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

The single cubane cluster ferredoxin (Fd) from the hyperthermophilic archae on Pyrococcus furiosus (Pf) possesses several unique properties when compar ed even to Fds from other hyperthermophilic archaea or bacteria. These incl ude an equilibrium molecular heterogeneity, a six- to seven-residue increas e in size, an Asp rather than the Cys as one cluster ligand, and a readily reducible disulfide bond. NMR assignments and determination of both seconda ry structure and tertiary contacts remote from the paramagnetic oxidized cl uster 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 (H-1 and N-15) and D14C (H-1 only) Fds with an int act disulfide bond and to the 4Fe oxidized WT Fd (H-1 and N-15) With a clea ved disulfide bond. All forms are shown to possess a long (13-member) alpha -helix, two beta-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 beta-sheet, that e xhibit extensions and form multiple tertiary contacts. All Pf Fd forms with an intact disulfide bond exhibit a dynamic equilibrium heterogeneity which is shown to modulate a hydrogen-bonding network in the hydrophobic core th at radiates from the Cys21-Cys48 disulfide bond and encompasses residues Ly s36, Val24, Cys21, and Cys17 and the majority of the long helix. The hetero geneity is attributed to population of the alternate S and R chiralities of the disulfide bond, each destabilized by steric interactions with the exte nded alpha-helix. Comparison of the chemical shifts and their temperature g radients 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 larger beta-sheet in Pf Fd, relative to other hyperthermostable Fds, more likely serves to destabilize the disulfide bond, and hence make i t more readily reducible, than to significantly increase protein thermostab ility.