Mechanism of the oxidation reaction of deoxyhemoglobin as studied by isolation of the intermediates suggests tertiary structure dependent cooperativity

Abstract

The intermediates in the oxidation of deoxyhemoglobin by ferricyanide in 0.1 M KCl, at 20 degrees C and three pH values, were studied by cryogenic techniques. Data analysis was carried out according to a simple four rate constant model, ignoring the functional heterogeneity of the subunits, to simulate the time courses of the oxidation reaction, as studied by the stopped-flow technique [Antonini et al., (1965) Biochemistry 4, 345], which show anticooperativity at neutral pH and cooperativity at alkaline pH. Data analysis according to a 12 rate constant model indicated that the rate of oxidation of the beta subunit in the first oxidation reaction was 4 times faster than the rate of oxidation of the alpha subunit at pH 6.2 and 12 times faster at pH 8.5. The reactions involving the alpha subunit were noncooperative except for the last oxidation step at acid and neutral pH, but were cooperative at alkaline pH. The reactions involving the beta subunit were partly noncooperative and partly anticooperative. These complex mechanistic patterns suggest that a simple two-state model requiring the concerted transition of the tertiary structures of the subunits from the T to the R conformation is not adequate to interpret the oxidation reaction and that tertiary structures contribute, positively and negatively, to cooperativity. A structural hypothesis is suggested to explain the difference in the reactivities of the alpha and beta subunits.

Oxygen Equilibrium Properties of Nickel(II)-Iron(II) Hybrid Hemoglobins Cross-Linked between 82.beta.1 and 82.beta.2 Lysyl Residues by Bis(3,5-dibromosalicyl)fumarate: Determination of the First Two-Step Microscopic Adair Constants for Human Hemoglobin

“…It has been well-known that the oxidation-reduction equilibrium of Hb is a cooperative process (Brunori et al, 1969), so that a significant part of the cooperative free energy could originate from a "cooperative" oxidation reaction of Hb. In contrast to oxygenation of Hb, however, equilibrium and kinetic measurements of the oxidation reaction of Hb have revealed that the difference in electron affinities of the subunits is very large and is significantly affected by pH, subunit assembly, and molecular forms (T or R) (Brunori et al, 1968(Brunori et al, , 1969Perrella et al, 1993). Furthermore, Ackers and his colleagues have assumed that dissociated dimers bind two ligands noncooperatively with a very high affinity in a similar manner to the dimeric oxygenation, although virtually nothing is known about the functional properties of the dimers in the cyanomet ligation system.…”

Erythrocytes are the major intravascular storage sites of nitrite in human blood

“…The principle of the method is that ferricyanide reacts with hemoglobin to form methemoglobin (Fe-II-hemoglobin ϩ ferricyanide 3 Fe-III-hemoglobin ϩ ferrocyanide). 28 Methemoglobin, unlike hemoglobin, does not react with nitrite to form nitrate nor does it reduce nitrite to NO. 29 Because the reaction of hemoglobin with nitrite represents the major mechanism for nitrite degradation, the addition of ferricyanide stabilizes nitrite in whole blood.…”

Bohr Effect in Hemoglobin Deoxy/Cyanomet Intermediates

“…The three components were then separated by cryofocusing at -25 OC on gel tubes for 15-20 h . The gel slices containing the components were eluted in 20 mM NaOH, 10 mM KCl, and 50% (v/v) pyridine, and the heme was assayed (Perrella et al, 1993). After a sample of the anaerobic mixture was quenched, the remaining mixture was exposed to oxygen to let the mixture reach rapidly a new equilibrium at which the three components should be present in statistical proportions.…”