Spectroscopic and Redox Studies of Valence-Delocalized [Fe<sub>2</sub>S<sub>2</sub>]<sup>+</sup> Centers in Thioredoxin-like Ferredoxins SubramanianSowmya DuinEvert C. E. J. FawcettSarah ArmstrongFraser A. MeyerJacques JohnsonMichael K. 2015 Reduced forms of the C56S and C60S variants of the thioredoxin-like <i>Clostridium pasteurianum</i> [Fe<sub>2</sub>S<sub>2</sub>] ferredoxin (<i>Cp</i>Fd) provide the only known examples of valence-delocalized [Fe<sub>2</sub>S<sub>2</sub>]<sup>+</sup> clusters, which constitute a fundamental building block of all higher nuclearity Fe–S clusters. In this work, we have revisited earlier work on the <i>Cp</i>Fd variants and carried out redox and spectroscopic studies on the [Fe<sub>2</sub>S<sub>2</sub>]<sup>2+,+</sup> centers in wild-type and equivalent variants of the highly homologous and structurally characterized <i>Aquifex aeolicus</i> ferredoxin 4 (<i>Aae</i>Fd4) using EPR, UV–visible–NIR absorption, CD and variable-temperature MCD, and protein–film electrochemistry. The results indicate that the [Fe<sub>2</sub>S<sub>2</sub>]<sup>+</sup> centers in the equivalent <i>Aae</i>Fd4 and <i>Cp</i>Fd variants reversibly interconvert between similar valence-localized <i>S</i> = 1/2 and valence-delocalized <i>S</i> = 9/2 forms as a function of pH, with p<i>K</i><sub>a</sub> values in the range 8.3–9.0, because of protonation of the coordinated serinate residue. However, freezing high-pH samples results in partial or full conversion from valence-delocalized <i>S</i> = 9/2 to valence-localized <i>S</i> = 1/2 [Fe<sub>2</sub>S<sub>2</sub>]<sup>+</sup> clusters. MCD saturation magnetization data for valence-delocalized <i>S</i> = 9/2 [Fe<sub>2</sub>S<sub>2</sub>]<sup>+</sup> centers facilitated determination of transition polarizations and thereby assignments of low-energy MCD bands associated with the Fe–Fe interaction. The assignments provide experimental assessment of the double exchange parameter, <i>B</i>, for valence-delocalized [Fe<sub>2</sub>S<sub>2</sub>]<sup>+</sup> centers and demonstrate that variable-temperature MCD spectroscopy provides a means of detecting and investigating the properties of valence-delocalized <i>S</i> = 9/2 [Fe<sub>2</sub>S<sub>2</sub>]<sup>+</sup> fragments in higher nuclearity Fe–S clusters. The origin of valence delocalization in thioredoxin-like ferredoxin Cys-to-Ser variants and Fe–S clusters in general is discussed in light of these results.