Cooperative role of Arg45 and His64 in the spectroscopic A(3) state of carbonmonoxy myoglobin: Molecular dynamics simulations, multivariate analysis,and quantum mechanical computations

The structural, dynamic, and electronic origin of the spectroscopically obs erved carbonmonoxy myoglobin (MbCO) A states has been investigated by using molecular dynamics to sample conformational space, multivariate analysis t o aid in structural interpretations, and quantum mechanics to compute ligan d stretch frequencies. Ten short (400 ps) and two longer time (1.2 ns) mole cular dynamics simulations, starting from five different crystallographic a nd solution phase structures centered in a 37 Angstrom radius sphere of wat er, were used to sample the native-fold of MbCO. Three discrete conformatio nal substates resulted where the primary structural differences corresponde d 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 minimization s and vibrational analysis on representative model geometries from each con formational substate. The A(0) state (out conformation) was determined to h ave both Arg45 and His64 removed from the heme pocket with negligible elect rostatic effect on the ligand. Alternatively, His64 was determined to induc e the redshifted frequencies characteristic of the A states (A(1-3)) by for ming 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 i n/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 w ith His64 in the in/N-delta conformation. The polarizing effect of the dist al histidine on the CO ligand (Delta v(comp) = -19.0 +/- 6.8 cm(-1)) was in creased by the positive charge on Arg45. Consequently, a new A-state model, which rationalizes the A3 state based upon the fluctuating electrostatic f ield generated by the gate-like dynamics of His64 and Arg45, is presented, which is consistent with previously reported time scales for substate inter conversion.