Electron Paramagnetic Resonance Studies of Spin-Labeled Hemoglobins and Their Implications to the Nature of Cooperative Oxygen Binding to Hemoglobin

Abstract

The spin label technique has been used to study human hemoglobins A, F, Zürich, and Chesapeake as a function of carbon monoxide saturation. The experimental results suggest that the changes in the electron paramagnetic resonance spectra of hemoglobin labeled with N -(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)iodoacetamide depend on the state of ligation of more than one heme group. For those hemoglobins with full or large cooperative ligand binding (such as A, F, and Zürich), there is a lack of isosbestic points in the spectra as a function of CO saturation. However, for those hemoglobins with little or no cooperative ligand binding (such as Chesapeake and methemoglobins), there is a sharp set of isosbestic points. These findings confirm and extend the early work of McConnell and co-workers. The absence of a set of isosbestic points in those hemoglobins with full cooperative ligand binding is consistent with the sequential model of Koshland, Némethy, and Filmer for cooperative oxygen binding to hemoglobin. The present results, with hemoglobin variants having known amino acid substitutions, also focus on the importance of the interactions among the amino acid residues located at α 1 -β 2 or α 2 -β 1 subunit contacts for the functioning of hemoglobin as an oxygen carrier. In addition, the resonance spectra of the spin label are very sensitive to small structural variations around the heme groups in the β- or γ-chains where the labels are attached. The results of the spin label experiment are discussed in relation to recent findings on the mechanism of oxygenation of hemoglobin from the nuclear magnetic resonance studies of this laboratory and the x-ray crystallographic analysis of Perutz and co-workers.

Dynamic Molecular Structure of DPPC-DLPC-Cholesterol Ternary Lipid System by Spin-Label Electron Spin Resonance

“…In this context the isosbesticity concept was applied in this work for determining boundaries in a phase diagram. The first application of isosbesticity to ESR was in the context of hemoglobin studies (Ogawa and McConnell, 1967), as were further studies (Asakura et al, 1972;Ho et al, 1970;Ohnishi et al, 1968). Once the absorptivities of the component species become dependent on the external variables, isosbestic points do not occur (Marriott and Griffith, 1974).…”

Conformation and cooperativity in hemoglobin

“…, covalently attached spin-labels in the \ß2 interface (Ogawa and McConnell, 1967;Deal et al, 1971;Ho et al, 1970), and carbon-13 resonances of mobile residues (Moon and Richards, 1972). The findings of these studies have been interpreted variously in light of X-ray crystallographic results (Perutz, 1970) as evidence in support of one of two theories of the mechanism of cooperativity-a concerted transition (Monod et al, 1965;Ogawa and Shulman, 1972) or generalized concerted transition (Ogata and McConnell, 1972) model, or a sequential (Koshland et al, 1968;Davis et al, 1971;Ogawa and McConnell, 1967;Deal et al, 1971) model.…”

“…Much of the spectroscopic evidence presented in support of the concerted models (Ogawa and Shulman, 1972) is open to the criticism that the techniques used were not sufficiently sensitive to permit detection of forms with intermediate structure. However, the evidence that such intermediate states must exist (Davis et al, 1971;Ogawa and McConnell, 1967;Ho et al, 1970), which has been obtained mainly from studies of changes at the «102 interface region, has been criticized on grounds that minute structural changes could occur within the subunits of the tetramer which do not affect the energetics of ligand binding (Ogawa and Shulman, 1972). The results of fluorine and phosphorus nuclear magnetic resonance (NMR) studies discussed in this communication (Huestis and Raftery, 1972a-d) three ligands.…”