Winslow S. Caughey

Mechanism for the autoxidation of hemoglobin by phenols, nitrite and "oxidant" drugs. Peroxide formation by one electron donation to bound dioxygen.

Abstract The reaction of HbO 2 with phenols to produce metHb shows inverse rate dependence upon [H + ], direct dependence upon [HbO 2 ] and [phenol], and a rate that correlates with the electron donor characteristics of the reagents. Thus, the availability of an electron from an external agent permits facile reduction of O 2 to O 2 = and the reaction of HbO 2 with phenols gives rise to metHb and peroxide as reaction products. In contrast, with nucleophiles such as azide O 2 is displaced as superoxide. Since reduction of bound O 2 is seen to occur only by reductive displacement or by reaction with a single electron donor, Hb apparently owes its normal resistance to autoxidation to the isolation of the binding site from electron donors and nucleophiles and not to an unique kind of iron-O 2 bonding. Such reasoning explains the effects of structural abnormality that render M-type Hbs susceptible to oxidation. Also the oxidation of HbO 2 upon exposure to “oxidant drugs” is explicable in terms of the drugs acting as one electron reducing agents towards bound dioxygen.

Elucidation of the mode of binding of oxygen to iron in oxyhemoglobin by infrared spectroscopy

Summary The infrared difference spectrum of packed human erythrocytes treated with 16 O 2 vs 18 O 2 or with 16 O 2 vs CO has a unique band at 1107 cm −1 assigned to 16 O- 16 O stretch for bound 16 O 2 . The frequency and intensity of this band prove non-linear end-on binding of O 2 to Fe(II) in oxyhemoglobin. An O-O bond order of ca. 1.5 is indicated. This is analagous to the change in bond order when CO, NO, and N 2 are similarly bound to iron. In consequence it seems unnecessary to use a bond description for O 2 bound to iron which is fundamentally different from that used for CO, NO, and N 2 . The preferred bonding description is . The strong covalent bonding between Fe and O 2 that results upon π-donation from Fe(II) to O 2 represents a quite sufficient reason for dioxygen to dissociate from oxyhemoglobin as O 2 rather than O − 2 and relegates the presence or absence of a nonpolar or hydrophobic environment to a minor role.

The mechanisms of hemoglobin autoxidation evidence for proton-assisted nucleophilic displacement of superoxide by anions

Human oxyhemoglobin (HbO2) in the presence of excess nucleophile (e.g., N3−, SCN−, F−, Cl−) is shown by visible and Soret spectra to form cleanly the oxidized metHb with the nucleophile as ligand. The rates, sensitive to pH and to both the concentration and the nucleophilicity of anionic nucleophile (N−), follow the rate law: rate = k[HbO2][N−][H+]. This autoxidation process thus appears to involve the nucleophilic displacement of superoxide from a protonated intermediate and can reasonably account for normal metHb formation in the erythrocyte where chloride can serve as the nucleophile. MetHb formation due to electron transfer agents (e.g. nitrite) which are normally not present can follow a different course such as direct electron transfer to bound dioxygen to form iron (III) peroxide. Abnormal amino acids or denaturation can provide increased access of nucleophile or electron transfer reactant and thus promote autoxidation.

Infrared evidence for the mode of binding of oxygen to iron of myoglobin from heart muscle

Infrared spectra for oxymyoglobin isolated from bovine heart muscle reveal non-linear end-on binding of O2 to Fe(II) similar, but not identical, to that found for oxyhemoglobins. Difference spectra for 16O2 Mb vs CO Mb and 16O2 Mb vs 18O2 Mb have a band at 1103 cm−1 assigned to bound 16O2. Human oxyhemoglobin A and other hemoglobins exhibited an analogous band at 1107 cm−1. A bonding description with strong covalent bonding between Fe(II) and O2 (i.e., ) thus applies to oxymyoglobin as well as to oxyhemoglobins. CO Mb and CO HbA give νCO bands at 1944 and 1951 cm−1 respectively with about 10-fold greater intensity than the O-O bands.

Bovine heart cytochrome c oxidase preparations contain high affinity binding sites for magnesium as well as for zinc, copper, and heme iron

Metal contents of six bovine heart cytochrome c oxidase preparations were determined by inductively coupled plasma atomic emission spectrometry (ICP-AES). Magnesium is present in consistent amounts reflected by Fe/Mg and Mg/Zn ratios of 2.06 +/- 0.18 and 1.10 +/- 0.14, respectively. More copper than iron is always present; the average Cu/Fe atom ratio is 1.27 +/- 0.10. Calcium is found in significant but variable amounts. Mg, Zn, Fe, and Cu are each bound with high affinity as shown by a resistance to removal upon dialysis against various media. The Cu in excess of Fe, the Mg, and the Zn may each have catalytic and/or structural roles in the oxidase. The observed metal stoichiometry suggests that a dimeric catalytic unit with 5 Cu, 4 Fe, 2 Zn, and 2 Mg may be present in the inner mitochondrial membrane to carry out O2 reduction and H+ pumping.

