Structural Changes in the Heme Proximal Pocket Induced by Nitric Oxide Binding to Soluble Guanylate Cyclase

Zhao, Yunde; Hoganson, Curtis W.; Babcock, Gerald T.; Marletta, Michael A.

doi:10.1021/bi9811563

Cited by 67 publications

(59 citation statements)

References 31 publications

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“…Nitric oxide exerts a repulsive trans effect upon binding to ferrous hemes; in proteins such as guanylate cyclase, distal NO binding results in rupture of the proximal histidine-iron bond. As reported previously, binding constants of imidazoles to H93G-NO can be measured by a UV/vis titration technique (6); similar results have recently been reported for a cavity mutant in a subunit fragment of guanylate cyclase (3).…”

Section: Effect Of Ser 92 On Im Exchange Ratessupporting

confidence: 78%

Hydrogen Bonding Modulates Binding of Exogenous Ligands in a Myoglobin Proximal Cavity Mutant

et al. 1999

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In the sperm whale myoglobin mutant H93G, the proximal histidine is replaced by glycine, leaving a cavity in which exogenous imidazole can bind and ligate the heme iron (Barrick, D. (1994) Biochemistry 33, 6545-6554). Structural studies of this mutant suggest that serine 92 may play an important role in imidazole binding by serving as a hydrogen bond acceptor. Serine 92 is highly conserved in myoglobins, forming a well-characterized weak hydrogen bond with the proximal histidine in the native protein. We have probed the importance of this hydrogen bond through studies of the double mutants S92A/H93G and S92T/H93G incorporating exogenous imidazole and methylimidazoles. 1 H NMR spectra reveal that loss of the hydrogen bond in S92A/H93G does not affect the conformation of the bound imidazole. However, the binding constants for imidazoles to the ferrous nitrosyl complex of S92A/H93G are much weaker than in H93G. These results are discussed in terms of hydrogen bonding and steric packing within the proximal cavity. The results also highlight the importance of the trans diatomic ligand in altering the binding and sensitivity to perturbation of the ligand in the proximal cavity.Exogenous molecules which serve a specific structural or functional role can be inserted into engineered protein cavities as an extension of site-directed mutagenesis for dissecting protein structure-function relationships in heme proteins (1-4). When a residue which ligates the heme iron (such as histidine) is mutated to a smaller, nonligating amino acid (typically alanine or glycine), exogenous molecules which may mimic natural amino acids side chains or bear nonnatural functionality can be introduced into the resulting cavity and incorporated into the protein structure. For example, in sperm whale myoglobin (Mb 1 ), the proximal histidine (His 93) is the sole covalent linkage between the polypeptide chain and the heme iron. In the mutant H93G, this residue has been replaced by glycine, creating a cavity in the proximal heme pocket. When H93G Mb is expressed in Escherichia coli in the presence of imidazole, the protein is isolated with an imidazole molecule occupying that cavity and serving as an axial ligand to the heme iron (1). This imidazole can be replaced with a variety of exogenous ligands via a simple dialysis technique, producing protein complexes that are distinguishable by many physical observables (2, 5-7). These proteins are denoted H93G(L), where L represents the bound proximal ligand, and they are hybrids of model compounds and site-directed mutants. As in model compounds, the heme ligand can be systematically varied beyond the limited range of the naturally occurring amino acids, but because the protein architecture is preserved, interactions between the polypeptide and the heme ligand can also be studied.The most striking difference between the structures of metaquoH93G(Im) and wild-type myoglobin (WTMb) is in the conformation of the proximal imidazole ring. The exogenous proximal imidazole of H93G(Im) is rotated ab...

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Section: Effect Of Ser 92 On Im Exchange Ratessupporting

confidence: 78%

Hydrogen Bonding Modulates Binding of Exogenous Ligands in a Myoglobin Proximal Cavity Mutant

et al. 1999

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“…It has been shown that with NO binding to sGC, a fivecoordinate heme complex with characteristic EPR spectrum with triplet splitting is observed (42,43). Indeed, with close examination of the observed NO-heme EPR spectra arising in ischemic myocardium, we observe that in addition to the large six-coordinate NO-Mb heme signal, a small component of five coordinate heme can be discerned, particularly with longer periods of ischemia (Fig.…”

Section: Fig 10supporting

confidence: 64%

Nitrosyl-Heme Complexes Are Formed in the Ischemic Heart

Tiravanti

Samouilov

Zweíer

2004

Journal of Biological Chemistry

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In addition to the generation from specific nitric-oxide (NO) synthases, NO formation from nitrite occurs in ischemic tissues, such as the heart. Although NO binding to heme-centers is the basis for NO-mediated signaling as occurs through guanylate cyclase, it is not known if this process is triggered with physiologically relevant periods of sublethal ischemia and if nitrite serves as a critical substrate. Therefore electron paramagnetic resonance studies were performed to measure nitrosylheme formation during the time course of myocardial ischemia and reperfusion and the role of nitrite in this process. Rat hearts were either partially nitrite-depleted by nitrite-free buffer perfusion or nitrite-enriched by preinfusion with 50 M nitrite. Ischemic hearts loaded with nitrite showed prominent spectra of six-coordinate nitrosyl-heme complexes, primarily NOmyoglobin, that increased as a function of ischemic duration, whereas in nonischemic-controls these signals were not seen. Total nitrosyl-heme concentrations within the heart were 6.6 ؎ 0.7 M after 30 min of ischemia. Nitrite-depleted hearts also gave rise to NO-heme signals during ischemia, but levels were 8-fold lower. Nitrite-mediated NO-heme complex formation during ischemia was associated with activation of guanylate cyclase. Upon reperfusion, the levels of NO-heme complexes decreased 3-fold by the first 15 min but remained elevated for over 45 min. The decrease in NO-heme complex levels was paralleled by the formation of nitrate, suggesting the oxidation of heme-bound NO upon reperfusion. Thus, nitrite-mediated NO-heme formation occurs progressively during ischemia, with these complexes serving as a store of NO with concordant activation of NO signaling pathways.

