Spectroscopic studies of myoglobin at low pH: heme ligation kinetics.

On the basis of the characterization of heme structure and ligation in equilibrium, we explore both proximal and distal ligation kinetics of myoglobin below pH 4. Upon photolysis of MbCO, a significant five-coordinate heme population is observed, with an intact iron-histidine bond that persists on the time scale of CO rebinding. Incomplete CO photolysis is attributed to a rapidly exchanging minority population of four-coordinate hemes, which leads to fast (greater than 10(10) s-1) geminate recombination. The possible relevance of such a mechanism at pH 7 is also noted. Using a novel experimental protocol, we observe the resonance Raman spectrum of partially photolyzed MbCO as a function of continuous wave illumination time (tau). Under extended illumination (tau approximately 35 ms at pH 3.4), there is a loss of intensity in the nu 4 region of the Raman spectrum and the iron-histidine mode is bleached from the spectrum of the five-coordinate photoproduct. In the Fe-CO stretching region of the CO-bound fraction, the intensity of the 526-cm-1 mode increases with tau at the expense of the 491-cm-1 mode. These changes are interpreted as being due to replacement of the proximal histidine ligand under continuous illumination. Complete relaxation to the pure four-coordinate deoxy heme structure observed in equilibrium is not observed even as tau----infinity, presumably since CO rebinding leads to acidification of the iron and its complexation with histidine. We propose a kinetic model to account for our results and discuss the implications for previous low-pH kinetics measurements.