Calcium ion downregulates soluble guanylyl cyclase activity: evidence for a two-metal ion catalytic mechanism

The inhibition of soluble guanylyl cyclase by Ca2+ has been kinetically characterized and the results support a two-metal-ion catalytic mechanism for formation of cGMP. Ca2+ reversibly inhibits both the basal and NO-stimulated forms of bovine lung soluble guanylyl cyclase. Inhibition is independent of the activator identity and concentration, revealing that Ca2+ interacts with a site independent of the heme regulatory site. Inhibition by Ca2+ is competitive with respect to Mg2+ in excess of substrate, with Kis values of 29 +/- 4 and 6.6 +/- 0.6 microM for the basal and activated states, respectively. Ca2+ inhibits noncompetitively with respect to the substrate MgGTP in both activity states. The qualitatively similar inhibition pattern and quantitatively different Ki values between the basal and NO-stimulated states suggest that the Ca2+ binding site undergoes some structural modification upon activation of the enzyme. The competitive nature of Ca2+ inhibition with respect to excess Mg2+ is consistent with a two-metal-ion mechanism for cyclization.     

Activation of soluble guanylyl cyclase at the leading edge during Dictyostelium chemotaxis

Dictyostelium contains two guanylyl cyclases, GCA, a 12-transmembrane enzyme, and sGC, a homologue of mammalian soluble adenylyl cyclase. sGC provides nearly all chemoattractant-stimulated cGMP formation and is essential for efficient chemotaxis toward cAMP. We show that in resting cells the major fraction of the sGC-GFP fusion protein localizes to the cytosol, and a small fraction is associated to the cell cortex. With the artificial substrate Mn2+/GTP, sGC activity and protein exhibit a similar distribution between soluble and particulate fraction of cell lysates. However, with the physiological substrate Mg2+/GTP, sGC in the cytosol is nearly inactive, whereas the particulate enzyme shows high enzyme activity. Reconstitution experiments reveal that inactive cytosolic sGC acquires catalytic activity with Mg2+/GTP upon association to the membrane. Stimulation of cells with cAMP results in a twofold increase of membrane-localized sGC-GFP, which is accompanied by an increase of the membrane-associated guanylyl cyclase activity. In a cAMP gradient, sGC-GFP localizes to the anterior cell cortex, suggesting that in chemotacting cells, sGC is activated at the leading edge of the cell.     

A-350619: a novel activator of soluble guanylyl cyclase

Nitric oxide (NO) is a key mediator in many physiological processes and one of the major receptors through which NO exerts its effects is soluble guanylyl cyclase. Guanylyl cyclase converts GTP to cyclic GMP as part of the cascade that results in physiological processes such as smooth muscle relaxation, neurotransmission, inhibition of platelet aggregation and immune response. The properties of A-350619, a novel soluble guanylyl cyclase activator, were examined to determine the modulatory effect on the catalytic properties of soluble guanylyl cyclase. A-350619 increased V(max) from 0.1 to 14.5 micromol/min/mg (145 fold increase), and lowered K(m) from 300 to 50 microM (6 fold decrease). When YC-1 (another sGC activator) and A-350619 were combined, a 156 fold increase in V(max) and a 5 fold decrease in Km were observed, indicating that the modulation of the enzyme brought about by YC-1 and A-350619 are not additive, suggesting a common binding site. Activation of soluble guanylyl cyclase by A-350619 was partially inhibited by ODQ, a specific inhibitor of soluble guanylyl cyclase by oxidation of the enzyme heme. YC-1 and A-350619 after pre-treatment with N-omega-nitro-L-arginine, an NO-synthase inhibitor, relaxed cavernosum tissue strips in a dose-dependent manner with EC(50) of 50 microM and 80 microM, respectively. Addition of SNP potentiated the relaxation effect of YC-1 and A-350619, shifting the dose-response curve to the left to 3 microM and 10 microM, respectively. Consistent with its biochemical activity, A-350619 (1 micromol/kg) alone induced penile erection in a conscious rat model. Activation of soluble guanylyl cyclase in cavernosum tissue as an alternate method of enhancing the effect of NO may provide a novel treatment of sexual dysfunction.     