REACTIONS OF OXYGEN WITH HEMOGLOBIN, CYTOCHROME C OXIDASE AND OTHER HEMEPROTEINS*

The key role of oxgyen in bioenergetics makes the processes whereby 0 2 is transported, stored, and utilized (reduced) of great interest. The hemeproteins hemoglobin, myoglobin, and cytochrome c oxidase participate in such processes. The structural features of hemoglobins and myoglobins are among the best understood of all proteins whereas the oxidase structure is more complex and far less clear. The oxygen reactions per se of these hemeproteins have remained the subject of much discussion and n o little controversy.’ Recent findings which tend to clarify these reactions are considered in this paper. X-ray crystallographic studies reveal the general features of the O2 binding site in hemoglobins and myoglobins, which have similar but not identical sites.’ “Normal” hemoglobins and myoglobins have protoheme as the iron porphyrin, histidine as a proximal (i.e., trans) ligand, and a passageway (pocket) through amino acid residues from the O2 binding site at the iron of the heme to the outer surface of the protein. A (distal) histidine is frequently, but not always, found positioned such that, upon binding t o iron, 0, fits between iron and the histidine that is the nearest neighboring amino acid residue. FIGURE 1 illustrates these general features based on x-ray data3 with the area where bound O2 “must be” indicated by dashed lines. Precise location of O2 cannot be shown

Potential roles of myoglobin autoxidation in myocardial ischemia-reperfusion injury.

The source(s) of reactive partially reduced oxygen species associated with myocardial ischemia/reperfusion injury remain unclear and controversial. Myoglobin has not been viewed as a participant but is present in relatively high concentrations in heart muscle and, even under normal conditions, undergoes reactions that generate met (Fe3+) species and also superoxide, hydrogen peroxide, and other oxidants, albeit slowly. The degree to which the decrease in pH and the freeing of copper ions, as well as the variations in pO2 associated with ischemia and reperfusion increase the rates of such myoglobin reactions has been investigated. Solutions of extensively purified myoglobin from bovine heart in 50 mM sodium phosphate buffer were examined at 37 degrees C. Sufficiently marked rate increases were observed to indicate that reactions of myoglobin can indeed contribute substantially to the oxidant stress associated with ischemia/reperfusion injury in myocardial tissues. These findings provide additional targets for therapeutic interventions.

Autoxidation reactions of hemoglobin A free from other red cell components: A minimal mechanism

Rates of autoxidation reactions are determined for normal human hemoglobin A preparations which are extensively purified to remove all other redox active red cell components. The effects of superoxide dismutase, catalase, and hydroxyl radical scavengers on the reaction provide evidence for superoxide formation as the rate determing step followed by fast reactions that involve peroxide and hydroxyl radical. These results support a minimum overall mechanism for heme iron(II) oxidation and dioxygen reduction to water. Side reactions also occur that result in the modification and precipitation of the protein moiety; catalase and hydroxyl radical scavengers reduce the extent of the side reactions. These studies provide insight into the basis of oxidant stress in the red cell.

Zinc is a constituent of bovine heart cytochrome c oxidase preparations

Cytochrome c oxidase preparations from bovine heart muscle contain 1 zinc per 2 irons. Metal contents of nine preparations determined by inductively coupled plasma atomic emission spectrometry (ICP-AES) show that Cu, Fe and Zn are the only metals present in significant amounts with average Cu/Fe, Fe/Zn, and Cu/Zn atom ratios of 1.3, 2.1 and 2.8, respectively. Removal of adventitious copper results in a Cu:Fe:Zn stoichiometry of 2:2:1. The zinc is tightly bound. Dialysis against a solution of 1,10-phenanthroline at pH 7.4 or an acidic buffer (pH 4.4) does not remove Zn. Dialysis against 0.8 M KCN at pH 10 causes partial loss of both Cu and Zn. This is the first evidence for the presence of Zn in a cytochrome c oxidase.

Infrared evidence for similar metal-dioxygen bonding in iron and cobalt oxyhemoglobins

Abstract The nature of the binding of O 2 to cobalt and to iron in reconstituted hemoglobins was compared by infrared spectroscopy. The proteins were obtained from globin of human hemoglobin A and iron(II) or cobalt(II) deuteroporphyrin IX. Infrared bands for bound O 2 appeared at 1106 and 1105 cm −1 for the iron and cobalt species respectively. These data thus provide direct evidence of strikingly similar bent end-on metal-dioxygen bonding ( ) for the two metals.