1H NMR Investigation of the Paramagnetic Cluster Environment in Pyrococcus furiosus Three-Iron Ferredoxin: Sequence-Specific Assignment of Ligated Cysteines Independent of Tertiary Structure

Abstract

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.

Spectroscopic Investigation of Selective Cluster Conversion of Archaeal Zinc-containing Ferredoxin fromSulfolobus sp. Strain 7

“…ferredoxin suggested splitting into two peaks, a1 and a2, at 30°C (Fig. 7E), as previously observed for the downfieldshifted resonances at around 24.5 ppm of the 3Fe form of P. furiosus monocluster ferredoxin (63)(64)(65). In addition, the new signal a1 at 24.0 -24.6 ppm showed Curie-type temperature dependence (Fig.…”

Investigation of exchange couplings in [Fe3S4]+ clusters by electron spin-lattice relaxation

“…However, the non-zero values for a 2 show that the common [82,83] assumption of equivalent exchange (J 12 = J 13 ; e9/e = 0) is somewhat of an oversimplification. This observation is of significance in refining paramagnetic NMR data [31].…”

“…In addition, Av-Fd I, Dg-Fd II, and aconitase have all been studied in detail by Mössbauer spectroscopy [11, 48±52]. Pf-Fd 3Fe has been studied by ENDOR [44] and by NMR [30,31], and mutants of this protein have been recently studied by EPR and MCD. Dg-Fd II has also been studied by NMR [34,51] and by saturation magnetization measurements [53].…”

1H NMR investigation of the secondary structure, tertiary contacts and cluster environment of the four-iron ferredoxin from the hyperthermophilic archaeon Thermococcus litoralis

“…In the absence of any suitable crystals, we have pursued ~H NMR characterization of the molecular and electronic structures of Pfand Tl Fds in both the 3Fe and 4Fe forms (Busse et al, 1992;Donaire et al, 1994;Teng et al, 1994;Gorst et al, 1995a). The molecules are sufficiently small to allow high-resolution NMR structural studies.…”

“…However, the paramagnetic ground states ([Fe 4-$4] +~, [FesS4] ~ and the extensive population of paramagnetic excited states ([Fe4S4] § at ambient temperatures (Poe et al, 1970;Cammack, 1992;Luchinat and Ciurli, 1993) induce effective relaxation, which severely complicates, but does not necessarily obviate, the use of appropriately tailored 1D and 2D NMR methods. Subsequent NMR studies, optimized for detecting the paramagnetically influenced cluster environment including the ligated cysteines, led to the proposal that, in contrast to previous approaches (Bertini et al, 1994;Donaire et al, 1994), the needed unambiguous assignments of the ligated cysteines can be obtained without any assumption regarding the tertiary structure of the protein (Gorst et al, 1995a). Subsequent NMR studies, optimized for detecting the paramagnetically influenced cluster environment including the ligated cysteines, led to the proposal that, in contrast to previous approaches (Bertini et al, 1994;Donaire et al, 1994), the needed unambiguous assignments of the ligated cysteines can be obtained without any assumption regarding the tertiary structure of the protein (Gorst et al, 1995a).…”