NMR determination of the global structure of the ¹¹³Cd derivative of desulforedoxin: Investigation of the hydrogen bonding pattern at the metal center

Abstract

Desulforedoxin (Dx) is a simple homodimeric protein isolated from Desulfovibrio gigas (Dg) containing a distorted rubredoxin‐like center with one iron coordinated by four cysteinyl residues (7.9 kDa with 36 amino acids per monomer). In order to probe the geometry and the H‐bonding at the active site of Dx, the protein was reconstituted with 113Cd and the solution structure determined using 2D NMR methods. The structure of this derivative was initially compared with the NMR solution structure of the Zn form (Goodfellow BJ et al., 1996, J Biol Inorg Chem 1:341‐353). Backbone amide protons for G4, D5, G13, L11 NH, and the Q14 NH side‐chain protons, H‐bonded in the X‐ray structure, were readily exchanged with solvent. Chemical shift differences observed for amide 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 also suggest that H‐bond lengths may vary between the Fe, Zn, and 113Cd forms. The H‐bonding pattern was further probed using a heteronuclear spin echo difference (HSED) experiment; the results confirm the presence of NH‐S H‐bonds inferred from D2O exchange data and observed in the NMR family of structures. The presence of “H‐bond mediated” coupling in Dx indicates that the NH‐S H‐bonds at the metal center have significant covalent character. The HSED experiment also identified an intermonomer “through space” coupling for one of the L26 methyl groups, indicating its proximity to the 113Cd center in the opposing monomer. This is the first example of an intermonomer “through space” coupling. Initial structure calculations produced subsets of NMR families with the S of C28 pointing away from or toward the L26 methyl: only the subset with the C28 sulfur pointing toward the L26 methyl could result in a “through space” coupling. The HSED result was therefore included in the structure calculations. Comparison of the Fe, Zn, and 113Cd forms of Dx suggests that the geometry of the metal center and the global fold of the protein does not vary to any great extent, although the H‐bond network varies slightly when Cd is introduced. The similarity between the H‐bonding pattern seen at the metal center in Dx, Rd (including H‐bonded and through space‐mediated coupling), and many zinc‐finger proteins suggests that these H‐bonds are structurally vital for stabilization of the metal centers in these proteins.

Structure of the 113Cd3β domains from Homarus americanus metallothionein-1: hydrogen bonding and solvent accessibility of sulfur atoms

“…The values obtained for 2h J HN-Cd coupling constants fall in the range 0.3-1.1 Hz (Table 2). These values are of similar magnitude to ones reported previously for rubredoxin and desulfodextrin [23,24,37]. A comparison of the coupling constants with the corresponding NHAEAEAES distances indicates that there is no direct correlation between the magnitude of 2h J HN-Cd and NHAEAEAES bond length.…”

Dynamics and Metal Exchange Properties of C4C4 RING Domains from CNOT4 and the p44 Subunit of TFIIH

“…18 For amide groups in such hydrogen bonds, 48 chemical shift changes presumably result from small changes in the hydrogen bond lengths caused by the increase in metal-sulphur bond length. 49,50 Smaller chemical shift changes may reflect minor structural rearrangements.…”

Cadmium – glutathione solution structures provide new insights into heavy metal detoxification

“…Nevertheless, the nature of the metal binding in the case of GSH remains subject to debate, as cadmium has been proposed to link to amide [15], carboxylates of both glycine [13,14] and glutamate residues [13,14,16] or the amine NH 2 lone pair [11,13,16]. 113 Cd NMR [12] was used to characterize this interaction, but without success, in contrast to many 113 Cd–protein experiments [17–19]. Simulation of theoretical chemical shifts [20,21] or EXAFS experiments [22] were also performed to analyse the cadmium(II) sphere of co‐ordination.…”

“…Cd NMR [12] was used to characterize this interaction, but without success, in contrast to many 113 Cd-protein experiments [17][18][19]. Simulation of theoretical chemical shifts [20,21] or EXAFS experiments [22] were also performed to analyse the cadmium(II) sphere of co-ordination.…”