REVERSIBLE AND NONDENATURING 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 reduce d protein, isolated after incubation with different metals under ident ical conditions (50 mu M protein, 1 mM metal, 1 h incubation) was foun d to contain amounts of foreign metals increasing with their thiophyli city, i.e. Cd2+ much greater than Zn2+ > Co2+. Little, if any, incorpo ration was observed for Ni2+, Cu2+, Mn2+ or in the absence of reductan t. The activity of substituted ferredoxins in a hydrogenase-coupled as say was proportional to the amount of residual iron, suggesting that t he residual iron is present in a population of intact active molecules rather than in partially substituted clusters distributed among indiv idual molecules. The cadmium-substituted ferredoxin did not contain ir on, but contained eight cadmium atoms and six labile sulfide atoms/mol . Folding of the isolated, substituted proteins was investigated by CD and H-1-NMR. Both techniques showed retention of the main structural features of the protein upon metal substitution. The rate and extent o f the substitution of iron by cadmium were essentially independent of pH, but were found to decrease with increasing ionic strength and to i ncrease with the cadmium concentration. In the cadmium-substituted pro tein, cadmium was replaced by iron upon incubation with iron and merca ptoethanol in the absence of dithionite. In the presence of dithionite , cadmium was not replaced by iron upon incubation of the cadmium-subs tituted protein with excess iron and mercaptoethanol. In competition e xperiments, incubation of iron-containing ferredoxin with stoichiometr ic amounts of cadmium in the presence of dithionite and excess iron an d mercaptoethanol resulted in quantifiable replacement of iron by cadm ium. Therefore, substitution of iron by cadmium was only achieved unde r reducing conditions, and was only reversible in the absence of stron g reductants.