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Mutants of sperm whale myoglobin were constructed at position 29 (B10 in helix
notation) to examine the effects of distal pocket size on the rates of ligand
binding and autooxidation. Leu29 was replaced with Ala, Val, and Phe using the
synthetic gene and Escherichia coli expression system of Springer and Sligar
(Springer, B. A., and Sligar, S. G. (1987) Proc. Natl. Acad. Sci. U. S. A. 84,
8961-8965). Structures of the ferric forms of Val29 and Phe29, and the oxy form
of Phe29 myoglobin were determined to 1.7 A by x-ray crystallography. The ferric
mutant proteins are remarkably isomorphous with the wild type protein except in
the immediate vicinity of residue 29. Thus, the protein structure in the distal
pocket of myoglobin can accommodate either a large "hole" (i.e. Ala or Val) or a
large side chain (i.e. Phe) at position 29 without perturbation of tertiary
structure. Phe29 oxymyoglobin is also identical to the native oxy protein in
terms of overall structure and interactions between the bound O2 and His64,
Val68, Phe43, and Ile107. The distance between the nearest side chain atom of
residue 29 and the second atom of the bound oxygen molecule is 3.2 A in the
Phe29 protein and 4.9 A in native myoglobin. The equilibrium constants for O2
binding to Ala29, Val29, and Leu29 (native) myoglobin are the same,
approximately 1.0 x 10(6) M-1 at 20 degrees C, whereas that for the Phe29
protein is markedly greater, 15 x 10(6) M-1. This increase in affinity is due
primarily to a 10-fold decrease in the O2 dissociation rate constant for the
Phe29 mutant and appears to be the result of stabilizing interactions between
the negative portion of the bound O2 dipole and the partially positive edge of
the phenyl ring. Increasing the size of residue 29 causes large decreases in the
rate of autooxidation of myoglobin: k(ox) = 0.24, 0.23, 0.055, and 0.005 h-1 for
Ala29, Val29, Leu29 (native), and Phe29 myoglobin, respectively, in air at 37
degrees C. Thus, the Leu29----Phe mutation produces a reduced protein that is
remarkably stable and is expressed in E. coli as 100% MbO2. The selective
pressure to conserve Leu29 at the B10 position probably represents a compromise
between reducing the rate of autooxidation and maintaining a large enough O2
dissociation rate constant to allow rapid oxygen release during respiration.
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