The Function and Properties of the Iron−Sulfur Center in Spinach Ferredoxin:Thioredoxin Reductase:  A New Biological Role for Iron−Sulfur Clusters

Abstract

Thioredoxin reduction in chloroplasts is catalyzed by a unique class of disulfide reductases which use a [2Fe-2S]2+/+ ferredoxin as the electron donor and contain an Fe-S cluster as the sole prosthetic group in addition to the active-site disulfide. The nature, properties, and function of the Fe-S cluster in spinach ferredoxin:thioredoxin reductase (FTR) have been investigated by the combination of UV/visible absorption, variable-temperature magnetic circular dichroism (MCD), EPR, and resonance Raman (RR) spectroscopies. The results indicate the presence of an S = 0 [4Fe-4S]2+ cluster with complete cysteinyl-S coordination that cannot be reduced at potentials down to -650 mV, but can be oxidized by ferricyanide to an S = 1/2 [4Fe-4S]3+ state (g = 2.09, 2.04, 2.02). The midpoint potential for the [4Fe-4S]3+/2+ couple is estimated to be +420 mV (versus NHE). These results argue against a role for the cluster in mediating electron transport from ferredoxin (Em = -420 mV) to the active-site disulfide (Em = -230 mV, n = 2). An alternative role for the cluster in stabilizing the one-electron-reduced intermediate is suggested by parallel spectroscopic studies of a modified form of the enzyme in which one of the cysteines of the active-site dithiol has been alkylated with N-ethylmaleimide (NEM). NEM-modified FTR is paramagnetic as prepared and exhibits a slow relaxing, S = 1/2 EPR signal, g = 2.11, 2.00, 1.98, that is observable without significant broadening up to 150 K. While the relaxation properties are characteristic of a radical species, MCD, RR, and absorption studies indicate at least partial cluster oxidation to the [4Fe-4S]3+ state. Dye-mediated EPR redox titrations indicate a midpoint potential of -210 mV for the one-electron reduction to a diamagnetic state. By analogy with the properties of the ferricyanide-oxidized [4Fe-4S] cluster in Azotobacter vinelandii 7Fe ferredoxin [Hu, Z., Jollie, D., Burgess, B. K., Stephens, P. J., & Münck, E. (1994) Biochemistry 33, 14475-14485], the spectroscopic and redox properties of NEM-modified FTR are interpreted in terms of a [4Fe-4S]2+ cluster covalently attached through a cluster sulfide to a cysteine-based thiyl radical formed on one of the active-site thiols. A mechanistic scheme for FTR is proposed with similarities to that established for the well-characterized NAD(P)H-dependent flavin-containing disulfide oxidoreductases, but involving sequential one-electron redox processes with the role of the [4Fe-4S]2+ cluster being to stabilize the thiyl radical formed by the initial one-electron reduction of the active-site disulfide. The results indicate a new biological role for Fe-S clusters involving both the stabilization of a thiyl radical intermediate and cluster site-specific chemistry involving a bridging sulfide.

An Iron-Sulfur Cluster in the C-terminal Domain of the p58 Subunit of Human DNA Primase

“…Like typical HiPIP clusters, the iron-sulfur cluster in p58C is only visible by EPR when oxidized, has an average g-value greater than two, is observed best below 30 K, and the signal does not readily saturate. Although the observed g-values deviate somewhat from the prototypical HiPIP cluster, such differences may be attributable to changes in the environment around the [4Fe-4S] cluster or distortion of the cluster by the protein environment (44,45). In summary, the biophysical data on p58C are consistent with assignment to the class of high potential iron proteins, thus adding to the growing list of DNA-processing proteins with this unique cofactor.…”

Ferredoxin/ferredoxin–thioredoxin reductase complex: Complete NMR mapping of the interaction site on ferredoxin by gallium substitution

“…The [4Fe-4S] cluster is in the 2+ state in purified FTR, and is EPR silent [24]. However, at room temperature low lying excited states are populated rendering the cluster paramagnetic [25].…”

Structural Basis of Redox Signaling in Photosynthesis: Structure and Function of Ferredoxin:thioredoxin Reductase and Target Enzymes

“…In analogy with known mechanisms, the reduction of thioredoxin proceeds via a mixed disulfide between thioredoxin and FTR (Staples et al 1996(Staples et al , 1998. Such an intermediate complex would cover one of the sides of the flat FTR molecule and the second electron for the reduction should be delivered by the next incoming ferredoxin, which has to dock on the opposite side of the flat disk-like heterodimer.…”