Abstract
Zinc centers play a key role as important structure determinants in a variety of proteins including ferredoxins (Fd). Here, we exploit the availability of two highly similar ferredoxin isoforms from the thermophile Sulfolobus metallicus, which differ in the residues involved in coordinating a His/Asp zinc site that ties together the protein core with its N-terminal extension, to investigate the ...
Abstract
Zinc centers play a key role as important structure determinants in a variety of proteins including ferredoxins (Fd). Here, we exploit the availability of two highly similar ferredoxin isoforms from the thermophile Sulfolobus metallicus, which differ in the residues involved in coordinating a His/Asp zinc site that ties together the protein core with its N-terminal extension, to investigate the effect of the absence of this site on ferredoxin folding. The conformational properties of the zinc-containing (FdA) and zinc-lacking (FdB) isoforms were investigated using visible absorption and tryptophan fluorescence emission. Fluorescence quenching studies, together with comparative modeling and molecular dynamics simulations, indicate that the FdB N-terminal extension assumes a fold identical to that of the Zn2+-containing isoform. The thermal stability of the isoforms was investigated in a broad pH range (2 < pH < 10), and at physiological pH conditions, both proteins unfold above 100 C. Surprisingly, the Zn2+-lacking isoform was always found to be more stable than its Zn2+-containing counterpart: a Delta T-m approximate to 9 degrees C is determined at pH 7, a difference that becomes even more significant at extreme pH values, reaching a Delta T-m approximate to 24 degrees C at pH 2 and 10. The contribution of the Zn2+ site to ferredoxin stability was further resolved using selective metal chelators. During thermal unfolding, the zinc scavenger TPEN significantly lowers the T-m in FdA (approximate to 10 C), whereas it has no effect in FdB. This shows that the Zn2+ site contributes to ferredoxin stability but that FdB has devised a structural strategy that accounts for an enhanced stability without using a metal cross-linker. An analysis of the FdB sequence and structural model leads us to propose that the higher stability of the zinc-containing ferredoxin results from van der Waals contacts formed between the residues that occupy the same spatial region where the zinc ligands are found in FdA. These favor the formation of a novel local stabilizing hydrophobic core and illustrate a strategy of natural fold design.
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