A novel antioxidant role for hemoglobin

The comproportionation of ferrylhemoglobin with oxyhemoglobin

Cecilia R Giulivi, Kelvin J A Davies

Research output: Contribution to journalArticle

187 Citations (Scopus)

Abstract

Ferrylhemoglobin (X-FeIV-OH, where X denotes an amino acid residue in the globin moiety) has long been suspected as a cytotoxic agent produced by the interaction of oxyhemoglobin (X-FeIIO2) or methemoglobin (X-FeIII) with H2O2 in red blood cells. To date, however, technical difficulties have prevented the identification and quantification of X-FeIV-OH. Oxyhemoglobin exposed to a continuous flux of H2O2 (generated at a rate of 120 μM/min during the glucose oxidase-catalyzed oxidation of glucose) was oxidized to (a) X-FeIV-OH when [X-FeIIO2] < 75 μM and (b) X-FeIII when [X-FeIIO2] > 75 μM (the production of X-FeIII proceeded with intermediate formation of X-FeIV-OH). The reduction of the X-FeIV-OH to X-FeIII could be explained by either of two alternative mechanisms: a O- 2-mediated X-FeIV-OH → X-FeIII transition or a comproportionation of X-FeIV-OH and X-FeIIO2 to yield X-FeIII (a process mediated by a tyrosine moiety in the hemoprotein). The low rate of X-FeIIO2 autoxidation plus the negligible decrease in the rate of X-FeIII formation in the presence of either native or heat-denatured superoxide dismutase or apoenzyme (1 μM) suggested that O- 2 does not contribute to the reduction of X-FeIV-OH. Moreover, the dependence of X-FeIII formation on X-FeIIO2 concentration, together with the results of O2 uptake and H2O2 consumption measurements, provide experimental evidence to support the comproportionation reaction. Comproportionation is apparently catalyzed by intermolecular electron transfer between tyrosine residues, since the reaction did not occur when tyrosine residues were blocked by acetylation. Intact red blood cells exposed to the same flow rate of H2O2 presented a spectral profile which could be explained as a transition from X-FeIIO2 to X-FeIII. The intermediate production of X-FeIV-OH was detected by adding Na2S (2 mM), which revealed a spectral profile identical with that obtained with purified X-FeIV-OH. Measurements of concentrations and relative rate constants for the reaction of various intracellular reductants (glutathione, NAD(P)H, uric acid, ascorbic acid) with X-FeIV-OH revealed that comproportionation of X-FeIV-OH with X-FeIIO2 is the favored reaction. Our results provide (to our knowledge) the first definitive evidence for X-FeIV-OH in intact red blood cells. The rapid comproportionation reaction between X-FeIV-OH and X-FeIIO2 (to produce X-FeIII) explains why X-FeIV-OH has been elusive to date. Moreover, the comproportionation reaction represents an important and novel antioxidant function for red blood cell X-FeIIO2, in which the cytotoxic X-FeIV-OH is quenched and the resultant X-FeIII is re-reduced by methemoglobin reductase.

Original languageEnglish (US)
Pages (from-to)19453-19460
Number of pages8
JournalJournal of Biological Chemistry
Volume265
Issue number32
StatePublished - Nov 15 1990
Externally publishedYes

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Methemoglobin
Oxyhemoglobins
Hemoglobins
Antioxidants
Blood
Erythrocytes
Cells
Tyrosine
Cytochrome-B(5) Reductase
ferrylhemoglobin
Apoenzymes
Acetylation
Glucose Oxidase
Globins
Reducing Agents
Cytotoxins
Uric Acid
NAD
Ascorbic Acid
Superoxide Dismutase

ASJC Scopus subject areas

  • Biochemistry

Cite this

A novel antioxidant role for hemoglobin : The comproportionation of ferrylhemoglobin with oxyhemoglobin. / Giulivi, Cecilia R; Davies, Kelvin J A.

In: Journal of Biological Chemistry, Vol. 265, No. 32, 15.11.1990, p. 19453-19460.

