Effects of Buried Ionizable Amino Acids on the Reduction Potential of Recombinant Myoglobin

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 Residue Val 68 , which is in van der Waals contact with the heme in Mb, has been replaced by Glu, Asp, and Asn.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 p H 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.