Identification of conformational substates in oxymyoglobin through the pH-Dependence of the low-temperature photoproduct yield

The effect of pH on the inner barrier to oxygen binding in myoglobin has been examined through a study of the pH-dependence of the low-temperature photoproduct yield for horse oxymyoglobin (MbO(2)). Chance et al. (Chance, M. R.; Courtney, S. H.; Chavez, M. D.; Ondrias, M. R.; Friedman, J. M. Biochemistry 1990, 29, 5537) have shown that the low-temperature photoproduct yield of MbO(2) is 0.50 +/- 0.05 and have suggested the existence of two conformational substates, where the ''photolyzable'' fraction has a barrier similar to MbCO and the ''unphotolyzable'' fraction represents a very low barrier or barrierless substate. Through optical spectroscopy, we show that the 10 K photoproduct yield decreases at low pH for MbO(2) (pH 7: 0.50 +/- 0.05; pH 5: 0.18 +/- 0.05) and cobalt-substituted MbO(2) (pH 7: 0.55 +/- 0.05; pH 4: 0.20 +/- 0.05), indicating distal pocket conformational changes that occur as pH is lowered which further populate a low barrier conformation. A comparison of the pH-dependence of (1) the MbO(2) photoproduct yield and (2) the A state population of carbonmonoxy myoglobin (MbCO) at cryogenic temperatures indicates that the conformational changes that give rise to the A(1) --> A(0) conformational substate transition in MbCO are functionally important in determining the inner barrier to oxygen binding. The pH-dependence of the A(1) --> A(0) state transition in MbCO has been attributed to protonation of the distal histidine, suggesting that the distal histidine also determines the conformational substate population in MbO(2), hence, the overall inner rebinding barrier. Further support of this theory comes from the photoproduct yield of the mutant, Mb(H64Q)O-2, where the hydrogen bond to the distal glutamine does not titrate with pH, which remains constant (0.50 +/- 0.05) from pH 4.8 to 9. The pH-dependence of the MbO(2) substate population may be structural or electronic in nature. Structurally, the pH-dependence of the distal pocket environment may lead to different ligand trajectories upon photolysis, affecting the barrier to rebinding. Electronically, the changes that occur in the distal pocket at low pH may cause a redistribution of electron density throughout the metal-porphyrin pi-electron system, yielding a fast-relaxing photoexcited state where the Fe-O bond is not ruptured. Finally, the pH versus photoproduct yield ''titration curve'' for oxymyoglobin was compared to cobalt-substituted oxymyoglobin (CoMbO(2)). We found a higher apparent pK for MbO(2) than CoMbO(2) indicating that protonation of the distal histidine is easier in ferrous oxymyoglobin. These results show direct communication between the pi-system of the metal and that of the distal histidine residue, and suggest a stronger hydrogen bond to the distal histidine in MbO(2) versus CoMbO(2).