Oxygen and Carbon Monoxide Equilibria and the Kinetics of Oxygen Binding by the Cooperative Dimeric Hemoglobin of Thyonella gemmated†

Oxygen and carbon monoxide equilibrium measurements have been made for the dimeric hemoglobin of Thyonella gemmata. For both ligands, the Hill number is ~ 1.4. The affinity for CO, however, is quite low, the median ligand activity at 20 °C, pH 7, being 1.3 μM. There is no apparent Bohr effect from pH 7 to pH 9. The replacement of 02 by CO gives an apparent value for M of 2.4. More unusual is the finding that this replacement reaction is also cooperative, the apparent Hill number being 1.4, suggesting that, for this hemoglobin, the HbO2 and HbCO conformations differ. HbO2 - HbCO difference spectra in the 290-nm region suggest that replacement of O2 by CO at the heme site is associated with the movement of a tryptophan from a nonpolar to a polar environment. The kinetics of oxygen dissociation from the hemoglobin of T. gemmata are biphasic. The dissociation rate constants for the hemes in the two polypeptide chains differ by a factor of about 5. Oxygen dissociation in the presence of CO is somewhat slower, but measurements at the HbO2-HbCO isosbestic wavelength show that a maximum of nearly 1/2 of the hemes are unliganded during the reaction. This shows that the predominant half-liganded oxyhemoglobin species does not provide a quickly reacting form for CO binding, since the maximum CO association constant can be only ~2 X 10-4 M-1 s_1. Oxygen-pulse experiments provide the clearest kinetic evidence for cooperativity in oxygen binding. The rate constants for oxygen dissociation from singly oxygenated forms must be greater than 5 times those for the fully oxygenated hemoglobin. The oxygen dissociation and oxygen-pulse measurements cannot be accommodated within a heterogeneous Adair model. In terms of an allosteric model, the rate constant for R→ T or for T→R for intermediates such as αO2-β is small (ti/2 is tens of milliseconds), or the R⇄ T equilibrium constant for the half-liganded form is ~ 1. The kinetics of oxygen association following HbCO photolysis are unusual in that the rate of oxygen binding is less than first power in oxygen. A model is proposed to account for these results. In this model, binding of oxygen to the initial pho-toproduct is some 30-40-fold slower than that to a second form, derived from the first by a conformational change, the t1/2 for which is ~1 ms.