Characterization of rhodanese-tetracyanonickelate. An active site complex that slows sulfur-free rhodanese conversion to inert conformers

The structure of the rhodanese-tetracyanonickelate (E·Ni(CN)₄^2-) complex has been characterized here in spectral and physical studies using urea as a structural perturbant. UV difference absorption, sedimentation velocity ultracentrifugation, fluorescence, and circular dichroism data show no significant conformational differences between sulfur-free rhodanese (E) and the E·Ni(CN)₄^2- complex. The urea-induced enzyme structural transition curves were noncoincident when different structural parameters were monitored. For E, the urea concentrations giving half-maximal change (C(m)) were: C(m) = 3.0 M for activity measurement; C(m) = 2.8 M for protein intrinsic fluorescence intensity; C(m) = 4.3 M for ellipticity at 220 nm; and C(m) = 3.3 M for wavelength of fluorescence emission maximum. For the E·Ni(CN)₄^2- complex, C(m) was shifted to a higher urea concentration relative to that found for E when activity (C(m) = 3.6 M) and native protein fluorescence (C(m) = 3.6 M) were the measured parameters but not when the wavelength of the emission maximum and ellipticity were monitored. Furthermore, urea-induced rhodanese structural changes were time-dependent and Ni(CN)₄^2- binding on E slowed enzyme inactivation that is associated with structural relaxations. These findings, that Ni(CN)₄^2- affects structural relaxations in rhodanese, are of particular interest in light of the recent suggestion that the E·Ni(CN)₄^2- complex mimics a normally inaccessible intermediate in catalysis.