600 zyxwvutsrqpo Biochemistry zyxwvut 1995, 34, 600-610 lH NMR Investigation of the Paramagnetic Cluster Environment in zyx Pyrococcus furiosus Three-Iron Ferredoxin: Sequence-Specific Assignment of Ligated Cysteines Independent of Tertiary Structure? Carol M. Gorst,+ You-Hsing Yeh,$ Quincy Teng,$,@ Luigi Calzolai,*sll Zhi-H. Zhou,l Michael zyxw W. W. Adams,l and Gerd N. La Mar*,$ Department of Chemistry, University zyxwvut of Califomia, Davis, Califomia 9561 6 and Department of Biochemistry, University of Georgia, Athens, Georgia 30602 Received August 26, 1994; Revised Manuscript Received November 4, 1994@ ABSTRACT: One- and two-dimensional 'H NMR data tailored to detect paramagnetically relaxed protons near the S zyxwvutsrqp = V2, three-iron-sulfur cluster of the ferredoxin from the hyperthennophile Pyrococcusfuriosus 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, 6316-63261. 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@) 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 zyxw NH and strongly relaxed C,H signals, while the two central Cys in that sequence will exhibit strongly relaxed peptide NH but weakly relaxed C,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 Cyss6 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, 693-7151. 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. Ferredoxins (Fds)' are small electron transfer proteins that contain one or two iron-sulfur clusters as redox center(s). The cluster architectures fall into two main classes, the two- iron or plant-type Fd which exhibit only localized valence (Le., 2Fe3+ for [Fe2S2I2+ in Fdox and 1Fe3+;1Fe2+ for [FezSz]'+ in Fdrd), and the cubane or bacterial-type Fd which This research was supported by grants from the National Science Foundation, DMB 90-04018 (G.N.L.), DMB 91-05150 (M.W.W.A.), and the National Institutes of Health, GM 45597 (M.W.W.A.) * Address correspondence to this author. *Department of Chemistry, University of Califomia, Davis, CA * Present address: Department of Chemistry,University of Georgia, I' Present address: Department of Chemistry, University of Siena, Department of Biochemistry, University of Georgia, Athens, GA @ Abstract published zyxwvutsrq in Advance ACS Abstracts, December 15, 1994. Abbreviations: Fd, ferredoxin; Hipip, high-potential iron-sulfur protein; PJ Pyrococcus furiosus; Dg, Desulfovibrio gigas; Pa, Pepto- coccus aerogenes; Bt, Bacillus thermoproteolyticus; Av, Azotobacter vinlandii; Cp, Clostridiumpasteurianum; Cau, Clostridium acidi unci; DSS, 2,2'-dimethyl-2-silapentane-5-sulfonate; ppm, parts per million; NOE, nuclear Overhauser effect; NOESY, 2D nuclear Overhauser spectroscopy; TOCSY, 2D total correlation spectroscopy; NMR, nuclear magnetic resonance. 95616. Athens, GA 30602. Siena, Italy. 30602. exhibit valence delocalization over pairs of iron in both their four-iron (Le., 4Fe2.5+ in [Fe4S4I2+ for Fd", 2Fe2,5+; 2Fe2+ in [Fe&]'+ for Fdred) and three-iron (3Fe3+ in [Fe3S4]'+ for Fd", 2Fe2.5+;1Fe3+ in [Fe3S4Io for Fdd) forms (Cammack et ai., 1977; Thompson, 1985; Beinert, 1990; Howard & Rees, 1991; Cammack, 1992; Munck et al., 1988). A schematic structure of a cubane cluster is illustrated in Figure 1. The related, but more oxidizing cubane cluster of high- potential iron-sulfur proteins, Hipip, exhibit the same ground state for Hipip'& as 4Fe FdoX, but with 2Fe2,5+; 2Fe3+ in for Hipipox(Cammack et al., 1977). The cluster environment therefore is modulated strongly by the protein matrix not only to vary the overall cluster redox potential but also to differentiate the redox properties of individual or pairs of iron atoms within the cluster. While the tendency for the cubane clusters to exhibit asymmetry appears to be an intrinsic property of even the symmetrical model com- pounds (Holm, 1977; Bominaar et al., 1994), this distortion is "locked in" by the asymmetric protein matrix in a fashion likely dictated by the need to control the flow of electrons (Langen et al., 1992). The mechanism by which the cluster environment modulates the location of the reducing electron may become clear when both the detailed molecular structure 0006-2960/95/0434-600$09.00/0 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA 0 1995 American Chemical Society