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 Residue Val68, which is in van der Waals contact with the heme in Mb, has been replaced by Glu, Asp, and Asn.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.

Site-directed mutants of human myoglobin have been prepared and characterized; each protein has a single surface-modifiable histidine (at position 48, 70, or 83). The proteins were .modified by covalent attachment of pentaammineruthenium (a5Ru) to the surface histidine and substitution of zinc mesoporphyrin IX diacid (ZnP) for the heme.Donor-acceptor separations (edge-edge distances d) in the modified proteins are 9.5 A, His70; 12.7 A, His48; and 15.5 A, His83. Rates of photoinduced electron transfer in these ruthenium-modified myoglobins were measured by transient absorption spectroscopy. The 3ZnP* -Ru3+ rate constants are 1.6 X IO' (His'lO), 7.2 X lo4 (His48), and 4.0 X lo2 s-I (His83) (-AGO = 0.82 eV); charge-recombination (Ru2+ -ZnP') rates are 1.1 X lo5 (His48) and 7.3 X lo2 s-I (His83) (-AGO = 0.96 eV).Activationless (maximum) rates assuming h = 1.3 eV are 7.2 X lo7 (His70), 3.3 X lo5 (His48). and 1.8 X lo3 s-l (His83). Distant electronic couplings, which limit the maximum rates in the modified myoglobins, have been analyzed along with data from Ru-modified cytochromes c in terms of a tunneling pathway model. Single dominant pathways adequately describe. the electronic couplings in cytochrome c but do not satisfactorily account for the myoglobin couplings. The correlation of electronic coupling with tunneling length for myoglobin is improved significantly by the inclusion of multiple pathways.

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