Electrostatic interactions in wild-type and mutant recombinant human myoglobins.
Residue Val68 in human myoglobin has been replaced by Asn, Asp, and Glu with site-directed mutagenesis. Purified proteins were characterized by isoelectric focusing and by absorption, CD, and NMR spectroscopy. These studies demonstrated that Mb is able to tolerate substitution of the buried hydrophobic residue Val68 by Asn, Asp, and Glu. In the metaquo derivatives of the Glu and Asp mutants, the negative charge at residue 68 is stabilized by a favorable Coulombic interaction with the heme iron. In the absence of this interaction, as in the metcyano and ferrous deoxy derivatives, the relatively nonpolar protein interior cannot stabilize an isolated buried negative charge, and the carboxylate is either protonated or stabilized via a salt bridge with the nearby distal histidine. Hence in the Asp and Glu mutant proteins, both reduction and cyanide binding are accompanied by proton uptake by the protein. The apoproteins were prepared and reconstituted with the chlorophyll derivative zinc pyrochlorophyllide a. Absorption and fluorescence spectra were quite similar for wild-type and all mutant proteins reconstituted with this derivative. These results do not support the point charge model for the red shifts observed in the spectra of chlorophylls associated with photosynthetic proteins. From the pH dependence of the absorption spectrum of zinc pyrochlorophyllide a in the Glu mutant, the apparent pKa of the buried glutamate residue was estimated to be 8.9. This increase of 4.4 pH units, over the value for Glu in aqueous solution, provides a measure of the polarity of the protein interior.
Varadarajan
R
R
Department of Chemistry, Stanford University, California 94305.
Lambright
D G
DG
Boxer
S G
SG
D003062:Codon / Q000235:genetics; D003486:Cyanides / Q000378:metabolism; D003545:Cysteine; D005822:Genetic Vectors; D006418:Heme / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008968:Molecular Conformation; D009154:Mutation; D009211:Myoglobin / Q000235:genetics* / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism; D013053:Spectrophotometry; D014633:Valine
D003062:Codon; D003486:Cyanides; D009211:Myoglobin; D011994:Recombinant Proteins; D006418:Heme; D014633:Valine; D003545:Cysteine