The solution structure of desulforedoxin, a simple iron-sulfur protein - An NMR study of the zinc derivative

The solution structure of desulforedoxin, a simple iron-sulfur protein - An NMR study of the zinc derivative, Goodfellow, B. J., Tavares P., Romao M. J., Czaja C., Rusnak F., Legall J., Moura I., and Moura J. J. G. , Journal of Biological Inorganic Chemistry, Aug, Volume 1, Number 4, p.341-354, (1996)


Desulforedoxin is a simple dimeric protein isolated from Desulfovibrio gigas containing a distorted rubredoxin-like center with one iron coordinated by four cysteinyl residues (7.9 kDa with a 36-amino-acid monomer). H-1 NMR spectra of the oxidized Dx(Fe3+) and reduced Dx(Fe2+) forms were analyzed. The spectra show substantial line broadening due to the paramagnetism of iron. However, very low-field-shifted resonances, assigned to H beta protons, were observed in the reduced state and their temperature dependence analyzed. The active site of Dx was reconstituted with zinc, and its solution structure was determined using 2D NMR methods. This diamagnetic form gave high-resolution NMR data enabling the identification of all the amino acid spin systems. Sequential assignment and the determination of secondary structural elements was attempted using 2D NOESY experiments. However, because of the symmetrical dimer nature of the protein standard, NMR sequential assignment methods could not resolve all cross peaks due to inter- and intra-chain effects. The X-ray structure enabled the spatial relationship between the monomers to be obtained, and resolved the assignment problems. Secondary structural features could be identified from the NMR data; an antiparallel beta-sheet running from D5 to V18 with a well-defined beta-turn around cysteines C9 and C12. The section G22 to T25 is poorly defined by the NMR data and is followed by a turn around V27-C29. The C-terminus ends up near residues V6 and Y7. Distance geometry (DG) calculations allowed families of structures to be generated from the NMR data. A family of structures with a low target function violation for the Dr monomer and dimer were found to have secondary structural elements identical to those seen in the X-ray structure. The amide protons for G4, D5, G13, L11 NH and Q14 NH epsilon amide protons, H-bonded in the X-ray structure, were not seen by NMR as slowly exchanging, while structural disorder at the N-terminus, for the backbone at E10 and for the section G22-T25, was observed. Comparison between the Fe and Zn forms of Dr suggests that metal substitution does not have an effect on the structure of the protein.


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