ABSTRACT
A solution molecular model for the conformationally dynamically heterogeneous Pyrococcus furiosus ferredoxin with an intact disulfide bond has been constructed on the basis of reported (1)H NMR spectral parameters using distance geometry and simulated annealing protocols. Conventional long-mixing time NOESY and H-bonding constraints have been augmented by previously reported short-mixing time NOESY, steady-state NOE, and cluster paramagnetism-induced relaxation. The family of 15 structures with inconsequential violations exhibited low rms deviations for backbone atoms for the overwhelming majority of the residues, including the cluster ligating loop with the unprecedented ligated Asp14. Larger rms deviations were observed across the disulfide bond, but closer inspection revealed that the 15 structures can be factored into 10 substructures exhibiting an "S" or right-handed disulfide orientation and 5 exhibiting an "R" or left-handed disulfide orientation. The remainder of the structure is indistinguishable for the two disulfide orientations but confirms stabilizing extensions of secondary structural elements in the lengthening of the long helix and both the lengthening and incorporation of a third strand into the beta-sheet involving the termini, with these extensions interacting strongly in a modular fashion through the rings of Tyr46 and Trp2. These extensions of stabilizing interactions in Pyrococcus furiosus Fd, however, lead to strong destabilization of the disulfide bond and destabilization of the highly conserved first and last beta-turns in the sequence. It is concluded that the structural alternations in Pyrococcus Fd relative to other hyperthermostable Fds are not to increase thermostability but to place "stress" on the disulfide bond and render it more reducible. The possible physiological implications of this unique reducible disulfide bond are discussed.
