Alteration of the reduction potential of the [4Fe-4S]2+/+ cluster of Azotobacter vinelandii ferredoxin I

The [4Fe-4S]2(+/+) cluster of Azotobacter vinelandii ferredoxin I (FdI) has an unusually low reduction potential (E-0') relative to other structurally similar ferredoxins. Previous attempts to raise that E-0' by modification of surface charged residues were unsuccessful, In this study mutants were designed to alter the E-0' by substitution of polar residues for nonpolar residues near the cluster and by modification of backbone amides, Three FdI variants, P21G;, I40N, and I40Q, were purified and characterized, and electrochemical E-0' measurements show that all had altered E-0' relative to native FdI, For P21G FdI and I40Q FdI, the E-0' increased by +42 and +53 mV, respectively validating the importance of dipole orientation in control of E-0'. Protein Dipole Langevin Dipole calculations based on models for those variants accurately predicted the direction of the change in E-0' while overestimating the magnitude, For I40N Pdl, initial calculations based on the model predicted a +168 mV change in E-0' while a -33 mV change was observed. The x-ray structure of that variant, which was determined to 2.8 Angstrom revealed a number of changes in backbone and side chain dipole orientation and in solvent accessibility, that were not predicted by the model and that were Likely to influence E-0'. Subsequent Protein Dipole Langevin Dipole calculations (using the actual I40N x-ray structures) did quite accurately predict the observed change in E-0'.