Regulation of soluble guanylate cyclase activity by porphyrins and metalloporphyrins

Alterations of the chemical structure of protoporphyrin IX markedly altered the activation of soluble guanylate cyclase purified from bovine lung. Hydrophobic side chains at positions 2 and 4 and vicinal propionic acid residues at positions 6 and 7 of the porphyrin ring (protoporphyrin IX, mesoporphyrin IX) were essential for maximal enzyme activation (Ka = 7-8 nM; Vmax = 6-8 mumol of cGMP/min/mg). Substitution of hydrophobic with polar groups (hematoporphyrin IX, coproporphyrin III), or with hydrogen atoms ( deuteroporphyrin IX), and methylation of propionate residues resulted in decreased enzyme stimulation. Stimulatory porphyrins increased the Vmax and the apparent affinities of enzyme for MgGTP and uncomplexed Mg2+. An open central core in the porphyrin ring was essential for enzyme activation. The pyrrolic nitrogen adduct, N-phenylprotoporphyrin IX, was inhibitory and competitive with protoporphyrin IX (KI = 73 nM). Similarly, metalloporphyrins inhibited enzymatic activity and ferro-protoporphyrin IX (KI = 350 nM), zinc-protoporphyrin IX (KI = 50 nM) and manganese-protoporphyrin IX (KI = 9 nM) were competitive with protoporphyrin IX. Inhibitory porphyrins and metalloporphyrins also prevented enzyme activation by S-nitroso-N- acetylpenicillamine and NO. Guanylate cyclase reconstituted with such porphyrins required higher concentrations of protoporphyrin IX for further activation and were not activated by NO. Thus, porphyrins, metalloporphyrins, and NO appeared to interact at a common binding site on guanylate cyclase. This common site is likely that which normally binds heme and, therefore, NO-heme when the heme-containing enzyme is exposed to NO. Thus, NO and nitroso compounds may react with enzyme-bound heme to generate a modified porphyrin which structurally resembles protoporphyrin IX in its interaction with guanylate cyclase.     

The Dictyostelium homologue of mammalian soluble adenylyl cyclase encodes a guanylyl cyclase

A new Dictyostelium discoideum cyclase gene was identified that encodes a protein (sGC) with 35% similarity to mammalian soluble adenylyl cyclase (sAC). Gene disruption of sGC has no effect on adenylyl cyclase activity and results in a >10-fold reduction in guanylyl cyclase activity. The scg- null mutants show reduced chemotactic sensitivity and aggregate poorly under stringent conditions. With Mn(2+)/GTP as substrate, most of the sGC activity is soluble, but with the more physiological Mg(2+)/GTP the activity is detected in membranes and stimulated by GTPgammaS. Unexpectedly, orthologues of sGC and sAC are present in bacteria and vertebrates, but absent from Drosophila melanogaster, Caenorhabditis elegans, Arabidopsis thaliana and Saccharomyces cerevisiae.     

Nucleotidyl cyclase activity of soluble guanylyl cyclase in intact cells

Soluble guanylyl cyclase (sGC) is activated by nitric oxide (NO) and generates the second messenger cyclic GMP (cGMP). Recently, purified sGC α₁β₁ has been shown to additionally generate the cyclic pyrimidine nucleotides cCMP and cUMP. However, since cyclic pyrimidine nucleotide formation occurred only the presence of Mn²⁺ but not Mg²⁺, the physiological relevance of these in vitro findings remained unclear. Therefore, we studied cyclic nucleotide formation in intact cells. We observed NO-dependent cCMP- and cUMP formation in intact HEK293 cells overexpressing sGC α₁β₁ and in RFL-6 rat fibroblasts endogenously expressing sGC, using HPLC–tandem mass spectrometry. The identity of cCMP and cUMP was unambiguously confirmed by HPLC–time-of-flight mass spectrometry. Our data indicate that cCMP and cUMP play second messenger roles and that Mn²⁺ is a physiological sGC cofactor.     

