x 58 kDa. It catalyses the oxidation of L-lactate into pyruvate and the electron transfer to cytochrome c in the mitochondrial intermembrane space. Each monomer is composed of a flavinmononucleotide (FMN) carrying domain and a 'b5-like' heme domain. The wild type structure has been described at a resolution of 2.4 A. We report here on the refined structure of the E. coli native recombinant flavocytochrome b2 from S cerevisiae inhibited by sulphite and that of two point mutants, Y143F and Y254F, in which pyruvate is bound to the active site. The crystals, obtained under very different conditions from those of the native enzyme, are isostructural (P 3(2) 2 1, a=b=164.5 A, c=114.0 A). In line with the similarities found to exist in the kinetic behaviour of the native and recombinant protein, few structural differences were observed here, and the crystallographic data further confirm the intrinsic mobility of the heme domain. The superimposable position of the aromatic rings of Phe 143 in the mutant Y143F and Tyr 143 in the native protein makes it seem unlikely that the aromatic ring may be directly involved in the intramolecular electron transfer. The fact that a very restricted number of domain interactions was observed in Y143F shows that Tyr 143 is one of the amino acids essential to the formation of the productive complex. In the Y143F mutant, the number of catalytically efficient complexes is probably drastically decreased, which will severely limit the rate of intramolecular election transfer. The structure of Y254F shows a reorientation of the substrate at the active site. Together with the kinetic results, this finding definitely excludes the possibility that Tyr 254 may act as general base and that the substrate may interact directly with Phe 254 in the mutant. The model between flavocytochrome b2 and cytochrome c will serve as a basis for designing suitable mutants of the amino acids involved either in the interaction or the electron transfer.