Tegoni M, Cambillau C
Laboratoire de Cristallisation et Cristallographie des Macromolécules Biologiques, CNRS-URA 1296, Faculté de Médecine Nord, Marseille, France.
Biochimie. 1994;76(6):501-14. doi: 10.1016/0300-9084(94)90174-0.
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.
来自酿酒酵母的黄素细胞色素b2是一种同源四聚体，分子量为4×58 kDa。它催化L-乳酸氧化为丙酮酸，并将电子传递至线粒体内膜间隙中的细胞色素c。每个单体由一个携带黄素单核苷酸（FMN）的结构域和一个“b5样”血红素结构域组成。野生型结构已在2.4 Å的分辨率下得到描述。我们在此报告了来自酿酒酵母的大肠杆菌天然重组黄素细胞色素b2在亚硫酸盐抑制下的精细结构，以及两个点突变体Y143F和Y254F的结构，其中丙酮酸结合在活性位点上。这些晶体是在与天然酶非常不同的条件下获得的，它们是同晶型的（P 3(2) 2 1，a = b = 164.5 Å，c = 114.0 Å）。鉴于天然蛋白和重组蛋白在动力学行为上存在相似性，在此观察到的结构差异很少，并且晶体学数据进一步证实了血红素结构域的内在流动性。突变体Y143F中的苯丙氨酸143的芳香环与天然蛋白中的酪氨酸143的芳香环可叠加的位置表明，芳香环不太可能直接参与分子内电子转移。在Y143F中观察到的结构域相互作用数量非常有限，这表明酪氨酸143是形成有效复合物所必需的氨基酸之一。在Y143F突变体中，催化效率高的复合物数量可能会大幅减少，这将严重限制分子内电子转移的速率。Y254F的结构显示了底物在活性位点处的重新定向。与动力学结果一起，这一发现明确排除了酪氨酸254可能作为通用碱以及底物可能在突变体中与苯丙氨酸254直接相互作用的可能性。黄素细胞色素b2与细胞色素c之间的模型将为设计参与相互作用或电子转移的氨基酸的合适突变体提供基础。