Nitric oxide-dependent allosteric inhibitory role of a second nucleotide binding site in soluble guanylyl cyclase

The mechanism of desensitization of the nitric oxide (NO) receptor (alpha1.beta1 isoform of soluble guanylyl cyclase, sGC) is not known. Models of the structure of alpha1.beta1, based on the x-ray crystal structure of adenylyl cyclase (AC) suggest the existence of a nucleotide-like binding site, in addition to the putative catalytic site. We have previously reported that mutating residues that coordinate Mg(2+)GTP (substrate) binding in alpha1.beta1 into those present in AC fully reverts GC activity to AC activity. The wild-type form of alpha1.beta1 (GC-wt) and the mutant form (AC-mut, alpha1R592Q.beta1E473K,C541D) were purified, and their sensitivities to various nucleotides were assessed. In using the AC-mut as well as other mutants that coordinate purine binding, we were able to distinguish allosteric inhibitory effects of guanine nucleotides from competitively inhibitory effects on catalytic activity. Here we report that several nucleotide analogs drastically alter sGC and AC-mut activity by acting at a second nucleotide site, likely pseudosymmetric to the catalytic site. In particular, Mg(2+)GTP gamma S and Mg(2+)ATP gamma S inhibited cyclase activity through a mixed, non-competitive mechanism that was only observable under NO stimulation and not under basal conditions. The non-competitive pattern of inhibition was not present in mutants carrying the substitution beta1D477A, the pseudosymmetric equivalent to alpha1D529 (located in the substrate-binding site and involved in substrate binding and catalysis), or with the double mutations alpha1E525K,C594D, the pseudosymmetric equivalent to beta1E473K,C541D. Taken together these data suggest that occupation of the second site by nucleotides may underlie part of the mechanism of desensitization of sGC.     

Hemoglobin mediated nitrite activation of soluble guanylyl cyclase

Nitrite has long been known to be vasoactive when present at large concentrations but it was thought to be inactive under physiological conditions. Surprisingly, we have recently shown that supraphysiological and near physiological concentrations of nitrite cause vasodilation in the human circulation. These effects appeared to result from reduction of nitrite by deoxygenated hemoglobin. Thus, nitrite was proposed to play a role in hypoxic vasodilation. We now discuss these results in the context of nitrite reacting with hemoglobin and effecting vasodilation and present new data modeling the nitric oxide (NO) export from the red blood cell and measurements of soluble guanylate cyclase (sGC) activation. We conclude that NO generated within the interior of the red blood cell is not likely to be effectively exported directly as nitric oxide. Thus, an intermediate species must be formed by the nitrite/deoxyhemoglobin reaction that escapes the red cell and effects vasodilation.     

Regulation of nitric oxide-responsive recombinant soluble guanylyl cyclase by calcium.

Calcium (Ca2+) and cyclic GMP (cGMP) subserve antagonistic functions that are reflected in their coordinated reciprocal regulation in physiological systems. However, molecular mechanisms by which Ca2+ regulates cGMP-dependent signaling remain incompletely defined. In this study, the inhibition of recombinant nitric oxide (NO)-stimulated soluble guanylyl cyclase (SGC) by Ca2+ was demonstrated. The alpha- and beta-subunits of recombinant rat SGC were heterologously coexpressed in HEK 293 cells which do not express NO synthase, whose Ca2+-stimulated activity can confound the effects of that cation on SGC. Ca2+ inhibited basal and NO-stimulated SGC in a concentration- and guanine nucleotide-dependent fashion. This cation inhibited SGC in crude cell extracts and immunopurified preparations. Ca2+ lowered both the Vmax and Km of SGC via an uncompetitive mechanism through direct interaction with the enzyme. In intact HEK 293 cells, increases in the intracellular Ca2+ concentration induced by ionomycin, a Ca2+ ionophore, and thapsigargin, which releases intracellular stores of that cation, inhibited NO-stimulated intracellular cGMP accumulation. Similarly, carbachol-induced elevation of the intracellular Ca2+ concentration inhibited NO-stimulated intracellular cGMP accumulation in HEK 293 cells. These data demonstrate that SGC behaves as a sensitive Ca2+ detector that may play a central role in coordinating the reciprocal regulation of Ca2+- and cGMP-dependent signaling mechanisms.     

