Characterization of the properties and roles of Fe-S centers in ferredoxins, glutaredoxins and radical S-adenosyl methionine enzymes
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The objectives of the research presented in this dissertation are to characterize the properties and to understand the role of Fe-S centers in [2Fe-2S]-ferredoxins, glutaredoxins and anaerobic sulfatase maturating enzymes (anSMEs), by using a combination of mutagenesis, analytical and spectroscopic studies. The spectroscopic techniques include UV-visible absorption, circular dichroism, variable temperature magnetic circular dichroism (VTMCD), EPR, Mössbauer, resonance Raman and MALDI-TOF MS. A detailed spectroscopic characterization of Cys-to-Ser variants of [2Fe-2S]-ferredoxin from Clostridium pasteurianum and Aquifex aeolicus were undertaken in order to characterize the electronic properties of valence-delocalized [Fe2S2]+ center, which constitutes the building block of higher-nuclearity Fe-S clusters. VTMCD and magnetization studies enabled the first ever measurement of the double exchange parameter (B) for a valence delocalized [2Fe-2S]+ cluster with sulfide bridging ligands. Spectroscopic studies also revealed that the [2Fe-2S]+ cluster in the valence-delocalized S = 9/2 state at room temperature converts, either partially or fully to valence-localized S = 1/2 state upon freezing. The results are interpreted to be an outcome of changes in solvent packing and H-bonding interactions near the reducible iron site that occur upon freezing and shift the equilibrium in favor of the valence-localized form. Studies carried out to understand the nature and function of Fe-S centers in Arabidopsis thaliana GrxS16, a monothiol glutaredoxin in chloroplast, revealed that recombinant GrxS16 is able to assemble a [2Fe-2S] cluster as-purified, and [2Fe-2S] and [4Fe-4S] clusters on in vitro reconstitution. The ability of GrxS16 to transfer both [2Fe-2S] and [4Fe-4S] cluster to plausible acceptor proteins suggests a role for S16 in the maturation of Fe-S proteins in chloroplast. To elucidate the nature of Fe-S centers and the mechanism of action of anSMEs, analytical and spectroscopic studies were undertaken on the wild-type and variant anSMEs from Clostridium perfringens and Bacteriodes thetaiotamicron. The results reveal that anSMEs can bind three [4Fe-4S] clusters, and activate both Cys-type and Ser-type sulfatases via radical mechanism initiated by the reductive cleavage of S-adenosyl methionine bound to the radical SAM cluster. The two additional [4Fe-4S] clusters are proposed to play a role in the activation of substrate and/or a relay that shuttles electron during enzymatic turnover.