Effects of buried ionizable amino acids on the reduction potential of recombinant myoglobin.
The temperature dependences of the reduction potentials (E degrees') of wild-type human myoglobin (Mb) and three site-directed mutants have been measured by the use of thin-layer spectroelectrochemistry. Residue Val68, which is in van der Waals contact with the heme in Mb, has been replaced by Glu, Asp, and Asn. The changes in E degrees' and the standard entropy (delta S degrees') and enthalpy (delta H degrees') of reduction in the mutant proteins were determined relative to values for wild type; the change in E degrees' at 25 degrees C was about -200 millivolts for the Glu and Asp mutants, and about -80 millivolts for the Asn mutant. At pH 7.0, reduction of Fe(III) to Fe(II) in the Glu and Asp mutants is accompanied by uptake of a proton by the protein. These studies demonstrate that Mb can tolerate substitution of a buried hydrophobic group by potentially charged and polar residues and that such amino acid replacements can lead to substantial changes in the redox thermodynamics of the protein.
Varadarajan
R
R
Department of Chemistry, Stanford University, CA 94305.
Zewert
T E
TE
Gray
H B
HB
Boxer
S G
SG
D001216:Asparagine; D001224:Aspartic Acid; D005971:Glutamates; D018698:Glutamic Acid; D006418:Heme / Q000378:metabolism; D006801:Humans; D009154:Mutation; D009211:Myoglobin / Q000378:metabolism*; D010084:Oxidation-Reduction; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism*; D013816:Thermodynamics; D014633:Valine
D005971:Glutamates; D009211:Myoglobin; D011994:Recombinant Proteins; D001224:Aspartic Acid; D018698:Glutamic Acid; D006418:Heme; D001216:Asparagine; D014633:Valine