Differential inhibition of adenylyl cyclase isoforms and soluble guanylyl cyclase by purine and pyrimidine nucleotides

Mammals express nine membranous adenylyl cyclase isoforms (ACs 1-9), a structurally related soluble guanylyl cyclase (sGC) and a soluble AC (sAC). Moreover, Bacillus anthracis and Bacillus pertussis produce the AC toxins, edema factor (EF), and adenylyl cyclase toxin (ACT), respectively. 2'(3')-O-(N-methylanthraniloyl)-guanosine 5'-[gamma-thio]triphosphate is a potent competitive inhibitor of AC in S49 lymphoma cell membranes. These data prompted us to study systematically the effects of 24 nucleotides on AC in S49 and Sf9 insect cell membranes, ACs 1, 2, 5, and 6, expressed in Sf9 membranes and purified catalytic subunits of membranous ACs (C1 of AC5 and C2 of AC2), sAC, sGC, EF, and ACT in the presence of MnCl(2). N-Methylanthraniloyl (MANT)-GTP inhibited C1.C2 with a K(i) of 4.2 nm. Phe-889 and Ile-940 of C2 mediate hydrophobic interactions with the MANT group. MANT-inosine 5'-[gamma-thio]triphosphate potently inhibited C1.C2 and ACs 1, 5, and 6 but exhibited only low affinity for sGC, EF, ACT, and G-proteins. Inosine 5'-[gamma-thio]triphosphate and uridine 5'-[gamma-thio]triphosphate were mixed G-protein activators and AC inhibitors. AC5 was up to 15-fold more sensitive to inhibitors than AC2. EF and ACT exhibited unique inhibitor profiles. At sAC, 2',5'-dideoxyadenosine 3'-triphosphate was the most potent compound (IC(50), 690 nm). Several MANT-adenine and MANT-guanine nucleotides inhibited sGC with K(i) values in the 200-400 nm range. UTP and ATP exhibited similar affinities for sGC as GTP and were mixed sGC substrates and inhibitors. The exchange of MnCl(2) against MgCl(2) reduced inhibitor potencies at ACs and sGC 1.5-250-fold, depending on the nucleotide and cyclase studied. The omission of the NTP-regenerating system from cyclase reactions strongly reduced the potencies of MANT-ADP, indicative for phosphorylation to MANT-ATP by pyruvate kinase. Collectively, AC isoforms and sGC are differentially inhibited by purine and pyrimidine nucleotides.     

Purification of soluble guanylyl cyclase from bovine lung by a new immunoaffinity chromatographic method

Soluble guanylyl cyclase was purified from bovine lung by an immunoaffinity chromatographic method using IgG fractions of antisera against a synthetic peptide of the C-terminus of the 70-kDa subunit of the enzyme. After anion-exchange chromatography, the enzyme was bound to an immunoaffinity column and was eluted with the synthetic peptide. This method allowed the convenient isolation of 2 mg of apparently homogeneous enzyme from 40 g cytosolic proteins. The enzyme had an apparent molecular mass of about 150 kDa and consisted of two subunits (70 kDa and 73 kDa) as determined by gel permeation fast protein liquid chromatography and SDS/PAGE. The basal activities determined in the presence of Mg2+ and Mn2+ were 10-20 nmol.min-1.mg-1 and 80-100 nmol.min-1.mg-1, respectively. The enzyme exhibited an ultraviolet-visible absorption spectrum typical for hemoproteins, with a Soret band at 430 nm. The purified enzyme was stimulated by NO-containing compounds. Maximal enzyme activities measured in the presence of sodium nitroprusside were 1.2-2.4 mumol.min-1.mg-1 (half-maximal effect of sodium nitroprusside at 1.3-1.9 microM) and 0.9-1.8 mumol.min-1.mg-1 (half-maximal effect at 0.28-0.41 microM sodium nitroprusside) in the presence of Mg2+ and Mn2+, respectively. The method developed for the large-scale purification of soluble guanylyl cyclase by immunoaffinity chromatography, using synthetic peptides for the elution of the enzyme, appears to be superior to previously described methods. As antibodies against synthetic peptides corresponding to deduced amino acid sequences of the respective protein are easily obtained, the described method may be suitable for a convenient large-scale purification of various proteins.     

Studying the structure and regulation of soluble guanylyl cyclase

Soluble guanylyl cyclase acts as the receptor for the signaling molecule nitric oxide. The enzyme consists of two different subunits. Each subunit shows the cyclase catalytic domain, which is also conserved in the membrane-bound guanylyl cyclases and the adenylyl cyclases. The N-terminal regions of the subunits are responsible for binding of the prosthetic heme group of the enzyme, which is required for the stimulatory effect of nitric oxide (NO). The five-coordinated ferrous heme displays a histidine as the axial ligand; activation of soluble guanylyl cyclase by NO is initiated by binding of NO to the heme iron and proceeds via breaking of the histidine-to-iron bond. Recently, a novel pharmacological and possibly physiological principle of guanylyl cyclase sensitization was demonstrated. The substance YC-1 has been shown to activate the enzyme independent of NO, to potentiate the effect of submaximally effective NO concentrations, and to turn carbon monoxide into an effective activator of soluble guanylyl cyclase.     

