Biochemical Characterization of HumanS-Nitrosohemoglobin

Patel, Rakesh P.; Hogg, Neil; Spencer, Netanya Y.; Kalyanaraman, Balaraman; Matalon, Sadis; Darley‐Usmar, Victor

doi:10.1074/jbc.274.22.15487

Cited by 130 publications

(57 citation statements)

References 33 publications

(54 reference statements)

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“…NO bioactivity is then exported out of RBCs through the anion exchange protein, band 3 (or AE1). Although detailed mechanisms of SNO (Snitrosothiol species) formation and NO bioactivity export from band 3 are still unclear (10,11), the theory provides an explanation for the preservation of NO bioactivity and highlights the importance of the RBC in preserving NO bioavailability, which has been suggested previously (12).…”

mentioning

confidence: 98%

Modulation of nitric oxide bioavailability by erythrocytes

Huang

Han

Hyduke

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

168

138

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Nitric oxide (NO) activates soluble guanylyl cyclase in smooth muscle cells to induce vasodilation in the vasculature. However, as hemoglobin (Hb) is an effective scavenger of NO and is present in high concentrations inside the red blood cell (RBC), the bioavailability of NO would be too low to elicit soluble guanylyl cyclase activation in the presence of blood. Therefore, NO bioactivity must be preserved. Here we present evidence suggesting that the RBC participates in the preservation of NO bioactivity by reducing NO influx. The NO uptake by RBCs was increased and decreased by altering the degree of band 3 binding to the cytoskeleton. Methemoglobin and denatured hemoglobin binding to the RBC membrane or cytoskeleton also were shown to contribute to reducing the NO uptake rate of the RBC. These alterations in NO uptake by the RBC, hence the NO bioavailability, were determined to correlate with the vasodilation of isolated blood vessels. Our observations suggest that RBC membrane and cytoskeleton associated NO-inert proteins provide a barrier for NO diffusion and thus account for the reduction in the NO uptake rate of RBCs. E xtensive studies have established the bioactivity of nitric oxide (NO) in vasoregulation through activation of soluble guanylyl cyclase (ref. 1 and references therein). On the other hand, NO is rapidly deactivated by oxygenated hemoglobin (HbO 2 ) and myoglobin (MbO 2 ) to form nitrate. As NO reacts rapidly (k ϳ 10 7 M Ϫ1 ⅐s Ϫ1) with HbO 2 and deoxygenated Hb (deoxyHb), infusion of cell-free normoxic Hb (hereafter, Hb denotes both HbO 2 and deoxyHb) at M levels into animal models, human subjects, or isolated blood vessels (2-5) causes significant vessel constriction due to NO scavenging. However, normal blood containing RBC-encapsulated Hb (rbcHb) at an equivalent concentration of about 10 mM shows insignificant NO reactivity under physiological conditions (4,6,7). This discrepancy is difficult to explain in view of the high permeability of NO through lipid bilayers. Thus, NO bioactivity in blood must be preserved by a yet unclear mechanism under physiological conditions. This problem recently was addressed by a NO bioactivity export theory (8). According to this theory, NO enters the RBC and preferentially binds with the free heme on Hb to form heme-nitrosylHb (HbNO) rather than being oxidized by O 2 -conjugated heme (9). HbNO then transfers the conjugated NO to ␤-93Cys to form S-nitrosoHb (9). NO bioactivity is then exported out of RBCs through the anion exchange protein, band 3 (or AE1). Although detailed mechanisms of SNO (Snitrosothiol species) formation and NO bioactivity export from band 3 are still unclear (10, 11), the theory provides an explanation for the preservation of NO bioactivity and highlights the importance of the RBC in preserving NO bioavailability, which has been suggested previously (12).In parallel to the NO bioactivity export theory, an independent, but not mutually exclusive, explanation that NO bioavailability is preserved by reducing interactions between NO and r...

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mentioning

confidence: 98%

Modulation of nitric oxide bioavailability by erythrocytes

Huang

Han

Hyduke

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

168

138

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“…Rates of transnitrosation by GSNO or other NO donors have been measured for small molecule thiols (14), bovine serum albumin (15), hemoglobin (16), and thioredoxin (17). However, the kinetic mechanism of transnitrosation has yet to be reported for any protein.…”

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confidence: 99%

S-Nitrosation of Glutathione Transferase P1-1 Is Controlled by the Conformation of a Dynamic Active Site Helix

Balchin

Wallace

Dirr

2013

Journal of Biological Chemistry

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Background: S-Nitrosation is an emerging post-translational modification that is not yet well understood on a molecular level. Results: We propose a mechanism for S-nitrosation of glutathione transferase P1-1 by S-nitrosoglutathione. Conclusion: Cys 101 is nitrosated in a single step, but Cys 47 nitrosation is limited by the rate of helix 2 opening. Significance: Detailing the mechanism of spontaneous transnitrosation is crucial to understanding how protein S-nitrosation is controlled.

