Reversible and non-denaturing replacement of iron by cadmium in Clostridium pasteurianum ferredoxin

Incubation of native, reduced Clostridium pasteurianum ferredoxin with different metals gave a range of modifications in the electronic and EPR spectrum of the protein, or made the signals disappear. The reduced protein, isolated after incubation with different metals under identical conditions (50 microM protein, 1 mM metal, 1 h incubation) was found to contain amounts of foreign metals increasing with their thiophylicity, i.e. Cd2+ >> Zn2+ > Co2+. Little, if any, incorporation was observed for Ni2+, Cu2+, Mn2+ or in the absence of reductant. The activity of substituted ferredoxins in a hydrogenase-coupled assay was proportional to the amount of residual iron, suggesting that the residual iron is present in a population of intact active molecules rather than in partially substituted clusters distributed among individual molecules. The cadmium-substituted ferredoxin did not contain iron, but contained eight cadmium atoms and six labile sulfide atoms/mol. Folding of the isolated, substituted proteins was investigated by CD and 1H-NMR. Both techniques showed retention of the main structural features of the protein upon metal substitution. The rate and extent of the substitution of iron by cadmium were essentially independent of pH, but were found to decrease with increasing ionic strength and to increase with the cadmium concentration. In the cadmium-substituted protein, cadmium was replaced by iron upon incubation with iron and mercaptoethanol in the absence of dithionite. In the presence of dithionite, cadmium was not replaced by iron upon incubation of the cadmium-substituted protein with excess iron and mercaptoethanol. In competition experiments, incubation of iron-containing ferredoxin with stoichiometric amounts of cadmium in the presence of dithionite and excess iron and mercaptoethanol resulted in quantifiable replacement of iron by cadmium. Therefore, substitution of iron by cadmium was only achieved under reducing conditions, and was only reversible in the absence of strong reductants.