Effects of Iron Nitrosylation on Sickle Cell Hemoglobin Solubility

Xu, Xiuli; Lockamy, Virginia; Chen, Kejing; Huang, Zhi; Shields, Howard; King, S. Bruce; Ballas, Samir K.; Nichols, James S.; Gladwin, Mark T.; Noguchi, Constance Tom; Schechter, Alan N.; Kim‐Shapiro, Daniel B.

doi:10.1074/jbc.m205350200

Cited by 22 publications

(12 citation statements)

References 42 publications

(41 reference statements)

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“…The percentage yield of each iron-nitrosylheme species during the reaction time course was similar to that observed by Hille et al when NO buffer was titrated into deoxyHb solution (25). Also, the iron-nitrosyl-heme spectral change observed during the reaction process, as shown in Figure 2C, was similar to the spectral change observed during deoxygenation and reoxygenation of substoichiometric iron-nitrosyl-Hb (26). This confirms that as the deoxyheme is converted to metheme and iron-nitrosyl-heme during the reaction, the T-to-R allosteric transition does indeed occur.…”

Section: Resultssupporting

confidence: 70%

“…EPR spectroscopy was conducted at temperatures of 132 K with a Bruker 4111 VT variable temperature unit and ESR-ER-200 D spectrometer set (Bruker BioSpin GmbH) at 9.43 GHz microwave frequency, 10 mW microwave power, 5 Gauss modulation amplitude, a time constant of 100 ms, and a collection time of 100 seconds for each scan. The EPR spectrum for iron-nitrosyl-Hb was deconvoluted to obtain the percentage of each species of nitrosyl-heme (penta- and hexacoordinate α heme and hexacoordinate β-nitrosyl-heme) using basis spectra as described previously (26).…”

Section: Methodsmentioning

confidence: 99%

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Enzymatic function of hemoglobin as a nitrite reductase that produces NO under allosteric control

Huang¹

2005

Journal of Clinical Investigation

469

511

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Hypoxic vasodilation is a fundamental, highly conserved physiological response that requires oxygen and/or pH sensing coupled to vasodilation. While this process was first characterized more than 80 years ago, the precise identity and mechanism of the oxygen sensor and mediators of vasodilation remain uncertain. In support of a possible role for hemoglobin (Hb) as a sensor and effector of hypoxic vasodilation, here we show biochemical evidence that Hb exhibits enzymatic behavior as a nitrite reductase, with maximal NO generation rates occurring near the oxy-to-deoxy (R-to-T) allosteric structural transition of the protein. The observed rate of nitrite reduction by Hb deviates from second-order kinetics, and sigmoidal reaction progress is determined by a balance between 2 opposing chemistries of the heme in the R (oxygenated conformation) and T (deoxygenated conformation) allosteric quaternary structures of the Hb tetramer -the greater reductive potential of deoxyheme in the R state tetramer and the number of unligated deoxyheme sites necessary for nitrite binding, which are more plentiful in the T state tetramer. These opposing chemistries result in a maximal nitrite reduction rate when Hb is 40-60% saturated with oxygen (near the Hb P 50 ), an apparent ideal set point for hypoxiaresponsive NO generation. These data suggest that the oxygen sensor for hypoxic vasodilation is determined by Hb oxygen saturation and quaternary structure and that the nitrite reductase activity of Hb generates NO gas under allosteric and pH control.

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Section: Resultssupporting

confidence: 70%

Section: Methodsmentioning

confidence: 99%

Enzymatic function of hemoglobin as a nitrite reductase that produces NO under allosteric control

Huang¹

2005

Journal of Clinical Investigation

469

511

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“…Partially liganded NO samples can result in the formation of α-nitrosylhemoglobin, in which the molecule retains a T-or deoxy quaternary state structure. Partially liganded HbS-NO does not significantly increase fiber solubility, as it does not stabilize or promote the formation of the R quaternary state [11]. These results call into question mechanisms, which require increased O 2 affinity as a consequence of HbNO formation.…”

