NMR DETERMINATION OF THE GLOBAL STRUCTURE OF THE CD-113 DERIVATIVE OFDESULFOREDOXIN - INVESTIGATION OF THE HYDROGEN-BONDING PATTERN AT THEMETAL CENTER

Desulforedoxin (Dr) is a simple homodimeric protein isolated from Desu lfovibrio gigas (Dg) containing a distorted rubredoxin-like center wit h one iron coordinated by four cysteinyl residues (7.9 kDa with 36 ami no acids per monomer). In order to probe the geometry and the H-bondin g at the active site of 2r, the protein was reconstituted with Cd-113 and the solution structure determined using 2D NMR methods, The struct ure of this derivative was initially compared with the NMR solution st ructure of the Zn form (Goodfellow BJ et al., 1996, J Biol Inorg Chern 1:341-353). Backbone amide protons for G4, D5, G13, LII NH, and the Q 14 NH side-chain protons, H-bonded in the X-ray structure, were readil y exchanged with solvent. Chemical shift differences observed for amid e protons near the metal center confirm the H-bonding pattern seen in the X-ray model (Archer M et al., 1995, J Mol Biol 251:690-702) and al so suggest that H-bond lengths may vary between the Fc, Zn, and Cd-113 forms. The H-bonding pattern was further probed using a heteronuclear spin echo difference (HSED) experiment; the results confirm the prese nce of NH-S H-bonds inferred from D2O exchange data and observed in th e NMR family of structures. The presence of ''H-bond mediated'' coupli ng in Dr indicates that the NH-S H-bonds at the metal center have sign ificant covalent character The HSED experiment also identified an inte rmonomer ''through space'' coupling for one of the L26 methyl groups, indicating its proximity to the Cd-113 center in the opposing monomer. This is the first example of an intermonomer ''through space'' coupli ng. Initial structure calculations produced subsets of NMR families wi th the S of C28 pointing away from or toward the L26 methyl: only the subset with the C28 sulfur pointing reward the L26 methyl could result in a ''through space'' coupling. The HSED result was therefore includ ed in the structure calculations. Comparison of the Fe, Zn, and Cd-113 forms of Dr suggests that the geometry of the metal center and the gl obal fold of the protein does not vary to any great extent, although t he I-I-bond network varies slightly when Cd is introduced, The similar ity between the I-I-bonding pattern seen at the metal center in Dr, Rd (including H-bonded and through space-mediated coupling), and many zi nc-finger proteins suggests that these H-bonds are structurally vital for stabilization of the metal centers in these proteins.