Soluble guanylate cyclase (sGC) serves as a receptor for the signaling agent nitric oxide (NO). sGC synthesis of cGMP is regulated by NO, GTP, ATP and allosteric activators such as YC-1. Guanylate cyclase and the adenylate cyclase activity of full-length sGC and the sGC catalytic domain constructs (α1catβ1cat) is reported here. ATP is a mixed-type inhibitor of cGMP production for both sGC and α1catβ1cat indicating that the C-terminus of sGC contains an allosteric nucleotide binding site. YC-1 did not activate α1catβ1cator compete with ATP inhibition of cGMP synthesis, which suggests that YC-1 and ATP bind to distinct sites. α1catβ1cat and NO-stimulated sGC also synthesize cAMP, but this activity is inhibited by ATP via non-competitive substrate inhibition, and by GTP via mixed-type inhibition. Additionally, the adenylate cyclase activity of purified sGC was inhibited by PC12 lysate, suggesting that an intracellular small molecule or protein regulates this activity in vivo.

Nitric oxide (NO) is the physiologically relevant activator of the mammalian hemoprotein soluble guanylate cyclase (sGC).The heme cofactor of ␣1␤1 sGC has a high affinity for NO but has never been observed to form a complex with oxygen. Introduction of a key tyrosine residue in the sGC heme binding domain ␤1(1-385) is sufficient to produce an oxygen-binding protein, but this mutation in the full-length enzyme did not alter oxygen affinity. To evaluate ligand binding specificity in fulllength sGC we mutated several conserved distal heme pocket residues (␤1 Val-5, Phe-74, Ile-145, and Ile-149) to introduce a hydrogen bond donor in proximity to the heme ligand. We found that the NO coordination state, NO dissociation, and enzyme activation were significantly affected by the presence of a tyrosine in the distal heme pocket; however, the stability of the reduced porphyrin and the proteins affinity for oxygen were unaltered. Recently, an atypical sGC from Drosophila, Gyc-88E, was shown to form a stable complex with oxygen. Sequence analysis of this protein identified two residues in the predicted heme pocket (tyrosine and glutamine) that may function to stabilize oxygen binding in the atypical cyclase. The introduction of these residues into the rat ␤1 distal heme pocket (Ile-145 3 Tyr and Ile-149 3 Gln) resulted in an sGC construct that oxidized via an intermediate with an absorbance maximum at 417 nm. This absorbance maximum is consistent with globin Fe II -O 2 complexes and is likely the first observation of a Fe II -O 2 complex in the full-length ␣1␤1 protein. Additionally, these data suggest that atypical sGCs stabilize O 2 binding by a hydrogen bonding network involving tyrosine and glutamine.Soluble guanylate cyclase (sGC) 3 is the most thoroughly characterized receptor for the gaseous signaling agent nitric oxide (NO). NO induced activation of sGC is critical to several physiological processes, including neurotransmission, vasodilation, and platelet aggregation (1-3). The importance of sGC to physiological function has been clearly demonstrated over the last decade (4, 5); however, much less is understood about the molecular mechanisms that regulate enzyme activity.sGC is a heterodimeric hemoprotein consisting of two homologous subunits, ␣ and ␤. The ␣1␤1 heterodimer is the most prevalent and commonly studied protein. Despite the same histidine ligated heme and iron oxidation state as found in the globins, sGC shows no measurable affinity for O 2 and therefore can selectively bind NO in the presence of O 2 (reviewed in Refs. 6, 7). Interestingly, not only does sGC discriminate against O 2 binding to the heme, but both the Fe II -unligated and Fe II -NO species are stable in an aerobic environment (8). This is in stark contrast to other hemoproteins that readily bind and ultimately react with O 2 (9 -12). The mechanism by which sGC discriminates against O 2 binding as well as the molecular events that lead to activation remain to be elucidated.There have been several proposals on the mechanism of ligand discr...

Eukaryotic nitric oxide (NO) signaling involves modulation in 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, chimeras were constructed 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-NOXs 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 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, heme pocket mutants (Pro118Ala and Ile145Tyr) were generated 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 inter-domain 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 containing proteins.

Lipid bilayer-coated mesoporous silica nanoparticles are unique core–shell nanomaterials currently being developed as drug delivery vehicles. To improve cargo loading and biocirculation, the pore structure and surface chemistry of the particle have been modified and well characterized. However, an understanding of cargo release mechanisms from cellular uptake pathways remains largely unexplored. Here, we present a study of the release mechanism of lipid bilayer-coated silica particles induced by endosomal-like pH change from 7.4 to 5.0. We found that this relatively small pH change produces rapid deformation of the supported lipid bilayer that ultimately results in holes in the membrane. Using a combination of dye release studies, wide-field and confocal fluorescence microscopies, and surface area modeling analysis, we determined that small blister-like structures are formed, which lead to lateral membrane displacement and hole formation. Possible mechanisms for the blister formation, which include curvature effects and interfacial interactions, are discussed.