Circular dichroism of hemoglobin and myoglobin.

While heme alone does not exhibit circular dichroism (CD) spectrum, it exhibits a prominent positive CD band in the Soret region when incorporated into apoglobin of myoglobin (Mb) and hemoglobin (Hb). The appearance of this optical activity is widely accepted to arise from the interactions between the heme and aromatic residues of the globin. However, the reversed heme orientation in Hb was found to exhibit a CD spectrum obviously different from that of native Hb, indicating that the interactions of side chains of heme with globin also contribute to the appearance of heme optical activity. We examined this possibility by comparing CD spectra of native Mb and those of Mb reconstituted with unnatural heme lacking the vinyl and/or propionate. Replacement of vinyls at the 2,4-positions with methyls induced a 30% decrease in CD intensity of the positive Soret CD band without changes of spectral shape. In contrast, the replacement of the propionate at the 6,7-positions with carboxylic acid groups resulted in almost complete disappearance of the Soret CD band. These results seem to suggest that interactions of heme side chains, especially 2,4-vinyls and 6,7-propionates, with globin, as well as the electronic coupling of the heme bands with those of intrinsic protein chromophores, contribute to the appearance of the prominent positive Soret CD band of Mb and Hb.