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

doi:10.1016/S0006-291X(73)80063-4

Biochemical and Biophysical Research Communications

Volume 55, Issue 1, 1 November 1973, Pages 91-95

https://doi.org/10.1016/S0006-291X(73)80063-4 Get rights and content

Summary

The infrared difference spectrum of packed human erythrocytes treated with ¹⁶O₂ vs ¹⁸O₂ or with ¹⁶O₂ vs CO has a unique band at 1107 cm⁻¹ assigned to ¹⁶O-¹⁶O stretch for bound ¹⁶O₂. The frequency and intensity of this band prove non-linear end-on binding of O₂ 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 N₂ are similarly bound to iron. In consequence it seems unnecessary to use a bond description for O₂ bound to iron which is fundamentally different from that used for CO, NO, and N₂. The preferred bonding description is

. The strong covalent bonding between Fe and O₂ that results upon π-donation from Fe(II) to O₂ represents a quite sufficient reason for dioxygen to dissociate from oxyhemoglobin as O₂ rather than O⁻₂ and relegates the presence or absence of a nonpolar or hydrophobic environment to a minor role.

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Citation Excerpt :
The precise electronic structure of the FeO2 bond has been debated since the 1936 papers on this subject were first published by Pauling and Coryell [29,30,34,35,128–134]. Infrared spectroscopy of the OO bond clearly supports formal reduction of bound oxygen to superoxide; thus the νOO frequency in Hb(O2) (1107 cm− 1) [135], Mb(O2) (1103 cm− 1) [136] and synthetic (porphinato)Fe(II)(O2) complexes (1150 cm− 1) [137] are clearly in a range expected for O2− (1150–1100 cm− 1) [29,137], and not for molecular O2 (1555 cm− 1) or peroxide (O22 −; 842 cm− 1) [29]. This is consistent with the formal superoxo model, Fe(III)+(O2−), proposed by H. G. Weiss [128,129].
Haemoglobin (Hb) is widely known as the iron-containing protein in blood that is essential for O₂ transport in mammals. Less widely recognised is that erythrocyte Hb belongs to a large family of Hb proteins with members distributed across all three domains of life—bacteria, archaea and eukaryotes. This review, aimed chiefly at researchers new to the field, attempts a broad overview of the diversity, and common features, in Hb structure and function. Topics include structural and functional classification of Hbs; principles of O₂ binding affinity and selectivity between O₂/NO/CO and other small ligands; hexacoordinate (containing bis-imidazole coordinated haem) Hbs; bacterial truncated Hbs; flavohaemoglobins; enzymatic reactions of Hbs with bioactive gases, particularly NO, and protection from nitrosative stress; and, sensor Hbs. A final section sketches the evolution of work on the structural basis for allosteric O₂ binding by mammalian RBC Hb, including the development of newer kinetic models. Where possible, reference to historical works is included, in order to provide context for current advances in Hb research.
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We have used x-ray crystallography to determine the structures of sperm whale myoglobin (Mb) in four different ligation states (unligated, ferric aquomet, oxygenated, and carbonmonoxygenated) to a resolution of better than 1.2 Å. Data collection and analysis were performed in as much the same way as possible to reduce model bias in differences between structures. The structural differences among the ligation states are much smaller than previously estimated, with differences of <0.25 Å root-mean-square deviation among all atoms. One structural parameter previously thought to vary among the ligation states, the proximal histidine (His-93) azimuthal angle, is nearly identical in all the ferrous complexes, although the tilt of the proximal histidine is different in the unligated form. There are significant differences, however, in the heme geometry, in the position of the heme in the pocket, and in the distal histidine (His-64) conformations. In the CO complex the majority conformation of ligand is at an angle of 18 ± 3° with respect to the heme plane, with a geometry similar to that seen in encumbered model compounds; this angle is significantly smaller than reported previously by crystallographic studies on monoclinic Mb crystals, but still significantly larger than observed by photoselection. The distal histidine in unligated Mb and in the dioxygenated complex is best described as having two conformations. Two similar conformations are observed in MbCO, in addition to another conformation that has been seen previously in low-pH structures where His-64 is doubly protonated. We suggest that these conformations of the distal histidine correspond to the different conformational substates of MbCO and MbO₂ seen in vibrational spectra. Full-matrix refinement provides uncertainty estimates of important structural parameters. Anisotropic refinement yields information about correlated disorder of atoms; we find that the proximal (F) helix and heme move approximately as rigid bodies, but that the distal (E) helix does not.
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This chapter presents the experimental approaches and interpretations of data for ligand spectra, microspectroscopy, thiol spectra, and amide I spectra. Methods using infrared (IR) spectroscopy provide important, often unique, means for the study of the reactions and structures of hemoglobins (Hb). The amide I bands, primarily because of the carbonyl groups in the peptide bonds that constitute the linkages between the amino acid residues of the Hb molecule, can be utilized for the qualitative and quantitative determination of α-helix, random, β-sheet, and turn secondary structures. Thus, the accurate measurement of an IR band at a given wavelength depends on the absorption of the medium as well as on protein concentration, intrinsic band intensity, and the distance through the sample traversed by the IR radiation. Improvements in IR instrumentation and in data analysis have greatly enhanced the sensitivity of the IR method applied to aqueous solutions.
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This chapter discusses the way metal ion environments in metalloproteins and metalloenzymes can be studied by using infrared (IR) spectroscopy. It explains experimental techniques and methods of analysis and interpretation by using selected examples. Fourier transform (FT) IR spectroscopy is mentioned only briefly. Although both IR and Raman spectroscopies provide information about transitions between two vibrational states, there are several advantages and disadvantages unique to each. IR and Raman spectroscopy are complementary, and both spectra should be obtained whenever possible. In fact, vibrational spectra of most of the biological compounds discussed in this chapter have been studied by IR as well as Raman spectroscopy. This chapter discusses IR spectra of axial ligands (CO, NO, O₂, CN^-, and N₃^-), which are bound to metalloproteins and metalloenzymes. IR spectra of metalloproteins and metalloenzymes themselves constitute another area that can be studied with FTIR spectroscopy, but their full potential as a probe of the structure of metal-containing biological systems has yet to be established. In this respect, there is no doubt that Raman spectroscopic techniques have proved to be much more informative. Nevertheless, several promising FTIR studies, particularly on the secondary structures of metalloproteins, have been carried out.

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

Summary

Ann. Rev. Biochem.

Hemoglobin and Myoglobin in Their Reactions with Ligands

Chem. Rev.

Inorg. Chem.

J. Amer. Chem. Soc.

Nature

J. Amer. Chem. Soc.