Disentangling First and Second Sphere Effects in Iron–Sulfur Cubanes

Cubane-type iron-sulfur clusters (Fe₄S₄) are some of the most versatile metallocofactors and, as such, among multiple functions, primarily responsible for mediating challenging electron transfers (ETs). Their efficient ET chemistry is enabled by a conflated interplay of cofactor-protein interactions, which can be categorized into the covalent first (1°) sphere ones and the noncovalent second (2°) sphere ones. The latter have remained particularly elusive, as they are difficult to observe and assess directly and independently. Accordingly, our understanding of these effects is hampered by their entangled nature. To address this, we herein leverage a systematic series of synthetic Fe₄S₄ complexes, which allows spectroscopically investigating 2° sphere electrostatic interactions and covalent 1° sphere interactions separately from one another. We expand the study of 1° sphere interactions with a histidine-type ligand in [Fe₄S₄]¹⁺ complexes to the [Fe₄S₄]²⁺ and [Fe₄S₄]³⁺ oxidation states, supporting the notion that 1° sphere interactions "fine-tune" the electronic/magnetic structure of these systems in a manner that persists at ambient temperatures. In contrast, scrutinizing the 2° sphere electric dipolar interactions in [Fe₄S₄]^1+,2+,3+ complexes revealed that although similar effects are observable at extremely low temperatures, no significant alteration of the clusters' gross electronic/magnetic structure persists at the temperatures relevant to enzyme function. These results thus not only systematically catalogue the influence of 1° sphere covalent and 2° sphere electrostatic interactions on the observables and properties of Fe₄S₄ complexes, but also establish a clear energetic distinction between the two. As such, they will facilitate identifying the elusive 2° sphere interactions in biological systems, while also strengthening our biophysical understanding of structure-function relationships in Fe₄S₄ cofactors.