| Summary: | Myoglobin is the subject of continuing investigations because of its ability to bind oxygen reversibly. This physiological role depends on the modulation of iron reactivity and heme affinity by the globin. The hypothesis that both of these factors are affected by residues in the proximal heme pocket is tested by site-directed mutagenesis of four proximal residues: Leu89(F4), His97(FG3), His99(FG5), and Leu104(G5). The structures of several mutant myoglobins have been determined by X-ray crystallography and then used to interpret the results of functional studies. These four proximal pocket residues support and maintain the structure of the porphyrin ring via steric and/or electrostatic interactions with the heme prosthetic group. In turn, stereochemical changes in the heme group induced by a proximal pocket mutation modulate heme-iron reactivity via changes in the displacement of the iron from the heme plane: a more accessible heme iron results in greater affinity for carbon monoxide and dioxygen. Leu89 and His97 are important surface residues that protect the hydrophobic heme pocket from hydration through steric and/or electrostatic interactions with the heme. Substitutions at either position with a small or polar residue expose the heme pocket to solvent and accelerate heme loss. Ile99 and Leu104 are located in the interior region of the heme pocket beneath the heme prosthetic group. Substitution with smaller or polar residues at positions 99 and 104 also results in water penetration of the heme pocket and promotion of heme loss. Furthermore, Leu104 is located between two sites previously found to bind xenon. Crystal structures of xenon-containing Leu104 mutant myoglobins support the hypothesis that changes in the volume accessible to photodissociated ligands affects the rates of primary and secondary recombination.
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