Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA.
Proc Natl Acad Sci U S A. 2009 Dec 22;106(51):21602-7. doi: 10.1073/pnas.0911083106. Epub 2009 Dec 9.
Nitric oxide (NO) regulates a number of essential physiological processes by activating soluble guanylate cyclase (sGC) to produce the second messenger cGMP. The mechanism of NO sensing was previously thought to result exclusively from NO binding to the sGC heme; however, recent studies indicate that heme-bound NO only partially activates sGC and additional NO is involved in the mechanism of maximal NO activation. Furthermore, thiol oxidation of sGC cysteines results in the loss of enzyme activity. Herein the role of cysteines in NO-stimulated sGC activity investigated. We find that the thiol modifying reagent methyl methanethiosulfonate specifically inhibits NO activation of sGC by blocking a non-heme site, which defines a role for sGC cysteine(s) in mediating NO binding. The nature of the NO/cysteine interaction was probed by examining the effects of redox active reagents on NO-stimulated activity. These results show that NO binding to, and dissociation from, the critical cysteine(s) does not involve a change in the thiol redox state. Evidence is provided for non-heme NO in the physiological activation of sGC in context of a primary cell culture of human umbilical vein endothelial cells. These findings have relevance to diseases involving the NO/cGMP signaling pathway.
一氧化氮 (NO) 通过激活可溶性鸟苷酸环化酶 (sGC) 产生第二信使 cGMP 来调节许多重要的生理过程。NO 感应的机制以前被认为完全是由于 NO 与 sGC 血红素结合;然而，最近的研究表明，血红素结合的 NO 仅部分激活 sGC，并且额外的 NO 参与最大 NO 激活的机制。此外，sGC 半胱氨酸的巯基氧化导致酶活性丧失。本文研究了半胱氨酸在 NO 刺激的 sGC 活性中的作用。我们发现，硫醇修饰试剂甲硫基甲烷硫代磺酸特异性地通过阻断非血红素结合位点来抑制 sGC 的 NO 激活，这定义了 sGC 半胱氨酸在介导 NO 结合中的作用。通过检查氧化还原活性试剂对 NO 刺激活性的影响，研究了 NO/半胱氨酸相互作用的性质。这些结果表明，NO 与关键半胱氨酸的结合和解离不涉及硫醇氧化还原状态的变化。在人脐静脉内皮细胞的原代细胞培养中，为非血红素 NO 在 sGC 的生理激活中提供了证据。这些发现与涉及 NO/cGMP 信号通路的疾病有关。
