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The influence of the distal pocket conformation on the structure and vibrations of the heme-CO bond in carbonmonoxy myoglobin (MbCO) is investigated by means of hybrid QM/MM calculations based on density functional theory combined with a classical force field. It is shown that the heme-CO structure (QM treated) is quite rigid and not influenced by the distal pocket conformation (MM treated). This excludes any relation between FeCO distortions and the different CO absorptions observed in the infrared spectra of MbCO (A states). In contrast, both the CO stretch frequency and the strength of the CO...His64 interaction are very dependent on the orientation and tautomerization state of His64. Our calculations indicate that the CO...N(epsilon) type of approach does not contribute to the A states, whereas the CO...H-N(epsilon) interaction is the origin of the A(1) and A(3) states, the His64 residue being protonated at N(epsilon). The strength of the CO...His64 interaction is quantified, in comparison with the analogous O(2)...His64 interaction and with the observed changes in the CO stretch frequency. Additional aspects of the CO...His64 interaction and its biological implications are discussed.

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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 Å 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 δ and the ligand (in/N δ conformation). The A 1,2 state was specifically assigned to the in/N δ conformation with Arg45 removed from His64 (∆ν 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 δ conformation. The polarizing effect of the distal histidine on the CO ligand (∆ν comp ) -19.0 ( 6.8 cm -1 ) was increased by the positive charge on Arg45. Consequently, a new A-state model, which rationalizes the A 3 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.

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The functional importance of large-scale motions and transitions of carbonmonoxy myoglobin (MbCO) conformational substates (CSs) has been studied by molecular dynamics (MD) and conformational flooding (CF) simulations. A flooding potential was constructed from an 800 ps MD trajectory of solvated MbCO to accelerate slower protein motions beyond the time scale of contemporary simulations. Two conformational transitions (tier-1 substates) resulting from seven principal molecular motions were assigned to the spectroscopic A 0 state (tier-0 substate) of MbCO, where His64 is solvated and not within the hydrophobic pocket binding site. The first computed conformational transition involves a distal pocket gate defined by the C and D helices and the interconnecting CD loop (residues 40-55). The gate-like motion is interpreted to regulate ligand access from the distal side of the heme. Simultaneously, a proximal pocket lever involving the F helix and surrounding EF and FG loops (residues 82-105) is found to shuttle the heme deep into the protein matrix (heme rmsd of 3.9 Å) as the distal pocket gate opened. The lever's effect on the heme motion is assumed to attract ligands into the heme pocket. The second major transition involves the compression and expansion of the cavity formed by the EF loop (residues 77-84) and the GH loop and H helix (residues 122-138). The motion is interpreted to modulate the hydrophobic pocket volume and regulate the ligand access from the proximal side of the heme. A third computed conformational transition was found to be a combination of the previous motions. For the first time, CF was applied in a series of room temperature simulations to accelerate molecular motions of the MbCO native fold and define the lower tier hierarchy of substate structure. The computed CSs and associated transitions coincide with previously suggested putative ligand escape pathways, and support a hierarchical description of protein dynamics and structure. A unified model that utilizes both mechanisms of distal His64 modulation (tier-0) and protein equilibrium fluctuations (tier-1) is presented to explain ligand diffusion in the MbCO dissociation reaction.

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