The accuracy of geometries is important for predicting the electronic properties of molecular systems using computational methods. A previous investigation has indicated that there exist two local minima for [4Fe–4S] clusters in proteins when optimized with a density functional theory (DFT) method. In this work, we performed a detailed study of two distinct local minima for [4Fe–4S] clusters in five proteins and two oxidation states, using combined quantum mechanical and molecular mechanical (QM/MM) methods.The MM part of the QM/MM calculations was performed with the Amber software, using the Amber ff14SB force fields. In the QM part, we employed nine different DFT methods, PBE, BP86, BLYP, B97D, TPSS, r2SCAN, TPSSh, B3LYP, and B3LYP*. All the functionals were combined withdef2-SV(P) or def2-TZVPDbasis sets.The QM system consisted of the Fe and S ions and the directly coordinated Cys groups modeled by CH3CH2S–, for the whole cluster.Weindicated that one local minimum (L state) has longer Fe–Fe distances than the other (S state) and that the L state is more stable for all cases studied. Also, our results showed that some DFT methods may only obtain the L state, while others may obtain both states. We recommend r2SCAN for optimizing [4Fe-4S] clusters in proteins, which gives the most accurate structures.
