Chain Inequivalence in Bovine Methemoglobin

Using pulse radiolysis, a single heme in the tetramer of bovine methemoglobin was reduced within a few microseconds to the ferro state, producing a valence intermediate. The kinetics of oxygen binding to the valence intermediate as well as the re‐oxidation of the ferro‐heme to the ferric state were studied as a function of pH. The kinetics of the oxygenation revealed the existence of two species, characterized by high and low affinities for oxygen that are associated with two quaternary structures (R and T, respectively). A sigmoidal curve representing a transition between the two states as a function of pH was derived. Above pH 7.7 only the R state could be observed, while below pH 6.5 the T state was dominant. The reaction between the valence intermediate and ferricyanide at pH 7.75 (R state) consisted of two (about) equal contributions (k₁= 23 × 10⁴M⁻¹ s⁻¹; k₂= 2.1 × 10⁴ M⁻¹ s⁻¹) attributed to the β and α subunits within the tetramer, respectively. At pH 6.3 (T state) a similar phenomenon was observed (k₁= 69 × 10⁴ M⁻¹ s⁻¹; k₂= 3.7 × 10⁴ M⁻¹ s⁻¹), indicating chain inequivalences both in the T and the R states of methemoglobin. In the presence of inositol hexakisphosphate the T → R transition, as monitored by oxygenation of the valence intermediate, was shifted up to a higher pH by about 0.35. Yet similar rate constants exhibiting similar chain inequivalences have been measured.