Cooperative role of Arg45 and His64 in the spectroscopic A3 state of carbonmonoxy myoglobin:: Molecular dynamics simulations, multivariate analysis, and quantum mechanical computations

The structural, dynamic, and electronic origin of the spectroscopically observed carbonmonoxy myoglobin (MbCO) A states has been investigated by using molecular dynamics to sample conformational space, multivariate analysis to aid in structural interpretations, and quantum mechanics to compute ligand stretch frequencies. Ten short (400 ps) and two longer time (1.2 ns) molecular dynamics simulations, starting from five different crystallographic and solution phase structures centered in a 37 Angstrom radius sphere of water, were used to sample the native-fold of MbCO. Three discrete conformational substates resulted where the primary structural differences corresponded to a variable strength nonbond interaction between His64, Arg45, and the bound ligand. To correlate the structures from the computed substates with the experimentally observed ligand stretch frequencies, Hartree-Fock theory with the 6-31G(d) basis set was used to carry out constrained minimizations and vibrational analysis on representative model geometries from each conformational substate. The A(0) state (out conformation) was determined to have both Arg45 and His64 removed from the heme pocket with negligible electrostatic effect on the ligand. Alternatively, His64 was determined to induce the redshifted frequencies characteristic of the A states (A(1-3)) by forming a weak hydrogen bond between its protonated N-delta and the ligand (in/N-delta conformation). The A(1,2) state was specifically assigned to the in/N-delta conformation with Arg45 removed from His64 (Delta v(comp) = -10.0 +/- 1.8 cm(-1)). The second and faster translational motion engaged Arg45 in an additional and cooperative electrostatic interaction with His64 that distinguished between the A(1,2) and A(3) states. The strongest red-shifted ligand stretch frequency (A(3) state) was computed when Arg45 interacted with His64 in the in/N-delta conformation. The polarizing effect of the distal histidine on the CO ligand (Delta v(comp) = -19.0 +/- 6.8 cm(-1)) was increased by the positive charge on Arg45. Consequently, a new A-state model, which rationalizes the A3 state based upon the fluctuating electrostatic field generated by the gate-like dynamics of His64 and Arg45, is presented, which is consistent with previously reported time scales for substate interconversion.