| In this thesis, the roles of the cofactors for folding and stability of flavodoxin and desulfoferredoxin proteins from Desulfovibrio desulfuricans , a sulfate-reducing bacterium has been addressed.; Flavodoxins are proteins with an α/β topology that non-covalently coordinate a flavin mononucleotide (FMN) cofactor. These proteins from two strains of Desulfovibrio desulfuricans, ATCC 27774 and ATCC 29577 have been studied. The two proteins have 75.3% sequence identity, with the FMN-binding site being fully conserved. In order to characterize the folding pathway, three spectroscopic techniques were used: Far UV-CD, to characterize the secondary structure changes, fluorescence, to monitor the environment around the aromatic residues and visible absorption to monitor the FMN environment. The equilibrium-unfolding mechanism of Desulfovibrio desulfuricans flavodoxin (ATCC 27774) involves a native-like intermediate. In contrast, the equilibrium-unfolding mechanism of Desulfovibrio desulfuricans flavodoxin (ATCC 29557) is two-state. FMN stays bound to the unfolded polypeptide in both cases (using GuHCI as the denaturant). Holo-flavodoxin is only somewhat more stable than apo-flavodoxin. For Desulfovibrio desulfuricans flavodoxin (ATCC 29577), the folding kinetics were also probed. The holo-protein exhibits two-state kinetic behavior, albeit an additional slower phase is present during folding at very low denaturant concentrations. The extrapolated folding time in water for holo-flavodoxin, ∼280 μs (pH 7.0, 20°C), is in good agreement with that predicted from the protein's native-state topology. Desulfovibrio desulfuricans apoflavodoxin (ATCC 29577) on the other hand, exhibits bi-phasic folding and unfolding kinetics. The extrapolated folding times in water for both phases are slower than for holo-flavodoxin. The apo-protein data can be explained by an essentially off-pathway kinetic intermediate.; Desulfoferredoxin (Dfx) is a homodimer with the monomers linked through β-strand interactions in two domains. Each domain contains an iron center: Fe-(S-Cys) 4 center in domain I and Fe-[S-Cys + (N-His)4] center in domain 1I. Unfolding of Dfx was monitored by fluorescence, visible absorption and far-UV CD. Equilibrium unfolding of Dfx involves a monomeric intermediate with native-like secondary structure. Only after polypeptide unfolds do the iron ions dissociate. It seems that the iron centers, the amino-acid composition and to a lesser extent, the dimeric structure are factors governing Dfx's high thermodynamic stability. |
