STRUCTURAL STUDIES ON RECOMBINANT AND POINT MUTANTS OF FLAVOCYTOCHROME B(2)

Flavocytochrome b(2) from S cerevisiae is a homotetramer with a molecu lar mass of 4 x 58 kDa. It catalyses the oxidation of L-lactate into p yruvate and the electron transfer to cytochrome c in the mitochondrial intermembrane space. Each monomer is composed of a flavinmononucleoti de (FMN) carrying domain and a 'b(5)-like' heme domain. The wild type structure has been described at a resolution of 2.4 Angstrom. We repor t here on the refined structure of the E coli native recombinant flavo cytochrome b(2) from S cerevisiae inhibited by sulphite and that of tw o point mutants, Y143F and Y254F, in which pyruvate is bound to the ac tive 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 Angstrom, c=114.0 Angstrom). In line with the similarities found to ex ist in the kinetic behaviour of the native and recombinant protein, fe w structural differences were observed here, and the crystallographic data further confirm the intrinsic mobility of the heme domain. The su perimposable position of the aromatic rings of Phe 143 in the mutant Y 143F 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 interactio ns 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 mut ant, the number of catalytically efficient complexes is probably drast ically decreased, which will severely limit the rate of intramolecular electron transfer. The structure of Y254F shows a reorientation of th e substrate at the active site. Together with the kinetic results, thi s 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 b(2) and cytochrome c will serve as a basis for designing suitable mutants of the aminoaci ds involved either in the interaction or the electron transfer.