Phe‐46(CD4) orients the distal histidine for hydrogen bonding to bound ligands in sperm whale myoglobin

Henry H. Lai, Tiansheng Li, Daniel S. Lyons, George N. Phillips, John S. Olson, Quentin H. Gibson

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The role of Phe‐46(CD4) in modulating the functional properties of sperm whale myoglobin was investigated by replacing this residue with Leu, Ile, Val, Ala, Trp, Tyr, and Glu. This highly conserved amino acid almost makes direct contact with the distal histidine and has been postulated to affect ligand binding. The overall association rate constants for CO, O2, and NO binding were little affected by decreasing the size of residue 46 step‐wise from Phe to Leu to Val to Ala. In contrast, the rates of CO, O2, and NO dissociation increased 4‐, 10‐, and 25‐fold, respectively, for the same series of mutants, causing large decreases in the affinity of myoglobin for all three diatomic gases. The rates of autooxidation at 37°C, pH 7.0 increased dramatically from ∼0.1–0.3 h−1 for wild‐type, Tyr‐46, and Trp‐46 myoglobins 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 O2 geminate recombination were measured using 35 ps and 9 ns laser excitation pulses. Decreasing the size of residue 46 causes significant decreases in the extent of both picosecond and nanosecond rebinding processes. High resolution structures of Leu‐46 and Val‐46 metmyoglobins, Val‐46 CO‐myoglobin, and Val‐46 deoxymyoglobin were determined by X‐ray crystallography. When Phe‐46 is replaced by Val, the loss of internal packing volume is compensated by (1) contraction of the CD corner toward the core of the protein, (2) movement of the E‐helix toward the mutation site, (3) greater exposure of the distal pocket to intruding solvent molecules, and (4) large disorder in the position of the side chain of the distal histidine (His‐64). In wild‐type myoglobin, the van der Waals contact between Cζ of Phe‐46 and Cβ of His‐64 appears to restrict rotation of the imidazole side chain. Insertion of Val at position 46 relieves this steric restriction, allowing the imidazole side chain to rotate about the Cα–Cβ bond toward the surface of the globin and about the Cβ–Cγ bond toward the space previously occupied by the native Phe‐46 side chain. This movement disrupts hydrogen bonding with bound ligands, causing significant decreases in affinity, and opens the distal pocket to solvent water molecules, causing marked increases 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 phenomena inhibit geminate recombination and can be correlated with molecular dynamics calculations. All of these results show that this mutation in the second shell of amino acids around the distal pocket can influence ligand binding significantly. © 1995 Wiley‐Liss, Inc.

Original languageEnglish
Pages (from-to)322-339
Number of pages18
JournalProteins: Structure, Function, and Bioinformatics
Issue number4
StatePublished - Aug 1995


  • distal histidine
  • ligand binding
  • myoglobin
  • site‐directed mutagenesis
  • X‐ray crystallography


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