H-1-NMR INVESTIGATION OF THE SECONDARY STRUCTURE, TERTIARY CONTACTS AND CLUSTER ENVIRONMENT OF THE 4-IRON FERREDOXIN FROM THE HYPERTHERMOPHILIC ARCHAEON THERMOCOCCUS-LITORALIS

The solution molecular structure of the four-iron ferredoxin (Fd) from the hyperthermophilic archaeon Thermococcus litoralis (Tl) has been i nvestigated by H-1 NMR spectroscopy. TOCSY and NOESY experiments in H2 O, tailored to detect both weakly and strongly relaxed resonances, tog ether with steady-state NOEs in both H2O and D2O, allowed the identifi cation of 58 of the 59 residues, with one residue near the paramagneti c center undetected. It is shown that the contact shifted and strongly relaxed signals for all four cysteines ligated to the paramagnetic cl uster can be assigned by standard backbone connectivities that do not require any assumptions about the tertiary structure. Secondary struct ural elements identified in Tl Fd are a three-stranded antiparallel be ta-strand involving the termini of the protein, a double beta-strand ( also antiparallel), two alpha-helices and four turns. The existence of a disulfide bridge between the nonligated cysteines is also proposed. Dipolar contacts observed in the NOESY maps and by steady-state NOEs between the ligated cysteines and the 'diamagnetic' protein matrix ind icate that the overall folding pattern of Tl Fd is very similar to tha t of the 3Fe ferredoxin from the mesophilic bacterium Desulfovibrio gi gas [Kissinger et al. (1991) J. Mol. Biol., 219, 693-723]. The influen ce of the paramagnetism of the cluster on the relaxation properties of the proton signals of nonligated residues near the cluster as well as on the ligated cysteines, correlates well with the proximity to the c luster iron(s), as predicted from the crystal structures for homologou s protons of other single-cluster ferredoxins. Finally, the potential role of the various identified structural factors in contributing to t he hyperthermostability of this protein is discussed.