INVESTIGATION OF METAL-ION UPTAKE REACTIVITIES OF [3FE-4S] CLUSTERS IN PROTEINS - VOLTAMMETRY OF COADSORBED FERREDOXIN AMINOCYCLITOL FILMS AT GRAPHITE-ELECTRODES AND SPECTROSCOPIC IDENTIFICATION OF TRANSFORMED CLUSTERS

Facile transformation of Fe-S clusters in proteins, as described by [3Fe-4S{0 + M2+ reversible [M3Fe-4S]2+ in which metal ion M enters the vacant subsite of a 3Fe cluster to complete a cubane-type structure, is identified and studied by a convenient and highly economical voltammetric procedure. The technique extends a recent discovery that ferredoxins coadsorb with aminocyclitols at a pyrolytic graphite ''edge- (PGE) electrode, giving a stable electroactive film, and is demonstrated by an investigation of cluster interconversions in Desulfovibrio africanus ferredoxin Ill (Fd 111). Cyclic voltammetric scanning (typically over the region 0 to -850 mV vs SHE) of a preformed film, transferred to an EGTA-containing buffer solution at pH 7, reveals well-defined voltammetric signals due to three redox couples. One of these (B') corresponds to the stable [4Fe-4S]2+/+ cluster; the other two (A' and C') are associated with the [3Fe-4S] cluster and assigned respectively as the normal 1+/0 couple and a chemically reversible two-electron process of as yet unestablished formulation. If the coated electrode is then transferred to stirred solutions devoid of EGTA but containing low concentrations of Fe2+, Zn2+, or Cd2+, reductive passage through couple A' ([3Fe-4S]+/0) initiates rapid changes. During subsequent cycles over the course of several seconds, waves A' and C' disappear simultaneously and are replaced by a new couple D'(M). Values of E-degrees' are as follows: D'(Fe), -393 +/- 10 mV; D'(Zn), -492 +/- 10 mV; D'(cd), -569 +/- 10 mV. The positions of the new couples correspond closely with cyclic voltammograms of Fd III undergoing transformations in the solution phase. Characterization by EPR and MCD spectroscopy identifies the latter species to be [4Fe-4S]2+/+, [Zn3Fe-4S]2+/+, and [Cd3Fe-4S]2+/+, respectively. The clusters [Zn3Fe-4S]2+ and [Cd3Fe-4S]2+ are shown to be isoelectronic with [3Fe-4S]0 and to have a ground electronic state S = 2 subject to a negative axial zero-field splitting. The reduced states [Zn3Fe-4S]+ and [Cd3Fe-4S]+ have a ground state spin S = 5/2 with characteristic EPR spectra. Rates and equilibria of metal ion uptake for protein molecules confined to the electrode surface depend upon the identity and concentration of the metal ion. Values of K(d), the equilibrium dissociation constant given by {M2+}{3Fe-4S]0}/{[M3Fe-4S]2+} are as follows: Fe, 30 +/- 15-mu-M; Zn, 1.6 +/- 1.0-mu-M; Cd, 0.8 +/- 0.5-mu-M. The affinity order Cd2+ greater-than-or-equal-to Zn2+ >> Fe2+ is thus established. The results demonstrate the feasibility of an intrinsic preference for Zn (a biologically abundant element) as compared to Fe, at the M subsite of [M3Fe-4S] clusters in proteins.