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“…Accordingly, NO would be preserved as iron nitrosyl Hb (HbNO) or SNO-Hb such that the total NO either bound or bonded to Hb would essentially remain constant during changes in oxygen tension, with NO migrating from the heme iron to the ␤-93 cysteine as oxygen tension increases, and conversely migrating from the cysteine to the heme as oxygen tension decreases (31). Although the NO transport model that includes allosterically controlled intramolecular transfer is attractive, it remains controversial (33)(34)(35)(36)(37)(38)(39)(40). In this article, we directly examine one aspect of the model, the ability of Hb to transfer NO from the ␤-93 cysteine to the heme and vice versa during cycles of oxygenation and deoxygenation.…”

mentioning

confidence: 99%

Measurements of nitric oxide on the heme iron and β-93 thiol of human hemoglobin during cycles of oxygenation and deoxygenation

Cho

Spencer

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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Nitric oxide has been proposed to be transported by hemoglobin as a third respiratory gas and to elicit vasodilation by an oxygenlinked (allosteric) mechanism. For hemoglobin to transport nitric oxide bioactivity it must capture nitric oxide as iron nitrosyl hemoglobin rather than destroy it by dioxygenation. Once bound to the heme iron, nitric oxide has been reported to migrate reversibly from the heme group of hemoglobin to the ␤-93 cysteinyl residue, in response to an oxygen saturation-dependent conformational change, to form an S-nitrosothiol. However, such a transfer requires redox chemistry with oxidation of the nitric oxide or ␤-93 cysteinyl residue. In this article, we examine the ability of nitric oxide to undergo this intramolecular transfer by cycling human hemoglobin between oxygenated and deoxygenated states. Under various conditions, we found no evidence for intramolecular transfer of nitric oxide from either cysteine to heme or heme to cysteine. In addition, we observed that contaminating nitrite can lead to formation of iron nitrosyl hemoglobin in deoxygenated hemoglobin preparations and a radical in oxygenated hemoglobin preparations. Using 15 N-labeled nitrite, we clearly demonstrate that nitrite chemistry could explain previously reported results that suggested apparent nitric oxide cycling from heme to thiol. Consistent with our results from these experiments conducted in vitro, we found no arterial͞venous gradient of iron nitrosyl hemoglobin detectable by electron paramagnetic resonance spectroscopy. Our results do not support a role for allosterically controlled intramolecular transfer of nitric oxide in hemoglobin as a function of oxygen saturation. How is the activity of the endothelium-derived relaxation factor, nitric oxide (NO), preserved if an abundant scavenger, hemoglobin (Hb), reacts so quickly with it? This question has puzzled many investigators ever since the identification of NO as the endothelium-derived relaxation factor was made (1-4). NO reacts with Hb via the following reactions:where Fe(II) and Fe(III) refer to the ferrous and ferric forms of the iron in the heme group of Hb, respectively. The rate constants for these reactions are 2.6 ϫ 10 7 M Ϫ1 ⅐s Ϫ1 at 20°C (5) and 6-9 ϫ 10 7 M Ϫ1 ⅐s Ϫ1 at 20°C (6, 7) for Eqs. 1 and 2, respectively. The second reaction destroys NO activity through the formation of nitrate. The activity of NO is potentially conserved by the reaction described by Eq. 1, but to function it must come off the heme and get out of the RBC (where the Hb concentration is Ϸ0.02 M in heme) before undergoing the fast reactions described by Eqs. 1 and 2. Thus, scavenging of NO in blood can be predicted to be a significant NO sink and poses the problem of how NO can function as the endothelium-derived relaxation factor (8).One possible resolution of this paradox would be that NO produced in endothelial cells acts only locally and is thus immune to scavenging by Hb, but a variety of evidence indicating increased vasoconstriction when free Hb or Hb substitutes are in...

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Biochemical Characterization of HumanS-Nitrosohemoglobin

Cited by 130 publications

References 33 publications

Modulation of nitric oxide bioavailability by erythrocytes

Modulation of nitric oxide bioavailability by erythrocytes

S-Nitrosation of Glutathione Transferase P1-1 Is Controlled by the Conformation of a Dynamic Active Site Helix

Measurements of nitric oxide on the heme iron and β-93 thiol of human hemoglobin during cycles of oxygenation and deoxygenation

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