A critical role for ATP in the stimulation of retinal guanylyl cyclase by guanylyl cyclase-activating proteins

It has been believed that retinal guanylyl cyclase (retGC), a key enzyme in the cGMP recovery to the dark state, is solely activated by guanylyl cyclase-activating proteins (GCAPs) in a Ca2+-sensitive manner. However, a question has arisen as to whether the observed GCAP stimulation of retGC is sufficient to account for the cGMP recovery because the stimulated activity measured in vitro is less than the light/GTP-activated cGMP phosphodiesterase activity. Here we report that the retGC activation by GCAPs is larger than previously reported and that a preincubation with adenine nucleotide is essential for the large activation. Under certain conditions, ATP is two times more effective than adenylyl imidodiphosphate (AMP-PNP), a hydrolysis-resistant ATP analog; however, this study mainly used AMP-PNP to focus on the role of adenine nucleotide binding to retGC. When photoreceptor outer segment homogenates are preincubated with AMP-PNP (EC50 = 0.65 +/- 0.20 mM), GCAP2 enhanced the retGC activity 10-13 times over the control rate. Without AMP-PNP, GCAP2 stimulated the control activity only 3-4-fold as in previous reports. The large activation is due to a GCAP2-dependent increase in Vmax without an alteration of retGC affinity for GCAP2 (EC50 = 47.9 +/- 2.7 nM). GCAP1 stimulated retGC activity in a similar fashion but with lower affinity (EC50 = 308 nM). In the AMP-PNP preincubation, low Ca2+ concentrations are not required, and retGC exists as a monomeric form. This large activation is accomplished through enhanced action of GCAPs as shown by Ca2+ inhibition of the activity (IC50 = 178 nM). We propose that retGC is activated by a two-step mechanism: a conformational change by ATP binding to its kinase homology domain under high Ca2+ concentrations that allows large enhancement of GCAP activation under low Ca2+ concentrations.     

Immunolocalization of soluble guanylyl cyclase subunits in rat kidney

Stimulation of soluble guanylyl cyclase (SGC) by nitric oxide (NO) results in the generation of cyclic guanosine monophosphate (cGMP). We recently described expression of abundant nitric oxide synthase, the enzyme by which NO is generated froml-arginine, in macula densa cells of rat kidney at the protein and mRNA level. In the present study we looked for possible targets of NO in the kidney. By light and electron microscopy, we applied polyclonal antisera against four subunits (₁,₂, ₁₂) of SGC in immunocytochemical studies of frozen sections of rat kidney. We demonstrate the presence of ₁-subunit in glomerular podocytes and of ₂-subunit in principal cells of the collecting duct. In both cell types a cytosolic localization was evident from ultrastructural analysis. Regarding the collecting duct, NO was shown by other authors to inhibit sodium reabsorption in cultured mouse cortical collecting duct principal cells. In podocytes NO may relax the contractile system of podocyte foot processes, the tone of which has been suggested to counteract the elastic distension of the capillary wall.     

Amphipathic alpha-helix mediates the heterodimerization of soluble guanylyl cyclase

Soluble guanylyl cyclase (soluble GC) is an enzyme consisting of alpha and beta subunits and catalyzes the conversion of GTP to cGMP. The formation of the heterodimer is essential for the activity of soluble GC. Each subunit of soluble GC has been shown to comprize three functionally different parts: a C-terminal catalytic domain, a central dimerization domain, and an N-terminal regulatory domain. The central dimerization domain of the beta(1) subunit, which contains an N-terminal binding site (NBS) and a C-terminal binding site (CBS), has been postulated to be responsible for the formation of alpha/ beta heterodimer. In this study, we analyzed heterodimerization by the pull-down assay using the affinity between a histidine tag and Ni(2+) Sepharose after co-expression of various N- and C-terminally truncated FLAG-tagged mutants of the alpha(1) subunit and the histidine-tagged wild type of the beta(1) subunit in the vaculovirus/Sf9 system, and demonstrated that the CBS-like sequence of the alpha(1) subunit is critical for the formation of the heterodimer with the beta(1) subunit and the NBS-like sequence of the alpha(1) subunit is essential for the formation of the enzymatically active heterodimer, although this particular sequence was not involved in heterodimerization. The analysis of the secondary structure of the alpha(1) subunit predicted the existence of an amphipathic alpha-helix in residues 431-464. Experiments with site-directed alpha(1) subunit mutant proteins demonstrated that the amphipathicity of the alpha-helix is important for the formation of the heterodimer, and Leu(463) in the alpha-helix region plays a critical role in the formation of a properly arranged active center in the dimer.     

