AZOTOBACTER-VINELANDII FERREDOXIN-I - ALTERATION OF INDIVIDUAL SURFACE-CHARGES AND THE [4FE-4S](2+ +) CLUSTER REDUCTION POTENTIAL/

The structures of Azotobacter vinelandii ferredoxin I (AvFdI) and Pept ococcus aerogenes ferredoxin (PaFd), near their analogous [4Fe-4S](2+/ +) clusters, are highly conserved (Backes, G., Mine, Y., Loehr, T M., Meyer, T E., Cusanovich, M. A., Sweeney, W V., Adman, E. T., and Sande rs-Loehr, J. (1991) J. Am. Chem. Sec. 11, 2055-2064). Despite these si milarities, the reduction potential (E(0')) of the AvFdI [4Fe-4S](2+/) cluster is more than 200 mV more negative than that of PaFd. We have tested the contribution that individual amino acid residues make to t he control of E(0') by converting residues in AvFdI into the correspon ding residue in PaFd. Four mutations involved substitutions of negativ ely charged surface residues with neutral residues and two involved su bstitution of buried hydrophobic residues. All AvFdI variants were cha racterized by x-ray crystallography, absorption, CD, EPR, and H-1 NMR spectroscopies and by electrochemical methods. For the F25I mutation, significant structural changes occurred that affected the EPR and H-1 NMR spectroscopic properties of AvFdI and had a minor influence on E(0 '). For all other mutations there were no changes in reduction potenti al. Thus we conclude, that variations in charged surface residues do n ot account for the observed differences in E(0') between the analogous [4Fe-4S](2+/+) cluster of PaFd and AvFdI. These differences are there fore most likely to be due to differences in solvent accessibility.