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Abstract

The catalytic moiety of nitrogenases contains two complex metalloclusters: The M-cluster (also called cofactor), where the catalytic reduction of substrates takes place, and the [Fe₈S₇] P-cluster responsible for electron transfer. Due to discrepancies between crystallography and EPR spectroscopy, the exact structure of the P-cluster in the VFe protein remains a topic of debate. Herein, we use an apo-form of VFe (which retains the P-cluster but lacks the FeVco) to study the VFe P-cluster. SDS-PAGE and NativePAGE showed a heterogeneous composition of the VFe and the apo-VFe samples with the presence of α₁β₂δ₂ and α₁β₂ complexes. The parallel mode EPR measurements of IDS oxidized MoFe, apo-MoFe, and VFe samples reveal a signal at g=12 associated with the two-electron oxidized state of the P-cluster (P²⁺) for all three samples, albeit with different intensities. In contrast, no P²⁺ was observed for IDS oxidized apo-VFe. Additionally, comparisons between apo-MoFe, apo-VFe and the model complex (NBu₄)₂[Fe₄S₄(SPh)₄] via EXAFS measurements showed that apo-VFe does not contain a fully formed [Fe₈S₇] P-cluster, but rather is comprised of fragmented iron-sulfur clusters. Our results point to a possible variation in the structure of the P-cluster in the different forms of the nitrogenase.