Sensitizing soluble guanylyl cyclase to become a highly CO-sensitive enzyme.

It took at least a decade to realize that the toxic gas NO is the physiological activator of soluble guanylyl cyclase (sGC), thereby acting as a signaling molecule in the nervous and cardiovascular systems. Despite its rather poor sGC-activating property, CO has also been implicated as a physiological stimulator of sGC in neurotransmission and vasorelaxation. Here, we establish YC-1 as a novel NO-independent sGC activator that potentiates both CO- and NO-induced sGC stimulation. As this potentiating effect is also observed with protoporphyrin IX which activates sGC independently of a gaseous ligand, we conclude that stabilization of the enzyme's active configuration is the underlying mechanism of YC-1's action. Moreover, the results obtained with YC-1 reveal that CO is capable of stimulating sGC to a degree similar to NO, and thus provide the molecular basis for CO functioning as a signaling molecule.     

Characterization of a novel type of endogenous activator of soluble guanylyl cyclase

Nitric oxide (NO) remains the only firmly established endogenous modulator of soluble guanylyl cyclase (sGC) activity, but physiological, structural, and biochemical evidence now suggests that in vivo regulation of sGC involves direct interaction with other factors. We searched for such endogenous modulators in human umbilical vein endothelial cells and COS-7 cells. The cytosolic fraction of both cell types stimulated the activity of semipurified sGC severalfold in the absence or presence of a saturating concentration of NO. The cytosolic factor was sensitive to proteinase K and destroyed by boiling, suggesting that it contains a protein component. Size exclusion chromatography revealed peaks of activity between 40 and 70 kDa. The sGC-activating effect was further purified by ion exchange chromatography. In the presence of the benzylindazole YC-1 or NO, the partially purified factor synergistically activated sGC, suggesting that this factor had a mode of activation different from that of YC-1 or NO. Four candidate activators were identified from the final purification step by matrix-assisted laser desorption ionization mass spectrometry analysis. Using an sGC affinity matrix, one of them, the molecular chaperone Hsp70, was shown to directly interact with sGC. This interaction was further confirmed by co-immunoprecipitation in lung tissues and by co-localization in smooth muscle cells. sGC and Hsp70 co-localized at the plasma membrane, supporting the idea that sGC can be translocated to the membrane. Hsp70 co-purifies with the sGC-activating effect, and immunodepletion of Hsp70 from COS-7 cytosol coincided with a marked attenuation of the sGC-activating effect, yet the effect was not rescued by the addition of pure Hsp70. Thus, Hsp70 is a novel sGC-interacting protein that is responsible for the sGC-activating effect, probably in association with other factors or after covalent modification.     

Aspartate 102 in the heme domain of soluble guanylyl cyclase has a key role in NO activation

Nitric oxide (NO) is involved in the physiology and pathophysiology of the cardiovascular and neuronal systems via activation of soluble guanylyl cyclase (sGC), a heme-containing heterodimer. Recent structural studies have allowed a better understanding of the residues that dictate the affinity and binding of NO to the heme and the resulting breakage of the bond between the heme iron and histidine 105 (H105) of the β subunit of sGC. Still, it is unknown how the breakage of the iron-His bond translates into NO-dependent increased catalysis. Structural studies on homologous H-NOX domains in various states pointed to a role for movement of the H105 containing αF helix. Our modeling of the heme-binding domain highlighted conserved residues in the vicinity of H105 that could potentially regulate the extent to which the αF helix shifts and/or propagate the activation signal once the covalent bond with H105 has been broken. These include a direct interaction of αF helix residue aspartate 102 (D102) with the backbone nitrogen of F120. Mutational analysis of this region points to an essential role of the interactions in the vicinity of H105 for heme stability and identifies D102 as having a key role in NO activation following breakage of the iron-His bond.