Research output: Contribution to journalArticle

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title = "A novel antioxidant role for hemoglobin: The comproportionation of ferrylhemoglobin with oxyhemoglobin",
abstract = "Ferrylhemoglobin (X-FeIV-OH, where X denotes an amino acid residue in the globin moiety) has long been suspected as a cytotoxic agent produced by the interaction of oxyhemoglobin (X-FeIIO2) or methemoglobin (X-FeIII) with H2O2 in red blood cells. To date, however, technical difficulties have prevented the identification and quantification of X-FeIV-OH. Oxyhemoglobin exposed to a continuous flux of H2O2 (generated at a rate of 120 μM/min during the glucose oxidase-catalyzed oxidation of glucose) was oxidized to (a) X-FeIV-OH when [X-FeIIO2] < 75 μM and (b) X-FeIII when [X-FeIIO2] > 75 μM (the production of X-FeIII proceeded with intermediate formation of X-FeIV-OH). The reduction of the X-FeIV-OH to X-FeIII could be explained by either of two alternative mechanisms: a O- 2-mediated X-FeIV-OH → X-FeIII transition or a comproportionation of X-FeIV-OH and X-FeIIO2 to yield X-FeIII (a process mediated by a tyrosine moiety in the hemoprotein). The low rate of X-FeIIO2 autoxidation plus the negligible decrease in the rate of X-FeIII formation in the presence of either native or heat-denatured superoxide dismutase or apoenzyme (1 μM) suggested that O- 2 does not contribute to the reduction of X-FeIV-OH. Moreover, the dependence of X-FeIII formation on X-FeIIO2 concentration, together with the results of O2 uptake and H2O2 consumption measurements, provide experimental evidence to support the comproportionation reaction. Comproportionation is apparently catalyzed by intermolecular electron transfer between tyrosine residues, since the reaction did not occur when tyrosine residues were blocked by acetylation. Intact red blood cells exposed to the same flow rate of H2O2 presented a spectral profile which could be explained as a transition from X-FeIIO2 to X-FeIII. The intermediate production of X-FeIV-OH was detected by adding Na2S (2 mM), which revealed a spectral profile identical with that obtained with purified X-FeIV-OH. Measurements of concentrations and relative rate constants for the reaction of various intracellular reductants (glutathione, NAD(P)H, uric acid, ascorbic acid) with X-FeIV-OH revealed that comproportionation of X-FeIV-OH with X-FeIIO2 is the favored reaction. Our results provide (to our knowledge) the first definitive evidence for X-FeIV-OH in intact red blood cells. The rapid comproportionation reaction between X-FeIV-OH and X-FeIIO2 (to produce X-FeIII) explains why X-FeIV-OH has been elusive to date. Moreover, the comproportionation reaction represents an important and novel antioxidant function for red blood cell X-FeIIO2, in which the cytotoxic X-FeIV-OH is quenched and the resultant X-FeIII is re-reduced by methemoglobin reductase.",
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N2 - Ferrylhemoglobin (X-FeIV-OH, where X denotes an amino acid residue in the globin moiety) has long been suspected as a cytotoxic agent produced by the interaction of oxyhemoglobin (X-FeIIO2) or methemoglobin (X-FeIII) with H2O2 in red blood cells. To date, however, technical difficulties have prevented the identification and quantification of X-FeIV-OH. Oxyhemoglobin exposed to a continuous flux of H2O2 (generated at a rate of 120 μM/min during the glucose oxidase-catalyzed oxidation of glucose) was oxidized to (a) X-FeIV-OH when [X-FeIIO2] < 75 μM and (b) X-FeIII when [X-FeIIO2] > 75 μM (the production of X-FeIII proceeded with intermediate formation of X-FeIV-OH). The reduction of the X-FeIV-OH to X-FeIII could be explained by either of two alternative mechanisms: a O- 2-mediated X-FeIV-OH → X-FeIII transition or a comproportionation of X-FeIV-OH and X-FeIIO2 to yield X-FeIII (a process mediated by a tyrosine moiety in the hemoprotein). The low rate of X-FeIIO2 autoxidation plus the negligible decrease in the rate of X-FeIII formation in the presence of either native or heat-denatured superoxide dismutase or apoenzyme (1 μM) suggested that O- 2 does not contribute to the reduction of X-FeIV-OH. Moreover, the dependence of X-FeIII formation on X-FeIIO2 concentration, together with the results of O2 uptake and H2O2 consumption measurements, provide experimental evidence to support the comproportionation reaction. Comproportionation is apparently catalyzed by intermolecular electron transfer between tyrosine residues, since the reaction did not occur when tyrosine residues were blocked by acetylation. Intact red blood cells exposed to the same flow rate of H2O2 presented a spectral profile which could be explained as a transition from X-FeIIO2 to X-FeIII. The intermediate production of X-FeIV-OH was detected by adding Na2S (2 mM), which revealed a spectral profile identical with that obtained with purified X-FeIV-OH. Measurements of concentrations and relative rate constants for the reaction of various intracellular reductants (glutathione, NAD(P)H, uric acid, ascorbic acid) with X-FeIV-OH revealed that comproportionation of X-FeIV-OH with X-FeIIO2 is the favored reaction. Our results provide (to our knowledge) the first definitive evidence for X-FeIV-OH in intact red blood cells. The rapid comproportionation reaction between X-FeIV-OH and X-FeIIO2 (to produce X-FeIII) explains why X-FeIV-OH has been elusive to date. Moreover, the comproportionation reaction represents an important and novel antioxidant function for red blood cell X-FeIIO2, in which the cytotoxic X-FeIV-OH is quenched and the resultant X-FeIII is re-reduced by methemoglobin reductase.

