SITE-DIRECTED MUTAGENESIS OF PHE-97 TO GLU IN AMICYANIN ALTERS THE ELECTRONIC COUPLING FOR INTERPROTEIN ELECTRON-TRANSFER FROM QUINOL METHYLAMINE DEHYDROGENASE

Conversion by site-directed mutagenesis of Phe 97 of amicyanin to Glu significantly decreases the rate constant for the electron-transfer re action from the quinol form of methylamine dehydrogenase to amicyanin. It is shown that the Delta G degrees and reorganizational energy (lam bda) associated with the electron-transfer reaction are unaffected by the mutation and that the decrease in the electron-transfer rate is at tributable completely to a decrease in the electronic coupling matrix element (H-AB). Phe 97 is not a part of the predicted pathway for elec tron-transfer from the tryptophan tryptophylquinone cofactor of MADH t o the copper of amicyanin. The most likely explanation for these resul ts is that the mutation of this residue at the protein-protein interfa ce causes an increase in the interprotein distance within the protein complex. The change in distance necessary to cause the observed reduct ion of H-AB is calculated assuming a range of beta values, and assumin g either solely a direct distance dependence or a pathway dependence, for the long-range electron-transfer reaction. Thermodynamic analysis of the association constants for complex formation reveal that the rea ction with the mutant amicyanin exhibits a large positive change in he at capacity whereas this is not observed in the reaction with the wild -type. This may be explained by the replacement of a hydrophobic resid ue with a polar residue at what is normally a hydrophobic protein-prot ein interface. The impact of these results on possible explanations fo r the relatively large reorganizational energy associated with this in terprotein electron-transfer reaction is also discussed.