Fundamental Electrostatics Explains Why Ferredoxin- and HiPIP-Type Proteins Favor Different Redox Transitions in [4Fe-4S]-Cys ₄ Clusters

[4Fe-4S] clusters in proteins typically occur in three distinct redox states: the oxidized state ([4Fe-4S]³⁺), the semireduced state ([4Fe-4S]²⁺), and the reduced state ([4Fe-4S]¹⁺). Consequently, proteins containing these clusters could exhibit two distinct redox transitions: the low-potential transition (LPT: [4Fe-4S]¹⁺/[4Fe-4S]²⁺) and the high-potential transition (HPT: [4Fe-4S]²⁺/[4Fe-4S]³⁺). However, individual proteins employ only one of them for their biological function. Despite sharing an identical inorganic cofactor, ferredoxins (Fds) consistently operate via the LPT, whereas high-potential iron-sulfur proteins (HiPIPs) exclusively use the HPT. In this study, we analyze the physical basis for the different behaviors of the two protein classes. Using continuum electrostatics, we calculate the redox potentials for both transitions across a representative selection of high-resolution protein structures from both classes. We find that transferring the cluster from water into the protein interior shifts both transitions to lower redox potentials, with the LPT experiencing a larger shift than the HPT. This larger shift is caused by the larger desolvation penalty due to the higher charge of the redox states involved in the LPT. As a result, the separation between the two transitions increases to about 1 V, matching values observed for model clusters in organic solvents. Protein background charges, including peptide bond dipoles, shift the redox potential of both transitions by nearly the same amount, tuning only one transition into the physiological redox window, while pushing the other transition outside of this window. These background charges stabilize the reduced state in both transitions, but the magnitude of this stabilization is substantially larger in Fds than in HiPIPs. Together, these effects position the native redox transition of each protein class at its physiological value, while shifting the non-native transition outside of the biologically accessible range.