H-1-NMR INVESTIGATION OF THE PARAMAGNETIC CLUSTER ENVIRONMENT IN PYROCOCCUS-FURIOSUS 3-IRON FERREDOXIN - SEQUENCE-SPECIFIC ASSIGNMENT OF LIGATED CYSTEINES INDEPENDENT OF TERTIARY STRUCTURE

One- and two-dimensional H-1 NMR data tailored to detect paramagnetica lly relaxed protons near the S = 1/2, three-iron-sulfur cluster of the ferredoxin from the hyperthermophile Pyrococcus furiosus are analyzed to sequence specifically assign the hyperfine shifted ligated cystein e signals, to determine the nature of the secondary structural element s on which these cysteines reside, and to define the tertiary contacts of the cluster with the remainder of the previously characterized sec ondary structure remote from the cluster [Teng, Q., Zhou, Z.-H., Busse , S. C., Howard, J. B., Adams, M. W. W., and La Mar, G. N. (1994) Bioc hemistry 33, 6316-6326]. Inspection of the geometry of the cluster lig ating cysteines in the six structurally characterized cubane ferredoxi n (Fd) clusters reveals a pattern of distances from the cluster iron(s ) that indicate that each Cys will exhibit one backbone proton that wi ll allow the detection of dipolar connectivities to the backbone of ad jacent residues. It is expected that the first and last of the Cys in the cluster consensus binding sequence will exhibit weakly relaxed pep tide NH and strongly relaxed C alpha H signals, while the two central Cys in that sequence will exhibit strongly relaxed peptide NH but weak ly relaxed C alpha H peaks. These dipolar contacts are clearly observe d for the three ligated Cys in 3Fe P. furiosus Fd, providing the first sequence specific assignment of ligated cysteines which do not explic itly require knowledge of the tertiary structure of the protein. This approach is proposed to have very general application to cubane ferred oxins. A combination of steady-state NOEs and short mixing time NOESY experiments demonstrate that Cys(17) is on a short helix through Leu(2 0) and that Cys(56) likely initiates a type I turn, as observed in the crystal structure of the 3Fe Fd for Desulfovibrio gigas [Kissinger, C . R., Sieker, L. C., Adman, E. T., and Jensen, L. H. (1991) J. Mel. Bi ol. 219, 693-715]. The observed relaxation rates of resolved or partia lly resolved signals are shown to correlate with their proximity to th e various iron in the cluster, as determined for the homologous residu es in D. gigas Fd, providing additional qualitative information on ter tiary contacts of the cluster.