MECHANISM OF THE OXIDATION REACTION OF DEOXYHEMOGLOBIN AS STUDIED BY ISOLATION OF THE INTERMEDIATES SUGGESTS TERTIARY STRUCTURE DEPENDENT COOPERATIVITY

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 cry ogenic techniques. Data analysis was carried out according to a simple four rate constant model, ignoring the functional heterogeneity of th e subunits, to simulate the time courses of the oxidation reaction, as studied by the stopped-flow technique [Antonini et al., (1965) Bioche mistry 4, 345], which show anticooperativity at neutral pH and coopera tivity at alkaline pH. Data analysis according to a 12 rate constant m odel indicated that the rate of oxidation of the beta subunit in the f irst 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 reac tions involving the alpha subunit were noncooperative except for the l ast oxidation step at acid and neutral pH, but were cooperative at alk aline pH. The reactions involving the beta subunit were partly noncoop erative and partly anticooperative. These complex mechanistic patterns suggest that a simple two-state model requiring the concerted transit ion of the tertiary structures of the subunits from the T to the R con formation is not adequate to interpret the oxidation reaction and that tertiary structures contribute, positively and negatively, to coopera tivity. A structural hypothesis is suggested to explain the difference in the reactivities of the alpha and beta subunits.