Kinetic and Thermodynamic Parameters of the Iron Spin‐State Transition in Human Aquomethemoglobin

The rate constants for the iron spin‐state transition in human aquomethemoglobin, as well as the corresponding thermodynamic parameters, have be determined at pH 6. klh, the rate constant for the low‐to‐high spin transition, varies between 242.6 s−1, and 480.6 s−1 in the temperature range 13–27°C; khl, the rate constant for the high‐to‐low spin transition, ‐varies between 57.4 s−1 and 69.4 s−2 over the same temperature range. The activation energies are 34.59 kJ mol−1 and 9.78 kJ mol−1 respectively, while the entropy change associated with the high‐to‐low spin transition is −99.7 J mol−1 K−1.The simple scheme postulated for formate binding to aquomethemoglobin [Okonjo, K. O. & Ilgenfritz, G. (1978) Arch. Biochem. Biophys. 189. 499–507] is shown to be inadequate. It is demonstrated that a scheme that includes a fast spin transition of the iron atoms, preceding formate binding, adequately accounts for formate binding to aquomethemoglobin in the T and R quaternary states.Evidence is presented to show that, in a given pure solvent, the spin‐state transition appears to be insensitive to differences in solute composition. By contrast, the spin transition appears to be quite sensitive to differences in solvent composition of ethylene glycol/water mixtures.In the presence of inositol hexakisphosphate. methemoglobin formate exhibits three relaxation times; the two with high amplitudes are affected by changes in inositol hexakisphosphate concentration. This indicates that inositol hexakisphosphate perturbs both the a and ß chains of methemoglobin and may be evidence to support the proposal that inositol hexakisphosphate, at acid pH. causes a quaternary structure transition in aquomethemoglobin.