Understanding the P-cluster of Vanadium Nitrogenase: an EPR and XAS study of the holo vs. apo forms of the enzyme.

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 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 XAS and 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 on the structure of the P-cluster in the different forms of the nitrogenase.