Cunane L M, Barton J D, Chen Z-W, Welsh F E, Chapman S K, Reid G A, Mathews F S
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Biochemistry. 2002 Apr 2;41(13):4264-72.
Flavocytochrome b(2) catalyzes the oxidation of L-lactate to pyruvate and the transfer of electrons to cytochrome c. The enzyme consists of a flavin-binding domain, which includes the active site for lacate oxidation, and a b(2)-cytochrome domain, required for efficient cytochrome c reduction. To better understand the structure and function of intra- and interprotein electron transfer, we have determined the crystal structure of the independently expressed flavin-binding domain of flavocytochrome b(2) to 2.50 A resolution and compared this with the structure of the intact enzyme, redetermined at 2.30 A resolution, both structures being from crystals cooled to 100 K. Whereas there is little overall difference between these structures, we do observe significant local changes near the interface region, some of which impact on amino acid side chains, such as Arg289, that have been shown previously to have an important role in catalysis. The disordered loop region found in flavocytochrome b(2) and its close homologues remain unresolved in frozen crystals of the flavin-binding domain, implying that the presence of the b(2)-cytochrome domain is not responsible for this positional disorder. The flavin-binding domain interacts poorly with cytochrome c, but we have introduced acidic residues in the interdomain interface region with the aim of enhancing cytochrome c binding. While the mutations L199E and K201E within the flavin-binding domain resulted in unimpaired lactate dehydrogenase activity, they failed to enhance electron-transfer rates with cytochrome c. This is most likely due to the disordered loop region obscuring all or part of the surface having the potential for productive interaction with cytochrome c.
黄素细胞色素b(2)催化L-乳酸氧化为丙酮酸，并将电子传递给细胞色素c。该酶由一个黄素结合结构域和一个b(2)-细胞色素结构域组成，前者包含乳酸氧化的活性位点，后者是高效还原细胞色素c所必需的。为了更好地理解蛋白质内和蛋白质间电子转移的结构和功能，我们已经确定了黄素细胞色素b(2)独立表达的黄素结合结构域的晶体结构，分辨率为2.50 Å，并将其与完整酶的结构进行了比较，后者在2.30 Å分辨率下重新测定，两种结构均来自冷却至100 K的晶体。虽然这些结构之间总体差异不大，但我们确实观察到界面区域附近有明显的局部变化，其中一些影响了氨基酸侧链，如Arg289，先前已证明其在催化中起重要作用。在黄素细胞色素b(2)及其紧密同源物中发现的无序环区域在黄素结合结构域的冷冻晶体中仍未解析，这意味着b(2)-细胞色素结构域的存在不是这种位置无序的原因。黄素结合结构域与细胞色素c的相互作用较弱，但我们在结构域间界面区域引入了酸性残基，以增强细胞色素c的结合。虽然黄素结合结构域内的L199E和K201E突变导致乳酸脱氢酶活性未受损，但它们未能提高与细胞色素c的电子传递速率。这很可能是由于无序环区域掩盖了全部或部分具有与细胞色素c进行有效相互作用潜力的表面。
