Direct metal ion substitution at the [M(SCys)(4)](2-) site of rubredoxin

The single Fe(II) in reduced rubredoxin from Clostridium pasteurianum was f ound to be quantitatively displaced by either Cd2+ or Zn2+ when a modest mo lar excess of the substituting metal salt was anaerobically incubated with the reduced rubredoxin under mild conditions, namely, room temperature, pH 5.4-8.4, and no protein denaturants. Under the same conditions, cadmium-for -zinc substitution was also achieved upon aerobic incubation of the zinc-su bstituted rubredoxin with a modest molar excess of Cd2+. Displacements of F e(II) from the reduced rubredoxin were not observed upon anaerobic incubati on with Ni2+, Co2+, or VO2+ salts, and no reaction with any of the divalent metal ions was observed for the oxidized [Fe(III)] rubredoxin. Fe(II) coul d not be re-inserted into the Zn- or Cd-substituted rubredoxins without res orting to protein denaturation. H-1 and Cd-113 NMR experiments showed that the cadmium-substituted rubredoxin prepared by the non-denaturing substitut ion method retained the pseudotetrahedral M(SCys)(4) coordination geometry and secondary structural elements characteristic of the native rubredoxin, and that "unzipping" of the beta-sheet did not occur during metal substitut ion. Rates of Fe(II) displacement by M2+ (M = Cd or Zn) increased with incr easing M2+/rubredoxin ratio, decreasing pH, and lower ionic strength. The s ubstitution rates were faster for M = Cd than for M = Zn. Rates of Cd2+ sub stitution into a V8A-mutated rubredoxin were significantly faster than for the wild-type protein. The side-chain of Vg is on the protein surface and c lose to the metal-ligating Cys42S gamma at the M(SCys)(4) site. Therefore, the rate-limiting step in the substitution process is suggested to involve direct attack of the [M(SCys)(4)](2-) site by the incoming M2+, without glo bal unfolding of the protein. Implications of these results for metal ion i ncorporation into rubredoxins in vivo are discussed.