AB - Ferrylhemoglobin (X-FeIV-OH, where X denotes an amino acid residue in the globin moiety) has long been suspected as a cytotoxic agent produced by the interaction of oxyhemoglobin (X-FeIIO2) or methemoglobin (X-FeIII) with H2O2 in red blood cells. To date, however, technical difficulties have prevented the identification and quantification of X-FeIV-OH. Oxyhemoglobin exposed to a continuous flux of H2O2 (generated at a rate of 120 μM/min during the glucose oxidase-catalyzed oxidation of glucose) was oxidized to (a) X-FeIV-OH when [X-FeIIO2] < 75 μM and (b) X-FeIII when [X-FeIIO2] > 75 μM (the production of X-FeIII proceeded with intermediate formation of X-FeIV-OH). The reduction of the X-FeIV-OH to X-FeIII could be explained by either of two alternative mechanisms: a O- 2-mediated X-FeIV-OH → X-FeIII transition or a comproportionation of X-FeIV-OH and X-FeIIO2 to yield X-FeIII (a process mediated by a tyrosine moiety in the hemoprotein). The low rate of X-FeIIO2 autoxidation plus the negligible decrease in the rate of X-FeIII formation in the presence of either native or heat-denatured superoxide dismutase or apoenzyme (1 μM) suggested that O- 2 does not contribute to the reduction of X-FeIV-OH. Moreover, the dependence of X-FeIII formation on X-FeIIO2 concentration, together with the results of O2 uptake and H2O2 consumption measurements, provide experimental evidence to support the comproportionation reaction. Comproportionation is apparently catalyzed by intermolecular electron transfer between tyrosine residues, since the reaction did not occur when tyrosine residues were blocked by acetylation. Intact red blood cells exposed to the same flow rate of H2O2 presented a spectral profile which could be explained as a transition from X-FeIIO2 to X-FeIII. The intermediate production of X-FeIV-OH was detected by adding Na2S (2 mM), which revealed a spectral profile identical with that obtained with purified X-FeIV-OH. Measurements of concentrations and relative rate constants for the reaction of various intracellular reductants (glutathione, NAD(P)H, uric acid, ascorbic acid) with X-FeIV-OH revealed that comproportionation of X-FeIV-OH with X-FeIIO2 is the favored reaction. Our results provide (to our knowledge) the first definitive evidence for X-FeIV-OH in intact red blood cells. The rapid comproportionation reaction between X-FeIV-OH and X-FeIIO2 (to produce X-FeIII) explains why X-FeIV-OH has been elusive to date. Moreover, the comproportionation reaction represents an important and novel antioxidant function for red blood cell X-FeIIO2, in which the cytotoxic X-FeIV-OH is quenched and the resultant X-FeIII is re-reduced by methemoglobin reductase.

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