J Biol Chem, Vol. 274, Issue 51, 36479-36487, December 17, 1999

Alteration of the Reduction Potential of the [4Fe-4S]^2+/+ Cluster of Azotobacter vinelandii Ferredoxin I^*

Kaisheng Chen, Gareth J. Tilley^§, Vandana Sridhar^¶, G. Sridhar Prasad^¶, C. David Stout^¶, Fraser A. Armstrong^§, and Barbara K. Burgess

From the  Department of Molecular Biology and Biochemistry, University of California, Irvine, California 92697, the ^§ Department of Chemistry, Oxford University, Oxford OX1 3QR, United Kingdom, and the ^¶ Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037

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 FdI, 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 Å, 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'.

The atomic coordinates and the structure factors (code 1b0v) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).