Characterization of rhodanese-tetracyanonickelate. An active site complex that slows sulfur-free rhodanese conversion to inert conformers.
The structure of the rhodanese-tetracyanonickelate (E X Ni(CN)2-4) 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 X Ni(CN)2-4 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 (Cm) were: Cm = 3.0 M for activity measurement; Cm = 2.8 M for protein intrinsic fluorescence intensity; Cm = 4.3 M for ellipticity at 220 nm; and Cm = 3.3 M for wavelength of fluorescence emission maximum. For the E X Ni(CN)2-4 complex, Cm was shifted to a higher urea concentration relative to that found for E when activity (Cm = 3.6 M) and native protein fluorescence (Cm = 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)2-4 binding on E slowed enzyme inactivation that is associated with structural relaxations. These findings, that Ni(CN)2-4 affects structural relaxations in rhodanese, are of particular interest in light of the recent suggestion that the E X Ni(CN)2-4 complex mimics a normally inaccessible intermediate in catalysis.
Chow
S F
SF
Horowitz
P
P
D001665:Binding Sites; D002942:Circular Dichroism; D003486:Cyanides; D004305:Dose-Response Relationship, Drug; D005470:Fluorometry; D009532:Nickel; D011487:Protein Conformation; D013056:Spectrophotometry, Ultraviolet; D013438:Sulfhydryl Compounds / Q000032:analysis; D013466:Sulfurtransferases; D013884:Thiosulfate Sulfurtransferase; D014461:Ultracentrifugation; D014508:Urea / Q000494:pharmacology
D003486:Cyanides; D013438:Sulfhydryl Compounds; C047503:tetracyanonickelate; D009532:Nickel; D014508:Urea; D013466:Sulfurtransferases; D013884:Thiosulfate Sulfurtransferase