Interaction of Soluble Guanylate Cyclase with YC-1: Kinetic and Resonance Raman Studies † (original) (raw)
Related papers
Chemistry & Biology, 1998
Background: Nitric oxide PNO) is used in biology as both an intercellular signaling agent and a cytotoxic agent. In signaling, submicromolar quantities of *NO stimulate the soluble isoform of guanylate cyclase (sGC) in the receptor cell. *NO increases the V,,, of this heterodimeric hemoprotein up to 400-fold by interacting with the heme moiety of sGC to form a 5-coordinate complex. Carbon monoxide (CO) binds to the heme to form a 6-coordinate complex, but only activates the enzyme 5-fold. YC-1 is a recently discovered compound that relaxes vascular smooth muscle by stimulating sGC. Results: In the presence of YC-1, CO activates sGC to the same specific activity as attained with *NO. YC-1 did not affect the NO-stimulated activity. The on-rate (ken) and off-rate (k,,) of CO for binding to sGC in the presence of YC-1 were determined by stopped-flow spectrophotometry. Neither the k,, nor the koif varied from values previously obtained in the absence of YC-I, indicating that YC-1 has no effect on the affinity of CO for the heme. In the presence of YC-1, the visible spectrum of the sGC-CO complex has a Soret peak at 423 nm, indicating the complex is B-coordinate. Conclusions: YC-1 has no effect on the affinity of CO for the heme of sGC. In the presence of YC-1, maximal activation of sGC by CO is achieved by formation of a 6-coordinate complex between CO and the heme indicating that cleavage of the Fe-His bond is not required for maximal activation of sGC.
Journal of Inorganic Biochemistry, 2004
Soluble guanylate cyclase (sGC), a physiological nitric oxide (NO) receptor, is a heme-containing protein and catalyzes the conversion of GTP to cyclic GMP. We found that 200 mM imidazole moderately activated sGC in the coexistence with 3-(5 0-hydroxymethyl-2 0-furyl)-1-benzylindazole (YC-1), although imidazole or YC-1 alone had little effect for activation. GTP facilitated this process. Resonance Raman spectra of imidazole complex of native sGC and CO-bound sGC (CO-sGC) have demonstrated that a simple heme adduct with imidazole at the sixth coordination position is not present for both sGC and CO-sGC below 200 mM of the imidazole concentration and that the Fe-CO stretching band (m Fe-CO) appears at 492 cm À1 in the presence of imidazole compared with 473 cm À1 in its absence. Both frequencies fall on the line of His-coordinated heme proteins in the m Fe-CO vs m CO plot. However, it is stressed that the CO-heme of sGC becomes apparently photo-inert in a spinning cell in the presence of imidazole, suggesting the formation of five-coordinate CO-heme or of six-coordinate heme with a very weak trans ligand. These observations suggest that imidazole alters not only the polarity of heme pocket but also the coordination structure at the fifth coordination side presumably by perturbing the heme-protein interactions at propionic side chains. Despite the fact that the isolated sGC stays in the reduced state and is not oxidized by O 2 , sGC under the high concentration of imidazole (1.2 M) yielded m 4 at 1373 cm À1 even after its removal by gel-filtration, but addition of dithionite gave the strong m 4 band at 1360 cm À1. This indicated that imidazole caused autoxidation of sGC.
Probing Soluble Guanylate Cyclase Activation by CO and YC-1 Using Resonance Raman Spectroscopy
Biochemistry, 2010
Soluble guanylate cyclase (sGC) is weakly activated by 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 ν FeC and ν CO bands from 473 and 1985 cm −1 to 487 and 1969 cm −1, respectively, and induces an additional ν FeC band, at 521 cm −1 , assigned to 5-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 ν 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 sGC-CO results in a conformational change that activates the protein. Specifically, YC-1 binding alters the heme geometry via peripheral non-bonded contacts, and also relieves an intrinsic electronic effect that diminishes 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 5-coordinate sGC-CO in addition to a conformationally distinct population of 6-coordinate sGC-CO. The loss of YC-1 activation in the sGC variants might involve a weakening of the heme-protein contacts which are thought to be critical to a YC-1-induced conformational change.
