Effects of buried ionizable amino acids on the reduction potential of recombinant myoglobin

Abstract

The temperature dependences of the reduction potentials (E °') 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 Residue Val68, which is in van der Waals contact with the heme in Mb, has been replaced by Glu, Asp, and Asn.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 °' and the standard entropy (δ S °') and enthalpy (δ H °') of reduction in the mutant proteins were determined relative to values for wild type; the change in E °' at 25 ° 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.