Spectroscopic and Redox Studies of Valence-Delocalized [Fe₂S₂]⁺ Centers in Thioredoxin-like Ferredoxins

Reduced forms of the C56S and C60S variants of the thioredoxin-like Clostridium pasteurianum [Fe₂S₂] ferredoxin (CpFd) provide the only known examples of valence-delocalized [Fe₂S₂]⁺ clusters, which constitute a fundamental building block of all higher nuclearity Fe–S clusters. In this work, we have revisited earlier work on the CpFd variants and carried out redox and spectroscopic studies on the [Fe₂S₂]^2+,+ centers in wild-type and equivalent variants of the highly homologous and structurally characterized Aquifex aeolicus ferredoxin 4 (AaeFd4) using EPR, UV–visible–NIR absorption, CD and variable-temperature MCD, and protein–film electrochemistry. The results indicate that the [Fe₂S₂]⁺ centers in the equivalent AaeFd4 and CpFd variants reversibly interconvert between similar valence-localized S = 1/2 and valence-delocalized S = 9/2 forms as a function of pH, with pK_a 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 S = 9/2 to valence-localized S = 1/2 [Fe₂S₂]⁺ clusters. MCD saturation magnetization data for valence-delocalized S = 9/2 [Fe₂S₂]⁺ 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, B, for valence-delocalized [Fe₂S₂]⁺ centers and demonstrate that variable-temperature MCD spectroscopy provides a means of detecting and investigating the properties of valence-delocalized S = 9/2 [Fe₂S₂]⁺ 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.