Biochemistry, 1998
When expressed in Escherichia coli, the heme domain [ 1(1-385)] of rat lung soluble guanylate cyclase (sGC) is isolated with a stoichiometric amount of bound heme [Zhao, Y., and Marletta, M. A. (1997) Biochemistry 36, 15959-15964]. Nitric oxide (NO) binding to the heme in 1(1-385) leads to cleavage of the Fe-His bond and formation of a five-coordinate NO-heme complex. Addition of imidazole to the five-coordinate NO complex shifts the Soret peak from 399 to 420 nm, which appears to result from the formation of a six-coordinate NO complex. Removal of the added imidazole by gel filtration results in formation of the five-coordinate NO complex once again. The EPR spectrum of the putative six-coordinate NO complex has nine distinct derivative-shaped lines (a triplet of triplets), which is the signature spectrum of a six-coordinate NO complex with two nitrogen atoms as the axial ligands. [ 15 N]Imidazole simplifies the six-coordinate NO complex EPR spectrum to six distinct derivative-shaped lines (a triplet of doublets), indicating that the other axial ligand in the six-coordinate NO complex is an imidazole molecule. These results show that NO binding to sGC not only leads to the cleavage of the Fe-His bond but also induces a conformational change which opens the heme proximal pocket large enough to accommodate an exogenous imidazole molecule. These observations have important implications for determining the NO activation mechanism of sGC.
Probing Domain Interactions in Soluble Guanylate Cyclase
Biochemistry, 2011
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 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, 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. † Funding was provided by NIH grant GM077365 to M.A.M.
Biochemistry, 2005
Resonance Raman (RR) spectra of soluble guanylate cyclase (sGC) reported by five independent research groups have been classified as two types: sGC 1 and sGC 2. Here we demonstrate that the RR spectra of sGC isolated from bovine lung contain only sGC 2 while both species are observed in the spectra of the CO-bound form (CO-sGC). The relative populations of the two forms were altered from an initial composition in which the CO-sGC 2 form predominated, with the Fe-CO (ν Fe-CO) and CO stretching modes (ν CO) at 472 and 1985 cm-1 , respectively, to a composition dominated by the CO-sGC 1 form with ν Fe-CO and ν CO at 488 and 1969 cm-1 , respectively, following the addition of a xenobiotic, YC-1. Further addition of a substrate, GTP, completed the change. GDP and cGMP had a significantly weaker effect, while a substrate analogue, GTP-γ-S, had an effect similar to that of GTP. In contrast, ATP had a reverse effect, and suppressed the effects of YC-1 and GTP. In the presence of both YC-1 and GTP, vinyl vibrations of heme were significantly influenced. New CO isotope-sensitive bands were observed at 521, 488, 363, and 227 cm-1. The 521 cm-1 band was assigned to the five-coordinate (5c) species from the model compound studies using ferrous iron protoporphyrin IX in CTAB micelles. Distinct from the 472 cm-1 species, both the 488 and 521 cm-1 species were apparently un-photodissociable when an ordinary Raman spinning cell was used, indicating rapid recombination of photodissociated CO. On the basis of these findings, binding of YC-1 to the heme pocket is proposed.
Structural Dynamics in the Guanylate Cyclase Heme Pocket after CO Photolysis
Journal of the American Chemical Society, 1999
Soluble guanylate cyclase (sGC) is highly activated by NO, whereas CO, a competing ligand, only weakly activates the enzyme. The fact that NO, but not CO, breaks the sGC heme Fe-proximal histidine bond, has been assumed to be the key step in the NO activation of sGC. In this paper, we investigate the response of the heme pocket of three forms of sGCsnative sGC, the homodimeric heme domain fragment [ 1(1-385)], and the proximal heme-ligand mutant of 1(1-385) [H105G(Im)]sto CO photolysis by using timeresolved resonance Raman spectroscopy to obtain better insight into the interaction of CO with sGC. Our results show that the heme pocket of native sGC assumes its equilibrium conformation within 10 ps after CO photolysis, while in 1(1-385) a 7 cm-1 upshift in ν(Fe-His) indicates a non-equilibrium conformation of the heme pocket, which relaxes with a time constant of 20 ns. In H105G(Im), a frequency downshift of 6 cm-1 is observed for ν(Fe-Im), and heme pocket relaxation has not fully occurred at 1 µs after CO photolysis. These differences can be explained by strain in the proximal heme pocket, which is large in sGC, smaller in 1(1-385), and greatly diminished in H105G(Im). We propose that the strain in the proximal heme pocket plays an important role in the regulation of sGC activation. A model for the activation of sGC is presented.
