Intramolecular translocation of the protein radical formed in the reaction of recombinant sperm whale myoglobin with H2O2.
A sperm whale myoglobin gene containing multiple unique restriction sites has been constructed in pUC 18 by sequential assembly of chemically synthesized oligonucleotide fragments. Expression of the gene in Escherichia coli DH5 alpha cells yields protein that is identical to native sperm whale myoglobin except that it retains the terminal methionine. Site-specific mutagenesis has been used to prepare all the possible tyrosine----phenylalanine mutants of the recombinant myoglobin, including the three single mutants at Tyr-103, -146, and -151, the three double mutants, and the triple mutant. All of the mutant proteins are stable except the Tyr-103 mutant. Introduction of a second mutation (Lys-102----Gln) stabilizes the Tyr-103 mutant. Absorption spectroscopy suggests that the active sites of the mutant proteins are intact. EPR and absorption spectroscopy show that all the proteins, including the triple mutant devoid of tyrosine residues, react with H2O2 to give a ferryl species and a protein radical. The presence of a protein radical in all the mutants suggests that the radical center is readily transferred from one amino acid to another. Cross-linking studies show, however, that protein dimers are only formed when Tyr-151 is present. Tyr-103, shown earlier to be the residue that primarily cross-links to Tyr-151 (Tew, D., and Ortiz de Montellano, P. R. (1988) J. Biol. Chem. 263, 17880-17886), is not essential for cross-linking. Electron transfer from Tyr-151 to the heme, which are 12 A apart, occurs in the absence of the intervening tyrosines at positions 103 and 146. The present studies show that the peroxide-generated myoglobin radical readily exchanges between remote loci, including non-tyrosine residues, but protein cross-linking only occurs when radical density is located on Tyr-151.
Wilks
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A
Department of Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco 94143-0446.
Ortiz de Montellano
P R
PR
D000818:Animals; D001692:Biological Transport; D003001:Cloning, Molecular; D003432:Cross-Linking Reagents; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005609:Free Radicals; D006861:Hydrogen Peroxide / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009211:Myoglobin / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015183:Restriction Mapping; D013057:Spectrum Analysis; D014907:Whales
D003432:Cross-Linking Reagents; D005609:Free Radicals; D009211:Myoglobin; D011994:Recombinant Proteins; D006861:Hydrogen Peroxide