Kinetics and mechanism of heme-induced refolding of human alpha-globin.
Hemoglobin alpha and beta chains are tightly packed, highly (75%) helical stable molecules. Removal of the heme results in unfolded (30% helical) unstable globin chains that can be refolded 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)alpha tryptophan; (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 decay, representing 80-85% of the total amplitude, marked the binding of hemin dicyanide to a relatively well-defined site at a rate constant of 3.3 x 10(7) M(-1) sec(-1), corresponding to a half-time of 10 msec at 2.4 muM 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 secondary and tertiary structures of the heme pocket were established, and the spatial relationship between the heme and the A12 tryptophan was fixed. A slower circular dichroism change, representing 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 measurements 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.
D002248:Carbon Monoxide; D002942:Circular Dichroism; D003486:Cyanides; D005914:Globins; D006418:Heme; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D010084:Oxidation-Reduction; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry
D003486:Cyanides; D046911:Macromolecular Substances; D006418:Heme; D002248:Carbon Monoxide; D005914:Globins