PHE-46(CB4) ORIENTS THE DISTAL HISTIDINE FOR HYDROGEN-BONDING TO BOUND LIGANDS IN SPERM WHALE MYOGLOBIN

The role of Phe-46(CD4) in modulating the functional properties of spe rm whale myoglobin was investigated by replacing this residue with Leu , De, Val, Ala, Trp, Tyr, and Glu. This highly conserved amino acid al most makes direct contact with the distal histidine and has been postu lated to affect ligand binding. The overall association rate constants for CO, O-2, and NO binding were little affected by decreasing the si ze of residue 46 step-wise from Phe to Leu to Val to Ala. In contrast, the rates of CO, O-2, and NO dissociation increased 4-, 10-, and 25-f old, respectively, for the same series of mutants, causing large decre ases in the affinity of myoglobin for all three diatomic gases. The ra tes of autooxidation at 37 degrees C, pH 7.0 increased dramatically fr om similar to 0.1-0.3 h(-1) for wild-type, Tyr-46, and Trp-46 myoglobi ns to 1.5, 5.2, 4.9, and 5.0 h(-1) for the Leu-46, Ile-46, Val-46, and Ala-46 mutants, respectively. Rates of NO and O-2 geminate recombinat ion were measured using 35 ps and 9 ns laser excitation pulses. Decrea sing the size of residue 46 causes significant decreases in the extent of both picosecond and nanosecond rebinding processes. High resolutio n structures of Leu-46 and Val-46 metmyoglobins, Val-46 CO-myoglobin, and Val-46 deoxymyoglobin were determined by X-ray crystallography. Wh en Phe-46 is replaced by Val, the loss of internal packing volume is c ompensated by (1) contraction of the CD corner toward the core of the protein, (2) movement of the E-helix toward the mutation site, (3) gre ater exposure of the distal pocket to intruding solvent molecules, and (4) large disorder in the position of the side chain of the distal hi stidine (His-64). In wild-type myoglobin, the van der Waals contact be tween C-zeta of Phe-46 and C-beta of His-64 appears to restrict rotati on of the imidazole side chain. Insertion of Val at position 46 reliev es this steric restriction, allowing the imidazole side chain to rotat e about the C-alpha-C-beta bond toward the surface of the globin and a bout the C-beta-C-gamma bond toward the space previously occupied by t he native Phe-46 side chain. This movement disrupts hydrogen bonding w ith bound ligands, causing significant decreases in affinity, and open s the distal pocket to solvent water molecules, causing marked increas es in the rate of autooxidation. The upward movement of the imidazole side chain also creates new space for photodissociated ligands and for incoming water molecules to approach the iron atom. Both of these phe nomena inhibit geminate recombination and can be correlated with molec ular dynamics calculations. All of these results show that this mutati on in the second shell of amino acids around the distal pocket can inf luence ligand binding significantly. (C) 1995 Wiley-Liss, Inc.