1MOA

A NOVEL SITE-DIRECTED MUTANT OF MYOGLOBIN WITH AN UNUSUALLY HIGH O2 AFFINITY AND LOW AUTOOXIDATION RATE

Summary for 1MOA

• Entry DOI: 10.2210/pdb1moa/pdb
• Descriptor: MYOGLOBIN, SULFATE ION, PROTOPORPHYRIN IX CONTAINING FE, ... (4 entities in total)
• Functional Keywords: oxygen storage
• Biological source: Physeter catodon (sperm whale)
• Total number of polymer chains: 1
• Total formula weight: 18111.73

Authors

Quillin, M.L.,Phillips Jr., G.N. (deposition date: 1994-12-01, release date: 1995-02-07, Last modification date: 2024-02-14)

Primary citation

Carver, T.E.,Brantley Jr., R.E.,Singleton, E.W.,Arduini, R.M.,Quillin, M.L.,Phillips Jr., G.N.,Olson, J.S.
A novel site-directed mutant of myoglobin with an unusually high O2 affinity and low autooxidation rate.
J.Biol.Chem., 267:14443-14450, 1992

PubMed Abstract: 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.

PubMed: 1629229

Experimental method

X-RAY DIFFRACTION (1.9 Å)