Electrochemical Potential and pH Dependences of [3Fe-4S] ↔ [M3Fe-4S] Cluster Transformations (M = Fe, Zn, Co, and Cd) in Ferredoxin III from Desulfovibrio africanus and Detection of a Cluster with M = Pb

Abstract

The 7Fe ferredoxin from Desulfovibrio africanus contains a reactive 3Fe cluster which incorporates Fe and other metals to form cubanes [M3Fe-4S] having non-cysteine ligation to the added metal (M). These reactions are addressed at controlled potentials in protein molecules adsorbed on a graphite electrode, thereby facilitating an appraisal of factors that control cluster interconversions. Electrochemical and spectroscopic methods have been used to establish connectivities among interconversions with different M, explore the influence of pH, and determine reactivities of specific cluster oxidation levels. Formation of clusters with M = Fe, Zn, and Co depends on ionization of a group with pK = 5.5 + 0.3, thus supporting congruence among products and suggesting common involvement of aspartate-14 which replaces a cysteine normally present in [4Fe-4S] binding motifs. The influence of potential is complex: rapid and reversible interconversion (M = Fe, Zn) occurs only between the states [M3Fe-4S]2+ and [3Fe-4S]0, with [3Fe-4S]1+ having little affinity for M. The [M3Fe-4S]1+ cubanes and hyperreduced [3Fe-4S]2- are relatively unreactive. Uptake and release are significantly more rapid for M = Zn compared to Fe. Among potentially intrusive metals, Pb has a particularly high affinity for the [3Fe-4S] cluster, but the product undergoes subsequent irreversible reactions. The studies provide complementary perspectives on factors influencing cluster stability or reactivity and on the feasibility and consequences of metal substitutions.

Solution NMR Structure of the Iron–Sulfur Cluster Assembly Protein U (IscU) with Zinc Bound at the Active Site

“…It has also been demonstrated for the [Fe-S] cluster binding protein, ferredoxin, that Zn 2C can be incorporated into these clusters. 49 Therefore, the possibility that zinc-ligation of IscU plays a physiological role cannot be ruled out. Based on the structural similarity with SufE and YgdK, the zinc-bound IscU structure appears to be a good structural model for a stabilized form of IscU.…”

Ferredoxin III of Desulfovibrio africanus: sequencing of the native gene and characterization of a histidine-tagged form

“…The pH dependence of the three redox couples measured over the pH range 5-8 was essentially identical to that of native Da FdIII [12] ; i.e. signals Ah and Bh only vary slightly with pH, whereas signal Ch shows strong pH-dependent behaviour, consistent with the two-electron reduction of [3Fe-4S]!…”

“…FdIII has been demonstrated for M l Fe(II), Zn(II), Cd(II), Tl(I), Cu(I), Co(II) and Pb(II), using both spectroscopic studies and direct electrochemical methods [3,11,12]. When Da FdIII is adsorbed as a film on a pyrolytic graphite edge (PGE) electrode, it is capable of responding rapidly to changes in solution conditions and displays facile cluster conversion.…”

“…When Da FdIII is adsorbed as a film on a pyrolytic graphite edge (PGE) electrode, it is capable of responding rapidly to changes in solution conditions and displays facile cluster conversion. This suggests strongly that the transformations occurring in this particular protein reflect intrinsic properties of the cluster species, rather than extrinsic (protein-structure-based) factors such as steric hindrance or inappropriate positioning of its ligand(s) [12]. In Da FdIII the product of the reaction of the [3Fe-4S]" + core with Fe(II) is a [4Fe-4S]# +/"+ cluster, with a reduced form which has an unusual electronic ground state with spin S l 3\2.…”

Spectroscopic Investigation of Selective Cluster Conversion of Archaeal Zinc-containing Ferredoxin fromSulfolobus sp. Strain 7

“…Site-directed mutagenesis has suggested that formation of the latter species requires the presence of non-ligating Cys 24 in the vicinity of a cluster sulfide (33). The difference between the oxidative degradation of ferredoxin from A. vinelandii and Sulfolobus sp.…”