Instituto de Bioquímica Vegetal y Fotosíntesis, CSIC and University of Sevilla, Spain.
FEBS J. 2013 Apr;280(8):1830-40. doi: 10.1111/febs.12207. Epub 2013 Mar 25.
l-galactono-1,4-lactone dehydrogenase (GALDH) catalyzes the terminal step of vitamin C biosynthesis in plant mitochondria. Here we investigated the communication between Arabidopsis thaliana GALDH and its natural electron acceptor cytochrome c (Cc). Using laser-generated radicals we observed the formation and stabilization of the GALDH semiquinone anionic species (GALDHSQ ). GALDHSQ oxidation by Cc exhibited a nonlinear dependence on Cc concentration consistent with a kinetic mechanism involving protein-partner association to form a transient bimolecular complex prior to the electron transfer step. Oxidation of GALDHSQ by Cc was significantly impaired at high ionic strength, revealing the existence of attractive charge-charge interactions between the two reactants. Isothermal titration calorimetry showed that GALDH weakly interacts with both oxidized and reduced Cc. Chemical shift perturbations for (1) H and (15) N nuclei of Cc, arising from the interactions with unlabeled GALDH, were used to map the interacting surface of Cc. For Arabidopsis Cc and yeast Cc, similar residues are involved in the interaction with GALDH. These residues are confined to a single surface surrounding the heme edge. The range of chemical shift perturbations for the physiological Arabidopsis Cc-GALDH complex is larger than that of the non-physiological yeast Cc-GALDH complex, indicating that the former complex is more specific. In summary, the results point to a relatively low affinity GALDH-Cc interaction, similar for all partner redox states, involving protein-protein dynamic motions. Evidence is also provided that Cc utilizes a conserved surface surrounding the heme edge for the interaction with GALDH and other redox partners.
NMR assignment of the backbone amide resonances of Arabidopsis CcRED has been deposited in BMRB database (BMRB accession number 18828). L-galactono-1,4-lactone dehydrogenase (L-galactono-1,4-lactone: ferricytochrome c oxidoreductase, EC 1.3.2.3).
l-半乳糖酸-1,4-内酯脱氢酶（GALDH）在植物线粒体中催化维生素 C 生物合成的终末步骤。在这里，我们研究了拟南芥 GALDH与其天然电子受体细胞色素 c（Cc）之间的通讯。使用激光产生的自由基，我们观察到 GALDH 半醌阴离子物种（GALDHSQ）的形成和稳定。Cc 对 GALDHSQ 的氧化作用随 Cc 浓度呈非线性依赖关系，这与一种动力学机制一致，该机制涉及在电子转移步骤之前形成蛋白质-伴侣缔合的短暂双分子复合物。在高离子强度下，Cc 对 GALDHSQ 的氧化作用明显受损，这表明两个反应物之间存在吸引力电荷相互作用。等温滴定量热法表明，GALDH 与氧化型和还原型 Cc 均弱相互作用。来自未标记 GALDH 相互作用的（1）H 和（15）N 核的 Cc 化学位移扰动用于绘制 Cc 的相互作用表面。对于拟南芥 Cc 和酵母 Cc，相似的残基参与与 GALDH 的相互作用。这些残基局限于围绕血红素边缘的单个表面。生理拟南芥 Cc-GALDH 复合物的化学位移扰动范围大于非生理酵母 Cc-GALDH 复合物的范围，这表明前者的复合物更具特异性。总之，结果表明 GALDH-Cc 相互作用的亲和力相对较低，与所有配体氧化还原态相似，涉及蛋白质-蛋白质动态运动。还提供了证据表明，Cc 利用围绕血红素边缘的保守表面与 GALDH 和其他氧化还原伴侣相互作用。
已将拟南芥 CcRED 骨干酰胺共振的 NMR 分配存入 BMRB 数据库（BMRB 编号 18828）。L-半乳糖酸-1,4-内酯脱氢酶（L-半乳糖酸-1,4-内酯：细胞色素 c 氧化还原酶，EC 1.3.2.3）。
