One- and two-dimensional 1H NMR data tailored to detect paramagnetically 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 cysteine signals, to determine the nature of the secondary structural elements on which these cysteines reside, and to define the tertiary contacts of the cluster with the remainder of the previously characterized secondary structure remote from the cluster [Teng, Q., Zhou, Z.-H., Busse, S.C., Howard, J.B., Adams, M. W. W., & La Mar, G. N. (1994) Biochemistry 33, 6316-6326]. Inspection of the geometry of the cluster ligating cysteines in the six structurally characterized cubane ferredoxin (Fd) clusters reveals a pattern of distances from the cluster iron(s) that indicate that each Cys will exhibit one backbone proton that will allow the detection of dipolar connectivities to the backbone of adjacent residues. It is expected that the first and last of the Cys in the cluster consensus binding sequence will exhibit weakly relaxed peptide NH and strongly relaxed C alpha H signals, while the two central Cys in that sequence will exhibit strongly relaxed peptide NH but weakly relaxed C alpha H peaks. These dipolar contacts are clearly observed for the three ligated Cys in 3Fe P. furiosus Fd, providing the first sequence specific assignment of ligated cysteines which do not explicitly require knowledge of the tertiary structure of the protein. This approach is proposed to have very general application to cubane ferredoxins. A combination of steady-state NOEs and short mixing time NOESY experiments demonstrate that Cys17 is on a short helix through Leu20 and that Cys56 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., & Jensen, L. H. (1991) J. Mol. Biol. 219, 693-715]. The observed relaxation rates of resolved or partially resolved signals are shown to correlate with their proximity to the various iron in the cluster, as determined for the homologous residues in D. gigas Fd, providing additional qualitative information on tertiary contacts of the cluster.
The problem of characterization of a region of an n-dimensional potential energy surface with maximization of the quality of representation for a given amount of computational effort is examined with the aid of well known theorems from numerical analysis. A choice of nonlinear grid and a representation of the potential expanded in Chebyshev polynomials is shown to be efficient. The strategy was applied to a two-dimensional analytical representation of a transition state and to the ground-state equilibrium geometry region of the Hartree-Fock potential energy surface obtained with split-valence basis sets for HzO, HzS, and H,Se. Results are reported for the equilibrium geometries and force constants for these molecules. Results are comparable to those obtained by others for HzO and H,S. A full set of values is reported for HzSe.
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