Ligand binding properties of myoglobin reconstituted with iron porphycene: Unusual O₂ binding selectivity against CO binding

Sperm whale myoglobin, an oxygen storage hemoprotein, was successfully reconstituted with the iron porphycene having two propionates, 2,7-diethyl-3,6,12,17-tetramethyl-13,16-bis(carboxyethyl)-porphycenatoiron. The physicochemical properties and ligand bindings of the reconstituted myoglobin were investigated. The ferric reconstituted myoglobin shows the remarkable stability against acid denaturation and only a low-spin characteristic in its EPR spectrum. The Fe(III)/Fe(II) redox potential (-190 mV vs NHE) determined by the spectroelectrochemical measurements was much lower than that of the wild-type. These results can be attributed to the strong coordination of His93 to the porphycene iron, which is induced by the nature of the porphycene ring symmetry. The O₂ affinity of the ferrous reconstituted myoglobin is 2600-fold higher than that of the wild-type, mainly due to the decrease in the O₂ dissociation rate, whereas the CO affinity is not so significantly enhanced. As a result, the O₂ affinity of the reconstituted myoglobin exceeds its CO affinity (M = K_CO/K_O2 < 1). The ligand binding studies on H64A mutants support the fact that the slow O ₂ dissociation of the reconstituted myoglobin is primarily caused by the stabilization of the Fe-O₂ σ-bonding. The IR spectra for the carbon monoxide (CO) complex of the reconstituted myoglobin suggest several structural and/or electrostatic conformations of the Fe-C-O bond, but this is not directly correlated with the CO dissociation rate. The high O₂ affinity and the unique characteristics of the myoglobin with the iron porphycene indicate that reconstitution with a synthesized heme is a useful method not only to understand the physiological function of myoglobin but also to create a tailor-made function on the protein.