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“…This dramatically emphasizes the role of the distal heme pocket structure, which leads in sGC to maximize the probability of NO interaction with Fe 2ϩ as shown by the very low base line (0.03), and it is plausible that the breaking of the iron-His bond, simultaneously to triggering the enzymatic activity, also changes the geometry of the heme pocket toward a more closed conformation, lowering the escape rate. This hypothetical closing of the distal side is supported by the observation that imidazole binds at the proximal side of the heme after NO ligation (35) and deserves further investigation.…”

Section: Figmentioning

confidence: 59%

“…The structural changes leading to the catalytic activation may include steps that cannot be simply reversed by rupture of the NO-Fe 2ϩ bond. After cleavage of the His-Fe 2ϩ bond triggered by NO ligation, an imidazole molecule can take its place at the proximal side of the heme pocket (35), showing that indeed structural changes are induced. The very large binding rate constant is almost diffusion-limited (k on Ͼ 1.4 ϫ 10 8 M Ϫ1 ⅐s Ϫ1 ; Ref.…”

Section: Figmentioning

confidence: 99%

Control of Nitric Oxide Dynamics by Guanylate Cyclase in Its Activated State

Négrerie

Bouzhir

Martin

et al. 2001

Journal of Biological Chemistry

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are assumed to induce conformational changes, which are the origin of the catalytic activation. At the molecular level, the activation and deactivation mechanisms are unknown, as is the dynamics of NO once in the heme pocket. Using ultrafast time-resolved absorption spectroscopy, we measured the kinetics of NO rebinding to sGC after photodissociation. The main spectral transient in the Soret band does not match the equilibrium difference spectrum of NO-liganded minus unliganded sGC, and the geminate rebinding was found to be monoexponential and ultrafast ( ‫؍‬ 7.5 ps), with a relative amplitude close to unity (0.97). These characteristics, so far not observed in other hemoproteins, indicate that NO encounters a high energy barrier for escaping from the heme pocket once the His-Fe 2؉ bond has been cleaved; this bond does not reform before NO recombination. The deactivation of isolated sGC cannot occur by only simple diffusion of NO from the heme; therefore, several allosteric states may be inferred, including a desensitized one, to induce NO release. Thus, besides the structural change leading to activation, a consequence of the decoupling of the proximal histidine may also be to induce a change of the heme pocket distal geometry, which raises the energy barrier for NO escape, optimizing the efficiency of NO trapping. The nonsingle exponential character of the NO picosecond rebinding coexists only with the presence of the protein structure surrounding the heme, and the single exponential rate observed in sGC is very likely to be due to a closed conformation of the heme pocket. Our results emphasize the physiological importance of NO geminate recombination in hemoproteins like nitric-oxide synthase and sGC and show that the protein structure controls NO dynamics in a manner adapted to their function. This control of ligand dynamics provides a regulation at molecular level in the function of these enzymes.Guanylate cyclase is involved in signal transduction pathways in which cGMP 1 acts as a second messenger in several types of cells (for reviews, see Refs. 1-4). In endothelial cells, sGC is activated by NO produced by the constitutive enzyme nitric-oxide synthase. The binding of NO induces a several hundred-fold increase of the sGC catalytic activity, leading to cGMP production from GTP. This heterodimeric enzyme possesses a heme in the ␤-subunit where NO binding takes place, while the ␣-subunit harbors the catalytic site, which binds the precursor substrate GTP. The second messenger cGMP then triggers the downstream events leading to smooth muscle relaxation.Regarding diatomic ligand binding, sGC differs from other hemoproteins like myoglobin or hemoglobin; while sGC displays a very high affinity for NO, its affinity for O 2 is very low, but the structural features giving rise to this property are unknown. As in Hb and Mb, the heme iron of sGC is bound to a His side chain (5, 6) but much more weakly. Upon binding of NO to the heme, the His-Fe 2ϩ bond is cleaved, as reflected by the change in Soret absorption posi...

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Structural Changes in the Heme Proximal Pocket Induced by Nitric Oxide Binding to Soluble Guanylate Cyclase

Cited by 67 publications

References 31 publications

Hydrogen Bonding Modulates Binding of Exogenous Ligands in a Myoglobin Proximal Cavity Mutant

Hydrogen Bonding Modulates Binding of Exogenous Ligands in a Myoglobin Proximal Cavity Mutant

Nitrosyl-Heme Complexes Are Formed in the Ischemic Heart

Control of Nitric Oxide Dynamics by Guanylate Cyclase in Its Activated State

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