Studies on reconstituted myoglobins and hemoglobins. I. Role of the heme side chains in the oxygenation of myoglobin.

Abstract

To clarify the functional role of the 2- and 4-side chains of heme in myoglobin oxygenation, we synthesized several new hemins carrying nonnatural side chains at positions 2 and 4, reconstituted myoglobins with them, and investigated their optical, ionization, and oxygen-binding properties. The absorption maxima for most of the reconstituted myoglobins except those reconstituted with hemins having carbonyl groups, no matter whether they are oxy-, deoxy-, or carbon monoxy-form, shifted by at most 20 nm toward shorter wavelengths than protoheme-myoglobin. The absorption spectrum is more affected by resonance effects than by inductive effects of the peripheral side chains of heme. Differences in optical and oxygenation properties between isomeric myoglobins carrying hemes with different side chains at positions 2 and 4 indicate that the 2- and 4-side chains of heme are functionally nonequivalent, as previously shown by Sono and Asakura [(1975) J. Biol. Chem. 250, 5227-5232] for monoformyl-monovinylheme myoglobins. Modification of the 4-side chain exerts greater influence on the oxygen affinity than that of the 2-side chain. The extrapolation method proposed by Sono and Asakura to correct for the protein factor seems to be applicable only as a special case and does not apply to the isomeric myoglobins studied by us. There was not correlation between pKa of the met-form and the oxygen pressure at half saturation, implying that the electronic effect of the side chains is not decisive for the oxygen affinity of myoglobin. The bulkiness of the 2- and 4-side chains, on the other hand, appear to have a certain relation to the affinity but is not a dominant factor. In addition to these factors, specific stereochemical considerations are required to explain all the oxygenation data for the various reconstituted myoglobins. We have proposed a stereochemical mechanism based on the movement of the heme group upon ligation of native myoglobin.