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Probing soluble guanylate cyclase activation by CO and YC-1 using resonance Raman spectroscopy.

ABSTRACT: Soluble guanylate cyclase (sGC) is weakly activated by carbon monoxide (CO) but is significantly activated by the binding of YC-1 to the sGC-CO complex. In this report, resonance Raman (RR) spectroscopy was used to study selected sGC variants. Addition of YC-1 to the sGC-CO complex alters the intensity pattern of RR bands assigned to the vinyl and propionate heme substituents, suggesting changes in the tilting of the pyrrole rings to which they are attached. YC-1 also shifts the RR intensity of the nu(FeC) and nu(CO) bands from 473 and 1985 cm(-1) to 487 and 1969 cm(-1), respectively, and induces an additional nu(FeC) band, at 521 cm(-1), assigned to five-coordinate heme-CO. Site-directed variants in the proximal heme pocket (P118A) or in the distal heme pocket (V5Y and I149Y) reduce the extent of YC-1 activation, along with the 473 cm(-1) band intensity. These lower-activity sGC variants display another nu(FeC) band at 493 cm(-1) which is insensitive to YC-1 addition and is attributed to protein that cannot be activated by the allosteric activator. The results are consistent with a model in which YC-1 binding to the sGC-CO complex results in a conformational change that activates the protein. Specifically, YC-1 binding alters the heme geometry via peripheral nonbonded contacts and also relieves an intrinsic electronic effect that weakens FeCO backbonding in the native, YC-1 responsive protein. This electronic effect might involve neutralization of the heme propionates via H-bond contacts or negative polarization by a distal cysteine residue. YC-1 binding also strains the Fe-histidine bond, leading to a population of the five-coordinate sGC-CO complex in addition to a conformationally distinct population of the six-coordinate sGC-CO complex. The loss of YC-1 activation in the sGC variants might involve a weakening of the heme-protein contacts that are thought to be critical to a YC-1-induced conformational change.

SUBMITTER: Ibrahim M

PROVIDER: S-EPMC2894327 | biostudies-literature | 2010 May

REPOSITORIES: biostudies-literature

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Probing soluble guanylate cyclase activation by CO and YC-1 using resonance Raman spectroscopy.

Ibrahim Mohammed M Derbyshire Emily R ER Marletta Michael A MA Spiro Thomas G TG

Biochemistry 20100501 18

Soluble guanylate cyclase (sGC) is weakly activated by carbon monoxide (CO) but is significantly activated by the binding of YC-1 to the sGC-CO complex. In this report, resonance Raman (RR) spectroscopy was used to study selected sGC variants. Addition of YC-1 to the sGC-CO complex alters the intensity pattern of RR bands assigned to the vinyl and propionate heme substituents, suggesting changes in the tilting of the pyrrole rings to which they are attached. YC-1 also shifts the RR intensity of ...[more]

PMID: 20353168

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Project description: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.

Dataset Information

Probing soluble guanylate cyclase activation by CO and YC-1 using resonance Raman spectroscopy.

Publications

Probing soluble guanylate cyclase activation by CO and YC-1 using resonance Raman spectroscopy.

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