John C. Maxwell

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.

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

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.

Properties of hemoglobin a and hemoglobin Zurich (β63 histidine→arginine): Quantitative evaluation of functional abnormalities in hemoglobins

Abstract The abnormal hemoglobin Zurich (β63 his→arg) exhibits abnormal properties. Thus, νCO occurs at 1951 cm−1 for HbACO while HbZCO shows bands at 1950 cm−1 and 1958 cm−1 for CO bound in α and β chains respectively (the βCOs are displaced less readily by O2). Acid catalyzed reductive displacement of superoxide by azide is slower on the β chain of HbZO2 than on the α chain under conditions where with HbAO2 both chains appear equally reactive. The one electron donor hydroquinone produces metHb and peroxide more rapidly from HbZO2 than from HbAO2. These property differences can be related to the β63 residue. Such studies provide generally useful probes of the structural basis for hemoglobin diseases.

[18] Infrared spectroscopy of ligands, gases, and other groups in aqueous solutions and tissue

Publisher Summary This chapter discusses the methods of infrared spectroscopy employed to study small ligands bound to metals in heme, copper, and other metalloproteins. Infrared spectroscopy is the form of vibrational spectroscopy. In the infrared experiment, light of the energy associated with a given vibration is absorbed. Infrared spectra of proteins can be used to explore relationships between the conformation and the frequencies of infrared bands, especially the amide bands I and II found in the region from 1680–1430 cm –l . In addition, difference infrared spectroscopy of proteins can be used to a limited extent to follow ionization equilibria of carboxylic acids 4,5 and hydrogen bonding of SH groups. The presence of water in biological materials restricts the measurement of infrared absorption. Water absorbs strongly in the infrared region—more strongly in some regions than in others. At least some radiation must pass through the material under observation at the wavelength of interest if an accurate absorption measurement is to be made. Infrared measurements on solutes are therefore more readily made in regions of low absorption by water than in regions of strong absorption. Recent advances in the design of infrared spectrometers of both dispersive and interferometer types, and in computer interfacing with the spectrometer have markedly increased sensitivity, resolution, and dataprocessing capabilities.

The utility of infrared spectroscopy as a probe of intact tissue: Determination of carbon monoxide and hemeproteins in blood and heart muscle

Abstract Infrared methods permit detection of CO within tissue under nearly physiological conditions. The CO stretch bands exhibit frequencies, band widths and intensities characteristic of the particular binding site with areas related to concentrations. For small volumes (

LIGAND BINDING TO HEMOGLOBINS: EFFECTS OF GLOBIN STRUCTURE

Present evidence shows that the α and β subunits of HbA bind O 2 , NO and CO as ligands with a stereochemistry bent somewhat from the axis of the heme plane. There is appreciable covalent (π) character to iron ligand bonding, with a net increase of electron density on the ligand in the order O 2 > NO > CO. In HbO 2 , based only upon the O–O stretch band frequency, iron(III) superoxide (O 2 − ) bonding could, but need not, be present. However, description of the bonding as iron(II) oxygenyl is more consistent with reactivities, as well as more reasonable theoretically. The CO stretch band is especially sensitive to changes in ligand environment; both frequencies (v CO ) and widths (δv ½ ) vary widely among hemeproteins. Nevertheless, v CO values for normal erythrocytes from mammals, birds, reptiles, and fish only vary −1 from the 1951 cm −1 of HbA CO. The consistency of v CO near 1951 cm −1 and of δv ½ near 8 cm −1 , for normal tetramers for all species, reflects the highly conserved nature of the O 2 binding site and suggests critical roles for the heme and globin structures involved. In HbA, the only amino acid residues in contact with bound ligand are the E7 His and E11 Val. The positions of these two residues, with respect to ligand, differ slightly in the two subunits; v CO is 1950.2 ± 0.2 and 1952.2 ± 0.2 cm −1 for α and β subunits, respectively, with a δv ½ of 7 cm −1 in each case. Hb Zurich has the E7 His of the β subunit replaced by Arg and, as a consequence, exhibits different ligand affinities, infrared and electronic spectra, and rates of oxidation. These differences from HbA may be most simply explained in terms of the relief from steric pressure by the βE7 residue on the ligand and in terms of greater access to the heme site. Hb Sydney, with Ala in place of the normal βE11 Val, experiences weaker, but detectable, effects, due to replacement of the two Val methyl groups adjacent to the bound ligand by less bulky hydrogens. In Hb Osler with β 145 Tyr → Asp, a substitution more remote from the heme, ligand bonding to iron appears unaffected, but a marked reduction of cooperativity and IHP effects on ligand binding and on oxidation reactions are noted. C–O stretch bands also appear near 1966 to 1969 cm −1 . Normally, these bands are of very low intensity with narrow width (δv ½ ca 8 cm −1 ), and represent additional types of bound CO. However, under denaturant “stress” conditions, much or all of the total C–O band intensity may appear as broad band (δv ½ > 15 cm −1 ) near 1968 cm −1 . Studies on these and other abnormal Hbs are thus contributing to an experimental elucidation of the normal and pathological roles of globin structures.