Section: Introductionmentioning

confidence: 89%

The role of β93 Cys in the inhibition of Hb S fiber formation

Knee

Roden

Flory

et al. 2007

Biophysical Chemistry

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Recent studies have suggested that nitric oxide (NO) binding to hemoglobin (Hb) may lead to the inhibition of sickle cell fiber formation and the dissolution of sickle cell fibers. NO can react with Hb in at least 3 ways: 1) formation of Hb(II)NO, 2) formation of methemoglobin, and 3) formation of S-nitrosohemoglobin, through nitrosylation of the β93 Cys residue. In this study, the role of β93 Cys in the mechanism of sickle cell fiber inhibition is investigated through chemical modification with N-ethylmaleimide. UV resonance Raman, FT-IR and electrospray ionization mass spectroscopic methods in conjunction with equilibrium solubility and kinetic studies are used to characterize the effect of β93 Cys modification on Hb S fiber formation. Both FT-IR spectroscopy and electrospray mass spectrometry results demonstrate that modification can occur at both the β93 and α104 Cys residues under relatively mild reaction conditions. Equilibrium solubility measurements reveal that singlymodified Hb at the β93 position leads to increased amounts of fiber formation relative to unmodified or doubly-modified Hb S. Kinetic studies confirm that modification of only the β93 residue leads to a faster onset of polymerization. UV resonance Raman results indicate that modification of the α104 residue in addition to the β93 residue significantly perturbs the α 1 β 2 interface, while modification of only β93 does not. These results in conjunction with the equilibrium solubility and kinetic measurements are suggestive that modification of the α104 Cys residue and not the β93 Cys residue leads to T-state destabilization and inhibition of fiber formation. These findings have implications for understanding the mechanism of NO binding to Hb and NO inhibition of Hb S fiber formation.

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“…Pure metHb is prepared by reacting hemoglobin with potassium ferricyanide (5 equivalents to 1 heme), and pure NOHb is prepared using an in-situ method of generating NO in solution. Excess sodium dithionite is added to deoxygenated hemoglobin and a stock solution of nitrite is added to the deoxyHb sample (63). The nitrite is reduced by dithionite to form NO, which binds quickly to the hemoglobin, allowing for stochiometric amounts of NOHb.…”

Section: Deconvolution Of Spectramentioning

confidence: 99%

Enhancing Nitrite Reductase Activity of Modified Hemoglobin: Bis-tetramers and Their PEGylated Derivatives

Lui

Kluger

2009

Biochemistry

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The clinical evaluation of stabilized tetrameric hemoglobin as alternatives to red cells revealed that the materials caused significant increases in blood pressure and related problems and this was attributed to the scavenging of nitric oxide and extravasation. The search for materials with reduced vasoactivity led to the report that conjugates of hemoglobin tetramers and polyethylene glycol (PEG) chains did not elicit these pressor effects. However, this material does not deliver oxygen efficiently due to its lack of cooperativity and high oxygen affinity, making it unsuitable as an oxygen carrier. It has been recently reported that PEGconjugated hemoglobin converts nitrite to nitric oxide at a faster rate than does the native protein, which may compensate for the scavenging of nitric oxide. It is therefore important to alter hemoglobin in order to enhance nitrite reductase activity while retaining its ability to deliver oxygen. If the beneficial effect of PEG is associated with the increased size reducing extravasation, this can also be achieved by coupling cross-linked tetramers to one another, giving materials with appropriate oxygen affinity and cooperativity for use as circulating oxygen carriers. In the present study it is shown that cross-linked bis-tetramers with good oxygen delivery potential have enhanced nitrite reductase activity with k obs = 0.70 M -1 s -1 (24 °C), compared to native protein and cross-linked tetramers, k obs = 0.25 M -1 s -1 and k obs = 0.52 M -1 s -1 , respectively, but are less active in reduction of nitrite than Hb-PEG5K 2 (k obs = 2.5 M -1 s -1 ). However, conjugation of four PEG chains to the bis-tetramer (at each β-Cys-93) produces a material with greatly increased nitrite reductase activity (k obs = 1.8 M -1 s -1 ) while retaining cooperativity (P 50 = 4.1, n 50 = 2.4). Thus, PEGylated bistetramers combine increased size and enhanced nitrite reductase activity expected for decreased vasoactivity with characteristics of an acceptable HBOC.

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Effects of Iron Nitrosylation on Sickle Cell Hemoglobin Solubility

Cited by 22 publications

References 42 publications

Enzymatic function of hemoglobin as a nitrite reductase that produces NO under allosteric control

Enzymatic function of hemoglobin as a nitrite reductase that produces NO under allosteric control

The role of β93 Cys in the inhibition of Hb S fiber formation

Enhancing Nitrite Reductase Activity of Modified Hemoglobin: Bis-tetramers and Their PEGylated Derivatives

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