Ferredoxins play crucial roles in biological electron transfer, but the details of how and where these electrons are transferred are poorly understood. We propose to carry out a detailed structure-function study of the 8Fe-8S low potential bacterial ferredoxins (Fd) by examining the kinetic and equilibrium behavior of a several 8Fe-8S ferredoxins with structural variants. These will be obtained from: 1) the several naturally occurring, homologous and sequenced 8Fe-8S Fd's 2) total apoprotein synthesis using solid state methods and 3) site specific mutagenesis of the C. pasteurianum Fd gene which has been cloned into E. coli. In nearly all previous studies only C. pasteurianum Fd has been examined. These Fd's serve as excellent models of other Fe4S4 or 2 Fe4S4 iron-sulfur proteins. As an indicator of function, we will determine the rates of reduction of these Fd's by small molecule reducing agents. Rates will be determined as a function of ionic strength, pH, and temperature. The kinetic-ionic strength experiments will permit us to use follow the influence of the Fd molecular dipole on the observed rates and thus determine the site of electron transfer. Equilibrium reduction potentials will be determined by spectroelectrochemical methods and correlated to specific structural changes. NMR difference spectroscopy will be used to verify the integrity of different Fd's as compared to the native C. pasteurianum Fd. We propose to change specific residues in the C. pasteurianum sequence that will alter electron transfer rates, in some cases with little or no change in cluster reduction potential. The reduction kinetic studies will be done as function of pH to determine if specific residues modulate electron transfer rates. Also the intramolecular self-exchange rates of these Fd's will be determined by NMR spectroscopy (i.e. analysis of peak broadening) in order to explore the influence of intervening amino acids and/or polypeptide between the clusters.
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