Unusual ligand discrimination by a myoglobin reconstituted with a hydrophobic domain-linked heme.

New, reconstituted horse heart myoglobins possessing a hydrophobic domain at the terminal of the two heme propionate side chains were constructed. The O2 and CO bindings for the reconstituted deoxymyoglobins were examined in detail by laser flash photolysis and stopped-flow rapid mixing techniques. The artificially created domain worked as a barrier against exogenous ligand penetration into the heme pocket, whereas the bound O2 was stabilized in the reconstituted myoglobin as well as in the native one. In contrast, the CO dissociation rate for the reconstituted myoglobin increased by 20-fold compared to the native protein, suggesting that the incorporation of the hydrophobic domain onto the heme pocket perturbs the distal-site structure of the reconstituted myoglobin. As a result, the substantial ligand selectivity for the reconstituted myoglobin significantly increases in favor of O2 over CO with the M' value (= KCO/KO2) of 0.88, whereas, to the best of our knowledge, there is no myoglobin mutant in which the O2 affinity exceeds the CO one. The present work concludes that the O2 selectivity of myoglobin over CO is markedly improved by chemically modifying the heme propionates without any mutation of the amino acid residues in the distal site.