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 Title: Myoglobin at pH 3: Dynamics of myoglobin with the iron-proximal histidine bond broken Author(s): Cowen, Benjamin Ring Doctoral Committee Chair(s): Frauenfelder, Hans Department / Program: Biophysics Discipline: Biophysics Degree Granting Institution: University of Illinois at Urbana-Champaign Degree: Ph.D. Genre: Dissertation Subject(s): Biophysics, General Abstract: Brunori and co-workers have shown that in Mb the proximal histidine (F8) protonates, causing the histidine-iron bond to break with a pK of 3.45. We measured the rebinding kinetics of CO to myoglobin (Mb) at pH3 in 75% glycerol/water in the Soret from 10K to 300K and 50 ns to 100 s. Below about 200K, the observed nonexponential kinetics is attributed to rebinding from the pocket, process I. The resulting distribution of enthalpic barriers for process I peaks at about 1 kJ/mol compared to 10 kJ/mol for Mb at pH7; the pre-exponential for Mb at pH3 is $\sim$10$\sp{11}$ compared to $\sim$10$\sp9$ s$\sp{-1}$ for Mb at pH7. The proximal histidine bond in Mb thus contributes significantly to both the enthalpic and entropic barriers at the heme. The enthalpy distribution does not fit the rebinding data for Mb at pH 3 as well as it does for Mb at pH 7 and neither does an entropy distribution. Slightly better fits were obtained by distributing both the entropic and the enthalpic barriers.The nanosecond rebinding data showed indirect evidence for a faster process in Mb at pH 3 and indeed a fast exponential process, I$\sp\*$, exists in protoheme. In collaboration with Prof. Dlott (Dept of Chemistry, Univ. of Illinois) we measured the rebinding kinetics on a picosecond timescale. The kinetics show only a power law behavior with no evidence for a fast exponential process. We also measured the rebinding kinetics of CO to Mb at pH 4 as a control experiment and showed that dramatic difference between Mb at pH 7 and pH 3 is due to the breaking of the proximal bond between pH 3 and pH 4. In collaboration with Dr. Friedman (AT&T Bell Labs) we measured the time-resolved Raman of MbCO at pH 3 after photolysis with 10 ns resolution at 80 K. The data show an absence of the $\nu\sb{\rm Fe-His}$ band proving that the proximal bond breaks within 10 ns (and probably within 30 ps) after photolysis and that the ligand rebinds to a protein with the proximal bond broken. Issue Date: 1990 Type: Text Language: English URI: http://hdl.handle.net/2142/23117 Rights Information: Copyright 1990 Cowen, Benjamin Ring Date Available in IDEALS: 2011-05-07 Identifier in Online Catalog: AAI9114213 OCLC Identifier: (UMI)AAI9114213
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