Characterization of [4Fe-4S] Cluster Vibrations and Structure in Nitrogenase Fe Protein at Three Oxidation Levels via Combined

(Av2) provides a rare opportunity to investigate a [4Fe-4S] cluster at three oxidation levels in the same protein environment. Here, we report the structural and vibrational changes of this cluster upon reduction using a combination of NRVS and EXAFS spectroscopies and DFT calculations. Key to this work is the synergy between these three techniques as each generates highly complementary information and their analytical methodologies are interdependent. Importantly, the spectroscopic samples contained no glassing agents. NRVS and DFT reveal a systematic 10−30 cm−1 decrease in Fe−S stretching frequencies with each added electron. The “oxidized ” [4Fe-4S]2+ state spectrum is consistent with and extends previous resonance Raman spectra. For the “reduced” [4Fe-4S]1+ state in Fe protein, and for any “all-ferrous ” [4Fe-4S]0 cluster, these NRVS spectra are the first available vibrational data. NRVS simulations also allow estimation of the vibrational disorder for Fe−S and Fe−Fe distances, constraining the EXAFS analysis and allowing structural disorder to be estimated. For oxidized Av2, EXAFS and DFT indicate nearly equal Fe−F