The kinetic mechanism of heme binding to human apohemoglobin.

Abstract

The formation of hemoglobin from free CO-heme and apoprotein is, at minimum, a two-step process. The first reaction involves very rapid equilibration of the heme groups with hydrophobic regions of the globin molecule. The spectrum of the resultant heme-globin complex is analogous to that of CO-heme incorporated into phospholipid membranes. The Soret absorbance maximum of this species is at about 413 nm, which suggests strongly that the iron-histidine bond has not been formed. The equilibrium dissociation constant for the formation of the heme-globin complex is 6.2 +/- 2 microM at pH 7.2 and 10 degrees C. This constant increases with increasing pH, with the addition of inositol hexaphosphate, with modification of the protoporphyrin vinyl groups, and with increasing glycerol concentration. The second step in hemoglobin formation is unimolecular and appears to involve a large conformational transition which results in iron-histidine bond formation and the appearance of a native CO-hemoglobin spectrum. The rate of this process is 500 +/- 150 s-1 at pH 7.2 and 10 degrees C and is little affected by changes in pH, the addition of organic phosphates, or heme modification. Increasing the solvent viscosity to about 10 c.p. by the addition of 50% glycerol causes a 10-fold decrease in the rate of this conformational change. This suggests that large protein movements, presumably folding around the heme, are involved in the final stages of hemoglobin formation. Iron-histidine bond formation does not appear to be a rate-limiting step.