Studies on reconstituted myoglobins and hemoglobins. II. role of the heme side chains in the oxygenation of hemoglobin

To clarify the functional role of the 2- and 4-side chains of heme in hemoglobin we prepared several hemins carrying nonnatural side chains at positions 2 and 4, reconstituted human adult hemoglobins with them, and investigated their optical and oxygen binding properties. The absorption maxima for all these reconstituted hemoglobins, no matter whether they are oxy-, deoxy-, or carbon monoxy-form, shifted toward shorter wavelengths than those for protoheme-hemoglobin. As in the case of myoglobin (Kawabe, K., et al. (1982) J. Biochem. 92, 1703-1712), the absorption spectrum is more significantly affected by resonance effects than by the inductive effects of the peripheral heme substituents. Contrary to the case of myo-globins, the spectral difference between pemptoheme-hemoglobin and isopemptoheme-hemoglobin and that between 2-isopropyl-4-vinyl-deuteroheme-hemoglobin and 2-vinyl-4-isopropyldeuteroheme-hemoglobin were very small. All the reconstituted hemoglobins used in this study showed higher oxygen affinity and reduced coopera-tivity in oxygen binding than native hemoglobin. We have shown that the 2- and 4-side chains are functionally nonequivalent and that modification of the 4-side chain exerts greater influence on oxygen affinity than modifying the 2-side chain. The magnitude of the Bohr effect and response to 2, 3-diphosphoglycerate and inositol hexaphosphate were reduced in the reconstituted hemoglobins. We have proposed a stereochemical mechanism based on constraint of heme movement before ligation against the tight distal side of the heme pocket.