Protein engineering of Azotobacter vinelandii ferredoxin I: Reduction potential control and electron-coupled proton transfer

This dissertation focuses on protein engineering study of a small [Fe-S] protein using site-directed mutagenesis methods.;Using A. vinelandii ferredoxin I (FdI) as a model system, site-directed mutant variants were designed to alter the reduction potential of the [4Fe-4S]2+/+ cluster of FdI. First, theoretical calculations were used as a guide to predict mutant variants that would have large reduction potential changes and the results proved that this approach was effective as long as accurate structure of the mutant variants were available. The second approach involved changing two Phe residues near the [4Fe-4S]2+/+ cluster of FdI to His residues. One of the mutant variants, F25H, has more than 200 mV change of reduction potential vs. the native protein, which is the largest change ever recorded on an [Fe-S] cluster with a single residue mutation. The third approach is based on sequence comparison between two group of ferredoxins that have more than 200 mV differences in their [4Fe-4S] 2+/+ cluster reduction potentials. Changing the residues from the low potential proteins to their counter parts in the high potential protein resulted in nearly 100 mV change in one of the mutant variants.;The rest of the dissertation involves a very detailed study to understand the mechanism of electron coupled proton transfer to the [3Fe-4S]+/0 cluster of FdI. Mutants were designed and detailed kinetic study by fast scan electrochemical experiments were carried out to eliminate several possible proton transfer pathways towards the cluster and results indicated that Asp15 was solely responsible for the proton transfer from the solvent to the buried cluster. Molecular dynamic simulation experiments also proved that Asp15 could deliver the proton to the cluster directly.