Kinetics and mechanism of heme-induced refolding of human

Hemoglobin a and , chains are tightly packed, highly (75%) helical stable molecules. Removal of the heme results in unfolded (30% helical) unstable globin chains that can be re- folded to the native conformation by recombination with heme. We have studied the kinetics of heme binding and the ensuing conformational changes by using three stopped-flow techniques: (i) fluorescence quenching, which monitors the spatial orientation and distance between the bound heme and the A12(14)a trypto- phan; (ii) absorption at the Soret band maxima, whose position and intensity depend on the local environment of the heme and the nature of the axial ligands; and (iii) far-UV circular dichroism, which directly gauges the recovery of secondary structure. The fluorescence quenching was biphasic: An initial second-order de- cay, representing 80-85% of the total amplitude, marked the bind- ing of hemin dicyanide to a relatively well-defined site at a rate constant of 3.3 x 107 M'1 sec1, corresponding to a half-time of 10 msec at 2.4 ,uM reactants. The Soret absorption and circular dichroism were also multiphasic, all three probes detecting a first- order process of half-time 25-40 sec, during which the final sec- ondary and tertiary structures of the heme pocket were estab- lished, and the spatial relationship between the heme and the A12 tryptophan was fixed. A slower circular dichroism change, rep- resenting two-thirds of the total backbone refolding, with a half- time of 116 sec, marked the full acquisition of the native subunit conformation. The results show that the residues of the heme pocket achieve or closely approach their final three-dimensional structure well before the entire chain is folded. These measure- ments represent a direct observation of the rate of prosthetic group-induced secondary structure formation and illustrate the advantages of multiple probe analysis in outlining a protein folding pathway.