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

Abstract

Azotobacter vinelandii nitrogenase Fe protein (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-Fe distances, while addition of one electron decreases the cluster symmetry. However, addition of the second electron to form the all-ferrous state induces significant structural change. EXAFS data recorded to k = 21 Å−1 indicates a 1:1 ratio of Fe-Fe interactions at 2.56 Å and 2.75 Å, a result consistent with DFT. Broken symmetry (BS) DFT rationalizes the interplay between redox state and the Fe-S and Fe-Fe distances as predominantly spin-dependent behavior inherent to the [4Fe-4S] cluster and perturbed by the Av2 protein environment.

An Iron–Sulfur Cluster in the Polymerase Domain of Yeast DNA Polymerase ε

“…The fitting data are summarized in Supplementary Table 1. The fitting results are similar to those obtained for other [4Fe-4S] clusters with a reduced iron center 14 . Fits to a [2Fe-2S] cluster gave consistently worse results, though the presence of a [2Fe-2S] cluster cannot entirely be ruled out based on the EXAFS data alone.…”

Low frequency dynamics of the nitrogenase MoFe protein via femtosecond pump probe spectroscopy — Observation of a candidate promoting vibration

“…The geometries, optimized at the PBE/LACV3P** level using GAUSSIAN 03 [54] software, were used for the analytic Hessian calculations, resulting in the DFT Raman intensities and other vibrational properties discussed in the text. We found the present and essentially equivalent setups to perform well for the vibrational dynamics of several iron-sulfur systems [4, 33, 55–58]. The analysis of the computed normal modes was done using an in-house Q-SPECTOR Python tool to model the Raman spectra.…”

Characterization of the [3Fe–4S]0/1+cluster from the D14C variant of Pyrococcus furiosus ferredoxin via combined NRVS and DFT analyses

“…1, C and D), nitrogenase Fe protein [4Fe-4S] cluster from Azotobacter vinelandii ( Av 2), and the [Fe 4 S 4 ](SPh) 4 model compound. 18, 19, 21 In what follows below, the DFT-computed spectra (Fig. 2, A–M, Fig.…”

“…1, D) and Av 2 proteins. 18, 19 For reduced [3Fe-4S] clusters, RR spectra have not been reported. The pronounced high-frequency maximum for the oxidized [3Fe-4S] D14C at 390 cm −1 downshifts to 378 cm −1 upon reduction, and the band at 362 cm −1 decreases in intensity; additionally, bands at 340 and 319 cm −1 appear.…”