Application of NMRD to Hydration of rubredoxin and a variant containing a (Cys-S)3FeIII(OH) site

Hydration of oxidized rubredoxin (FeIII(S-Cys)4 center) was investigated by 1H and 17O relaxation measurements of bulk water as a function of the applied magnetic field (nuclear magnetic relaxation dispersion). Oxidized rubredoxin showed an increased water 1H relaxation profile with respect to the diamagnetic gallium derivative or reduced species. Analysis of the data shows evidence of exchangeable proton(s) ;4.0–4.5 A˚ from the metal ion, the exchange time being longer than 1010 s and shorter than 105 s. The correlation time for the proton-electrons interaction is 7 3 1011 s and is attributed to the effective electron relaxation time. Its magnitude is consistent with the large signal linewidths of the protein donor nuclei, observed in high resolution NMR spectra. For reduced rubredoxin, such correlation time is proposed to be smaller than 1011 s. 17O relaxation measurements suggest the presence of at least one long-lived protein-bound water molecule. Analogous relaxation measurements were performed on the C6S rubredoxin variant, whose iron(III) center has been previously shown to be coordinated to three cysteine residues and a hydroxide ion above pH 6. 1H nuclear magnetic relaxation dispersion profiles indicate increased hydration with respect to the wild-type.