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

Michele Perrella, Richard I. Shrager, Marilena Ripamonti, Giacomo Manfredi, Robert L. Berger, Luigi Rossi-Bernardi

Research output: Contribution to journalArticlepeer-review

Abstract

The intermediates in the oxidation of deoxyhemoglobin by ferricyanide in 0.1 M KCl, at 20 °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 β subunit in the first oxidation reaction was 4 times faster than the rate of oxidation of the a subunit at pH 6.2 and 12 times faster at pH 8.5. The reactions involving the α subunit were noncooperative except for the last oxidation step at acid and neutral pH, but were cooperative at alkaline pH. The reactions involving the β 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 α and β subunits.

Original languageEnglish
Pages (from-to)5233-5238
Number of pages6
JournalBiochemistry
Volume32
Issue number19
Publication statusPublished - 1993

ASJC Scopus subject areas

  • Biochemistry

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