Kinetics of Carbon Monoxide Binding to the Cooperative Dimeric Hemoglobin from Thyonella gemmata. Analysis of Carbon Monoxide Equilibrium Results†

The CO association reaction for the dimeric hemoglobin of Thyonella gemmata is biphasic with rate constants of 2 x 103 and 1 x 104 M-1 s-1 among the slowest known for a hemoglobin. The kinetic heterogeneity almost certainly derives from differences in the α- and β-chain heme environments since 90% of the hemoglobin migrated as a single electrophoretic component and the more rapid kinetic phase contributed about 48% to the absorbance change. The activation enthalpies for the CO association reaction are 7.5 kcal/mol (rapid heme) and 9.0 kcal/mol (slow heme). The value for horse hemoglobin is 8.3-8.6 kcal/mol corresponding to a CO association constant of 1.7 x 105 M-1 s-1. These findings suggest that the CO binding site in Thyonella hemoglobin is even more restricted than that in horse hemoglobin. Stopped-flow and flash-photolysis results were identical. Although the hemoglobin is cooperative in CO binding, there is no evidence for a quickly reacting (Hb*) form of the protein. Tandem flash-photolysis experiments show that the heme that is rapidly reacting toward CO is also the more rapid in oxygen association and dissociation. Stopped-flow studies on partially CO-saturated hemoglobin show that intermediate liganded forms are present in significant amounts and allow all equilibrium constants to be determined in a heterogeneous Adair model. The Adair model can account satisfactorily for all existing CO equilibrium and kinetic results and predicts that the dissociation of CO from half-liganded forms should be about 10 times faster than that from Hb-(CO)2. A heterogeneous allosteric model using observed rate constants assumed to pertain to the R form gave L = 150 and c = 0.0207 and predicted a 48-fold enhancement in the CO off rate (T form vs. R form). If there exists a quickly reacting CO form (Hb*) with an association constant only 10 times that which is observed, but which reverts to the T form more rapidly than CO binds to Hb* (thus accounting for its nonappearance), L = 15 479, c = 0.00242, the enhancement in the CO dissociation rate is 41, and the equilibrium ratio of R to T for half-liganded hemoglobin is 0.02. A larger value for the hypothetical Hb* association constant leads to an even larger value for the allosteric constant L.