Department of Molecular and Cell Biology, University of California-Berkeley, CA 94720, USA.
Biochemistry. 2011 May 24;50(20):4281-90. doi: 10.1021/bi200341b. Epub 2011 May 3.
Eukaryotic nitric oxide (NO) signaling involves modulation of cyclic GMP (cGMP) levels through activation of the soluble isoform of guanylate cyclase (sGC). sGC is a heterodimeric hemoprotein that contains a Heme-Nitric oxide and OXygen binding (H-NOX) domain, a Per/ARNT/Sim (PAS) domain, a coiled-coil (CC) domain, and a catalytic domain. To evaluate the role of these domains in regulating the ligand binding properties of the heme cofactor of NO-sensitive sGC, we constructed chimeras by swapping the rat β1 H-NOX domain with the homologous region of H-NOX domain-containing proteins from Thermoanaerobacter tengcongensis, Vibrio cholerae, and Caenorhabditis elegans (TtTar4H, VCA0720, and Gcy-33, respectively). Characterization of ligand binding by electronic absorption and resonance Raman spectroscopy indicates that the other rat sGC domains influence the bacterial and worm H-NOX domains. Analysis of cGMP production in these proteins reveals that the chimeras containing bacterial H-NOX domains exhibit guanylate cyclase activity, but this activity is not influenced by gaseous ligand binding to the heme cofactor. The rat-worm chimera containing the atypical sGC Gcy-33 H-NOX domain was weakly activated by NO, CO, and O(2), suggesting that atypical guanylate cyclases and NO-sensitive guanylate cyclases have a common molecular mechanism for enzyme activation. To probe the influence of the other sGC domains on the mammalian sGC heme environment, we generated heme pocket mutants (Pro118Ala and Ile145Tyr) in the β1 H-NOX construct (residues 1-194), the β1 H-NOX-PAS-CC construct (residues 1-385), and the full-length α1β1 sGC heterodimer (β1 residues 1-619). Spectroscopic characterization of these proteins shows that interdomain communication modulates the coordination state of the heme-NO complex and the heme oxidation rate. Taken together, these findings have important implications for the allosteric mechanism of regulation within H-NOX domain-containing proteins.
真核生物一氧化氮（NO）信号转导通过激活可溶性鸟苷酸环化酶（sGC）来调节环鸟苷酸（cGMP）水平。sGC 是一种异源二聚体血红素蛋白，包含一个血红素-NO 和氧结合（H-NOX）结构域、一个 Per/ARNT/Sim（PAS）结构域、一个卷曲螺旋（CC）结构域和一个催化结构域。为了评估这些结构域在调节 NO 敏感 sGC 血红素辅因子配体结合特性中的作用，我们通过交换来自 Thermoanaerobacter tengcongensis、霍乱弧菌和秀丽隐杆线虫的 H-NOX 结构域同源区域，构建了嵌合体大鼠β1 H-NOX 结构域与 Thermoanaerobacter tengcongensis、霍乱弧菌和秀丽隐杆线虫（TtTar4H、VCA0720 和 Gcy-33，分别）中的 H-NOX 结构域包含蛋白。通过电子吸收和共振拉曼光谱对配体结合的表征表明，其他大鼠 sGC 结构域影响细菌和蠕虫 H-NOX 结构域。对这些蛋白质中环鸟苷酸生成的分析表明，含有细菌 H-NOX 结构域的嵌合体具有鸟苷酸环化酶活性，但这种活性不受血红素辅因子与气态配体结合的影响。含有非典型 sGC Gcy-33 H-NOX 结构域的大鼠-蠕虫嵌合体被 NO、CO 和 O(2) 弱激活，表明非典型鸟苷酸环化酶和 NO 敏感鸟苷酸环化酶具有共同的酶激活分子机制。为了探测其他 sGC 结构域对哺乳动物 sGC 血红素环境的影响，我们在β1 H-NOX 构建体（残基 1-194）、β1 H-NOX-PAS-CC 构建体（残基 1-385）和全长α1β1 sGC 异源二聚体（β1 残基 1-619）中生成了血红素口袋突变体（Pro118Ala 和 Ile145Tyr）。这些蛋白质的光谱特征表明，结构域间的通讯调节血红素-NO 配合物的配位状态和血红素氧化速率。总之，这些发现对 H-NOX 结构域包含蛋白的变构调节机制具有重要意义。
