Probing Domain Interactions in Soluble Guanylate Cyclase

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• 作者：Emily R. Derbyshire ; Michael B. Winter ; Mohammed Ibrahim ; Sarah Deng ; Thomas G. Spiro ; Michael A. Marletta
• 刊名：Biochemistry
• 出版年：2011
• 出版时间：May 24, 2011
• 年：2011
• 卷：50
• 期：20
• 页码：4281-4290
• 全文大小：958K
• 年卷期：v.50,no.20(May 24, 2011)
• ISSN：1520-4995

文摘

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鈥揘itric 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 尾1 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鈥搘orm chimera containing the atypical sGC Gcy-33 H-NOX domain was weakly activated by NO, CO, and O₂, 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 尾1 H-NOX construct (residues 1鈥?94), the 尾1 H-NOX-PAS-CC construct (residues 1鈥?85), and the full-length 伪1尾1 sGC heterodimer (尾1 residues 1鈥?19). Spectroscopic characterization of these proteins shows that interdomain communication modulates the coordination state of the heme鈥揘O 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.