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

Abstract

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 myoglobins, 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 cooperativity 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.