Metal binding to the tetrathiolate motif of desulforedoxin and related polypeptides

Desulforedoxin and the N-terminus of desulfoferrodoxin share a 36 amino aci d domain containing a (Cys-S)(4) metal binding site. Recombinant forms of d esulforedoxin, an N-terminal fragment of desulfoferrodoxin, and two desulfo redoxin mutant proteins were reconstituted with Fe3+ Cd2+, and Zn2+ and rel ative metal ion affinities assessed by proton titrations. Protons compete w ith metal for protein ligands, a process that can be followed by monitoring the optical spectrum of the metal-protein complex as a function of pH. For all polypeptides, Fe3+ bound with the highest affinity, whereas the affini ty of Zn2+ was greater than Cd2+ in desulforedoxin and the N-terminal fragm ent of desulfoferrodoxin, but this order was reversed in desulforedoxin mut ant proteins. Metal binding in both mutants was significantly impaired. Fur thermore, the Fe3+ complex of both mutants underwent a time-dependent bleac hing process which coincided with increased reactivity of cysteine residues to Ellman's reagent and concomitant metal dissociation. It is hypothesized that this results from an autoredox reaction in which Fe3+ is reduced to F e2+ with attendant oxidation of ligand thiols.