Secondary structure extensions in Pyrococcus furiosus ferredoxin destabilize the disulfide bond relative to that in other hyperthermostable ferredoxins. Global consequences for the disulfide orientational heterogeneity

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The single cubane cluster ferredoxin (Fd) from the hyperthermophilic archaeon Pyrococcus furiosus (Pf) possesses several unique properties when compared even to Fds from other hyperthermophilic archaea or bacteria. These include an equilibrium molecular heterogeneity, a six- to seven-residue increase in size, an Asp rather than the Cys as one cluster ligand, and a readily reducible disulfide bond. NMR assignments and determination of both secondary structure and tertiary contacts remote from the paramagnetic oxidized cluster of Pf 3Fe Fd with an intact disulfide bond reported previously (Teng Q., Zhou, Z. H., Smith, E. T., Busse, S.C., Howard, J. B. Adams, M. W. W., and La Mar, G. (1994) Biochemistry 33, 6316-6328) are extended here to the 4Fe oxidized cluster WT (1H and 15N) and D14C (1H only) Fds with an intact disulfide bond and to the 4Fe oxidized WT Fd (1H and 15N) with a cleaved disulfide bond. All forms are shown to possess a long (13-member) α-helix, two β-sheets (one double-, one triple-stranded), and three turns outside the cluster vicinity, each with tertiary contacts among themselves as found in other Fds. While the same secondary structural elements, with similar tertiary contacts, are found in other hyperthermostable Fds, Pf Fd has two elements, the long helix and the triple- stranded β-sheet, that exhibit extensions and form multiple tertiary contacts. All PfFd forms with an intact disulfide bond exhibit a dynamic equilibrium heterogeneity which is shown to modulate a hydrogen-bonding network in the hydrophobic core that radiates from the Cys21-Cys48 disulfide bond and encompasses residues Lys36, Va124, Cys21, and Cys17 and the majority of the long helix. The heterogeneity is attributed to population of the alternate S and R chiralities of the disulfide bond, each destabilized by steric interactions with the extended α-helix. Comparison of the chemical shifts and their temperature gradients reveals that the molecular structure of the protein with the less stable R disulfide resembles that of the Fd with a cleaved disulfide bond. Both cluster architecture (3Fe vs 4Fe) and ligand mutation (Cys for Asp14) leave the disulfide orientational heterogeneity largely unperturbed. It is concluded that the six- to seven-residue extension that results in a longer helix and largerβ-sheet in Pf Fd, relative to other hyperthermostable Fds, more likely serves to destabilize the disulfide bond, and hence make it more readily reducible, than to significantly increase protein thermostability.

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This article was published in Biochemistry, Volume 38, Issue 25, Pages 8167-8178.

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