Calculated pH-dependent population and protonation of carbon-monoxy-myoglobin conformers

X-ray structures of carbonmonoxymyoglobin (MbCO) are available for differen t pH values. We used conventional electrostatic continuum methods to calcul ate the titration behavior of MbCO in the pH range from 3 to 7. For our cal culations, we considered five different x-ray structures determined at pH v alues of 4, 5, and 6. We developed a Monte Carlo method to sample protonati on states and conformations at the same time so that we could calculate the population of the considered MbCO structures at different pH values and th e titration behavior of MbCO for an ensemble of conformers. To increase the sampling efficiency, we introduced parallel tempering in our Monte Carte m ethod. The calculated population probabilities show, as expected, that the x-ray structures determined at pH 4 are most populated at low pH, whereas t he x-ray structure determined at pH 6 is most populated at high pH, and the population of the x-ray structures determined at pH 5 possesses a maximum at intermediate pH. The calculated titration behavior is in better agreemen t with experimental results compared to calculations using only a single co nformation. The most striking feature of pH-dependent conformational change s in MbCO-the rotation of His-64 out of the CO binding pocket-is reproduced by our calculations and is correlated with a protonation of His-64, as pro posed earlier.