Differences between two active forms of CO-bound soluble guanylate cyclase in the presence of activators and substrate and their populations revealed by resonance Raman spectroscopy (original) (raw)
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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, 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
Interactions of soluble guanylate cyclase with diatomics as probed by resonance Raman spectroscopy
Journal of Inorganic Biochemistry, 2005
Soluble guanylate cyclase (sGC, EC 4.6.1.2) acts as a sensor for nitric oxide (NO), but is also activated by carbon monoxide in the presence of an allosteric modulator. Resonance Raman studies on the structure-function relations of sGC are reviewed with a focus on the CO-adduct in the presence and absence of allosteric modulator, YC-1, and substrate analogues. It is demonstrated that the sGC isolated from bovine lung contains one species with a five-coordinate (5c) ferrous high-spin heme with the Fe-His stretching mode at 204 cm À1 , but its CO adduct yields two species with different conformations about the heme pocket with the Fe-CO stretching (m Fe-CO) mode at 473 and 489 cm À1 , both of which are His-and CO-coordinated 6c ferrous adducts. Addition of YC-1 to it changes their population and further addition of GTP yields one kind of 6c (m Fe-CO = 489 cm À1) in addition to 5c CO-adduct (m Fe-CO = 521 cm À1). Under this condition the enzymatic activity becomes nearly the same level as that of NO adduct. Addition of c-S-GTP yields the same effect as GTP does but cGMP and GDP gives much less effects. Unexpectedly, ATP cancels the effects of GTP. The structural meaning of these spectroscopic observations is discussed in detail.
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.
Interaction of Soluble Guanylate Cyclase with YC-1: Kinetic and Resonance Raman Studies †
Biochemistry, 2000
The enzyme-soluble guanylate cyclase (sGC), which converts GTP to cGMP, is a receptor for the signaling agent nitric oxide (NO). YC-1, a synthetic benzylindazole derivative, has been shown to activate sGC in an NO-independent fashion. In the presence of carbon monoxide (CO), which by itself activates sGC approximately 5-fold, YC-1 activates sGC to a level comparable to stimulation by NO alone. We have used kinetic analyses and resonance Raman spectroscopy (RR) to investigate the interaction of YC-1 and CO with guanylate cyclase. In the presence of CO and 200 µM YC-1, the V max /K m GTP increases 226-fold. While YC-1 does not perturb the RR spectrum of the ferrous form of baculovirus/Sf9 cell expressed sGC, it induces a shift in the Fe-CO stretching frequency for the CO-bound form from 474 to 492 cm-1. Similarly, YC-1 has no effect on the RR spectrum of ferrous 1 1-385 , the isolated sGC heme-binding domain, but shifts the ν(Fe-CO) of CO-1 1-385 from 478 to 491 cm-1 , indicating that YC-1 binds in heme-binding region of sGC. In addition, the CO-bound forms of sGC and 1 1-385 in the presence of YC-1 lie on the ν(Fe-CO) vs ν(C-O) correlation curve for proximal ligands with imidazole character, which suggests that histidine remains the heme proximal ligand in the presence of YC-1. Interestingly, YC-1 does not shift ν(Fe-CO) for the CO-bound form of H105G(Im), the imidazole-rescued heme ligand mutant of 1 1-385. The data are consistent with binding of CO and YC-1 to the sGC hemebinding domain leading to conformational changes that give rise to an increase in catalytic turnover and a change in the electrostatic environment of the heme pocket.
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.
Biochemistry, 2012
Soluble guanylyl cyclase (sGC), the key enzyme for the formation of second messenger cyclic GMP (cGMP), is an authentic sensor for nitric oxide (NO). Binding of NO to sGC leads to strong activation of the enzyme activity. Multiple molecules and steps of NO binding to sGC have been implicated, but the target of the second NO and the detailed binding mechanism remain controversial. In this study, we used 15 NO and 14 NO and anaerobic sequential mixing-freeze quench EPR to unambiguously confirm that the heme Fe is the target of the second NO. Linear dependence on NO concentrations up to 600 s −1 for the observed rate of the second step of NO binding not only indicates that the binding site of the second NO is different from that in the first step, i.e. the proximal site of the heme, but also support a concerted mechanism in which the dissociation of the His105 proximal ligand occurs simultaneously with the binding of the second NO molecule. Computer modeling successfully predicts the kinetics of formation of a set of fivecoordinate NO complexes with the ligand on either the distal or proximal site and supports a selective release of NO from the distal side of the transient bis-NO sGC complex. Thus, as has been demonstrated with cytochrome c', a five-coordinate NO-sGC containing a proximal NO is formed after the binding of the second NO.
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
Identification of residues crucially involved in soluble guanylate cyclase activation
2006
The ubiquitous heterodimeric nitric oxide (NO) receptor soluble guanylate cyclase (sGC) plays a key role in various signal transduction pathways. Binding of NO takes place at the prosthetic heme moiety at the N-terminus of the b 1-subunit of sGC. The induced structural changes lead to an activation of the catalytic C-terminal domain of the enzyme and to an increased conversion of GTP into the second messenger cyclic GMP (cGMP). In the present work we selected and substituted different residues of the sGC heme-binding pocket based on a sGC homology model. The generated sGC variants were tested in a cGMP reporter cell for their effect on the enzyme activation by heme-dependent (NO, BAY 41-2272) stimulators and hemeindependent (BAY 58-2667) activators. The use of these experimental tools allows the enzyme's heme content to be explored in a non-invasive manner. Asp 44 , Asp 45 and Phe 74 of the b 1-subunit were identified as being crucially important for functional enzyme activation. b 1 Asp 45 may serve as a switch between different conformational states of sGC and point to a possible mechanism of action of the heme dependent sGC stimulator BAY 41-2272. Furthermore, our data shows that the activation profile of b 1 IIe 145 Tyr is unchanged compared to the native enzyme, suggesting that Tyr 145 does not confer the ability to distinguish between NO and O 2. In summary, the present work further elucidated intramolecular mechanisms underlying the NOand BAY 41-2272-mediated sGC activation and raises questions regarding the postulated role of Tyr 145 for ligand discrimination.
Molecular steps in soluble guanylate cyclase activation
BMC Pharmacology, 2005
Soluble guanylate cyclase (sGC) is a hemoprotein that is selectively activated by specifically binding NO. Once activated sGC synthesizes cyclic GMP from GTP which then triggers reactions essential to animal physiology. sGC essentially functions as a selective sensor for NO. sGC belongs to a recently identified group of proteins termed the H-NOX family (Heme Nitric oxide/OXygen binding proteins) that includes bacterial counterparts from aerobic and anaerobic organisms . Based on our recent structure of a family member [3], a molecular basis for the ligand discrimination against O 2 in NO-regulated sGCs has been established. Further studies have pointed towards O 2 -regulated sGCs in C. elegans . NO binding to the heme remains as a key molecular activation step; however, it has become clear that activation and deactivation are regulated in a complex manner . In the accepted model shown below, NO binds to the sGC heme, activating the enzyme after conversion to the 5coordinate nitrosyl complex.