9728793 Miller Comparison of Mn-substituted Fe-superoxide dismutase (Mn-sub-(Fe)(SOD) with Mn-SOD will provide a test that Fe-sub-Mn-SOD and Mn-sub-(Fe)SOD are inactive due to a mismatch between the E( tuning applied by the protein and that appropriate to the metal ion. Experimental determination of the degree of protonation of the coordinated solvent in the two different proteins by resonance Raman spectroscopy and ESEEM will indicate the extent to which the proteins could modulate E( by manipulating the protonation state of coordinated solvent. Spectroscopic and electrochemical studies of mutant (Fe)SOD containing the conserved Gln characteristic of Mn-SODs and mutant (Mn)SOD containing the Gln characteristic of Fe-SODs, with each of the metal ions bound, will provide reciprocal quantitations of the effects on the E( of this conserved amino acid difference between Mn- and Fe-SODs. Observation of the conserved Gln's side chain directly by 15N NMR or indirectly by ESEEM will probe the polarity of this residue's hydrogen bonds with key ingredients of SOD's catalytic activity: Tyr 34 and the coordinated solvent. Random mutagenesis of (Mn)SOD and screening for activity in the presence of Fe but not Mn will afford an unbiased selection of "convertant" SODs that have acquired activity with Fe. Amino acid sequence determination and biophysical and electrochemical characterizations will then reveal what amino acids confer activity on Fe, by what chemical mechanisms and at what cost to activity with Mn. Finally, solution of the X-ray crystal structures of Mn-SOD and Fe-sub-(Mn)SOD will show how the protein structure depends on the metal ion identity. Thus, the work will identify general principles governing metal ion activity in proteins and examples of how they are implemented by particular amino acids in SOD. Mn- and Fe-specific superoxide dismutases (SODs) will be used to study how proteins confer specific activity on bound metal ions. Comparisons between inactive Fe-substituted-(Mn)SOD an d active Fe-SOD, and between the inactive Mn-sub-(Fe)SOD and active Mn-SOD should highlight differences that may be correlated with activity. It is anticipated that metal ion substitution in existing enzymes may afford a rapid route to novel enzymes with useful properties. Therefore, knowledge of the different protein factors that determine the activity of bound metal ions is crucial to ability to design metallo-enzymes with desired catalytic activity. ***
