An ab initio investigation of a model heme prosthetic group based on the carbonmonoxy myoglobin (MbCO) 1MBC X-ray structure reproduces the large off-perpendicular distortions of the Fe—C—O unit reported for the protein. The distortion is mainly caused by the nonequilibrium orientation of the proximal residue and not by the distal residue: inclusion of the distal residue in a supermolecule calculation has a smaller effect on the Fe—C—O geometry. If such a mechanism primarily determines the Fe—C—O distortion in the protein itself, then the large strain energies implied by the Fe—C—O geometries in the X-ray structures are delivered by the protein tertiary structure, via the proximal residue, and not by the mobile distal side chain, as had been previously proposed. The structure—function relationship, as revealed by the X-ray structure, would then be clarified. Distortion of the Fe—C—O geometry is largely determined by the proximal residue, and so Fe—C—O is nonperpendicular even in the His64Gly mutant. Thedistal residue isnot subject to a large repulsive interaction with the carbonyl ligand; thus, its orientation in the solvated protein can be determined by weaker attractive electrostatic interactions, as inferred from recent experimental studies of distal residue mutant myoglobins. This result removes the need to invoke a large stabilization of the distal side chainorientation by a rigid hydrogen-bonding network, an interpretation of the physiological structure-function relationship that was at odds with the X-ray Bfactors and the mobility of surface residue side chains expected under physiological conditions.