Biochemistry, 2012
Soluble guanylate cyclase (sGC) is a hemecontaining enzyme that senses nitric oxide (NO). Formation of a heme Fe−NO complex is essential to sGC activation, and several spectroscopic techniques, including electron paramagnetic resonance (EPR) spectroscopy, have been aimed at elucidating the active enzyme conformation. Of these, only EPR spectra (X-band ∼9.6 GHz) have shown differences between low-and high-activity Fe−NO states, and these states are modeled in two different heme domain truncations of sGC, β1(1−194) and β2(1−217), respectively (Derbyshire et al., Biochemistry 2008, 47, 3892−3899). The EPR signal of the lowactivity sGC Fe−NO complex exhibits a broad lineshape that has been interpreted as resulting from site-to-site inhomogeneity, and simulated using g strain, a continuous distribution about the principal values of a given g tensor. This approach, however, fails to account for visible features in the X-band EPR spectra as well as the g anisotropy observed at higher microwave frequencies. Herein we analyze X-, Q-, and D-band EPR spectra and show that both the broad lineshape and the spectral structure of the sGC EPR signal at multiple microwave frequencies can be simulated successfully with a superposition of only two distinct g tensors. These tensors represent different populations that likely differ in Fe−NO bond angle, hydrogen bonding, or the geometry of the amino acid residues. One of these conformations can be linked to a form of the enzyme with higher activity. S oluble guanylate cyclase (sGC) catalyzes the formation of cyclic guanylate monophosphate (cGMP) from guanylate triphosphate (GTP). The synthesized cGMP is a secondary messenger for, and a critical step in, neuronal signaling, platelet aggregation, and vasodilation in mammals. 1−6 Binding of the radical diatomic gas nitric oxide (NO) to the heme cofactor is a key determinant of enzyme activation. sGC is a heterodimer that exists primarily of α1β1 subunits. 7 The β-subunit contains a heme-nitric oxide/oxygen binding (H−NOX) domain at the N-terminus, and a catalytic domain at the C-terminus. The αsubunit also contains a C-terminal catalytic domain, but does not bind heme. The heme cofactor in the β subunit is ligated by histidine, like in most globins, but it does not bind oxygen, and is stable in the ferrous-heme state. Upon NO binding, a sixcoordinate intermediate conformation forms until the Fe−His bond breaks, producing a five-coordinate Fe II −NO complex. 8−13 The α1β1 sGC five-coordinate NO complex is known to exhibit low-activity in the presence of stoichiometric NO and GTP, and high-activity in the presence of the small molecule activator YC-1 or excess NO and GTP. 14,15 In addition to the ubiquitously expressed α1 and β1 subunits, a β2 subunit exists which is expressed largely in the kidney. N-terminal truncations of both β1 and β2 have been prepared and shown to bind heme and NO. 16
Biochemistry, 2010
Modulation of soluble guanylate cyclase (sGC) activity by nitric oxide (NO) involves two distinct steps. Low level activation of sGC is achieved by the stoichiometric binding of NO (1-NO) to the heme cofactor, while much higher activation is achieved by the binding of additional NO (xsNO) at a non-heme site. Addition of the allosteric activator YC-1 to the 1-NO form leads to activity comparable to xsNO state. In this study the mechanisms of sGC activation were investigated using electronic absorption and resonance Raman (RR) spectroscopic methods. RR spectroscopy confirmed that the 1-NO form contains 5-coordinate NO-heme and showed that the addition of NO to the 1-NO form has no significant effect on the spectrum. In contrast, addition of YC-1 to either the 1-NO or xsNO forms alters the RR spectrum significantly, indicating a protein-induced change in the heme geometry. This change in the heme geometry was also observed when BAY 41-2272 was added to the xsNO form. Bands assigned to bending and stretching motions of the vinyl and propionate substituents change intensity in a pattern suggesting altered tilting of the pyrrole rings to which they are attached. In addition, the N-O stretching frequency increases, with no change in the Fe-NO frequency, an effect modeled via DFT calculations as resulting from a small opening of the Fe-N-O angle. These spectral differences demonstrate different mechanisms of activation by synthetic activators, such as YC-1 and BAY 41-2272, and excess NO. † This work was supported financially by NIH grants GM033576 (TGS) and GM077365 (MAM) Supporting Information Available. Complete reference 23 ; Mulliken charges calculated for 5-coordinate (NO)Fe(II)P under varying Fe-NO angle; RR spectra of full-length WT sGC containing one NO (1-NO), excess NO (xsNO), and the 14 NO-15 NO difference bands, covering the ν 4 and ν 7 regions; RR spectra of β1(1-385) in the presence of 1-NO, xsNO and YC-1. This material is available free of charge via the Internet at
Primary response of the sGC heme binding domain to the cleavage of the Fe-His bond
Bioinformation, 2008
Soluble guanylate cyclase (sGC) is an important heme sensor protein. Regulation of the status of heme in the heme binding domain (or HNOX domain) by various gaseous activators can increase the catalytic efficiency of the cyclase domain. Several studies have demonstrated that the full activation of sGC is directly related to the cleavage of the Fe-His bond of the HNOX domain. To expand the primary response of the sGC HNOX domain to the cleavage event, a structural model of the sGC HNOX domain was constructed using homology modeling and the Fe-His bond was released at 6 ns of a 10-ns molecular dynamics simulation. An instant increment of Calpha-RMSD over L2 (Loop2, residues 124-130) was found after the cleavage of the Fe-His bond, which was consistent with the principle component analysis (PCA). The energy analysis results suggest that the motions of L2 are energetic. Based on the results, energetic conformational transformation of L2 is identified as the primary response of the sGC H...