title: Spectroscopic studies of myoglobin at low pH: heme ligation kinetics.
abstract: 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.
authors.affiliation: Department of Physics, Northeastern University, Boston, Massachusetts 02115.
authors.forename: J T
authors.initials: JT
authors.lastname: Sage
authors.forename: P S
authors.initials: PS
authors.lastname: Li
authors.forename: P M
authors.initials: PM
authors.lastname: Champion
mesh_terms: D000818:Animals; D006418:Heme / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007501:Iron / Q000737:chemistry; D007700:Kinetics; D008786:Metmyoglobin / Q000737:chemistry; D009211:Myoglobin / Q000737:chemistry*; D010782:Photolysis; D013059:Spectrum Analysis, Raman; D014907:Whales
chemical_list: D009211:Myoglobin; D008786:Metmyoglobin; D006418:Heme; D006639:Histidine; D007501:Iron