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. : 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. : 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.