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 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.

Keywords: EPR; EXAFS; FeS cluster; Nitrogenase; P-cluster; XAS.

Figure 1

Rendering of the vanadium nitrogenase system, including the structures of the three metallocofactors (right). Iron is colored in orange, sulfur in yellow, carbon in gray, vanadium in purple, oxygen in red, and nitrogen in blue. The figures were generated using PDB IDs 5N6Y and 6Q93.

Figure 2

A) SDS‐PAGE Novex™ 4–20 %, Tris‐Glycine and B) NativePAGE™ 3–12 %, Bis‐Tris of MoFe, apo‐MoFe, VFe, and apo‐VFe. Red arrows indicate main species and orange arrows indicate additional bands.

Figure 3

Schematics of the proposed clusters observed on the SDS‐PAGE and NativePAGE. (A) MoFe complex with α₂β₂‐subunit configuration. (B) VFe with α₂β₂δ₂ and α₁β₂δ₂‐subunit configurations. The gels also suggested the presence of VnfJ bound to these complexes, thereby forming VnfJ‐α₂β₂δ₂ and VnfJ‐α₁β₂δ₂. (C) apo‐MoFe complex with α₂β₂‐subunit configuration. The gels also suggested the presence of NafY bound to this complex, thereby forming NafY‐α₂β₂. (D) apo‐VFe with α₂β₂ and α₁β₂‐subunit configurations.

Figure 4

Perpendicular‐mode X‐band EPR of VFe (black) and apo‐VFe (purple) proteins. Both samples were at a concentration of 140 μM and were reduced with 5 mM DT. EPR conditions: temperature 14 K; microwave frequency 9.63 GHz; microwave power 5 mW; modulation amplitude 7.46 G. Each trace is the average of 5 scans.

Figure 5

(A) X‐band perpendicular mode EPR of VFe (black) and apo‐VFe (purple). (B) X‐band parallel mode EPR of MoFe (orange), VFe (black), apo‐MoFe (green) and apo‐VFe (purple). All samples were at a concentration of 140 μM and oxidized by 10x excess of IDS. EPR conditions: temperature, 14 K; microwave frequency, 9.63 GHz (perpendicular‐mode) and 9.32 GHz (parallel‐mode); microwave power, 5 mW (perpendicular‐mode) and 200 mW (parallel‐mode); modulation amplitude, 7.46 G; Each trace is an average of 10 scans.

Figure 6

X‐band EPR spectra of VFe protein oxidized with thionine as indicated in the graph. EPR spectra measured in perpendicular‐mode (A) and parallel‐mode (B). EPR conditions: protein concentration 140 μM, temperature 14 K; microwave frequency, 9.63 GHz (perpendicular‐mode) and 9.32 GHz (parallel‐mode); microwave power, 5 mW (perpendicular‐mode) and 200 mW (parallel‐mode); modulation amplitude, 7.46 G; Each trace is the average of 10 scans.

Figure 7

X‐band EPR spectra of apo‐VFe protein oxidized with thionine as indicated in the graph. EPR spectra measured in perpendicular‐mode (A) and parallel‐mode (B). EPR conditions: protein concentration 140 μM, temperature 14 K; microwave frequency, 9.63 GHz (perpendicular‐mode) and 9.32 GHz (parallel‐mode); microwave power, 5 mW (perpendicular‐mode) and 200 mW (parallel‐mode); modulation amplitude, 7.46 G; Each trace is the average of 10 scans.

Figure 8

X‐ray absorption spectra of (NBu₄)₂[Fe₄S₄(SPh)₄] (yellow), apo‐VFe (purple), apo‐MoFe (green). Non‐phase shifted EXAFS Fourier transform over a k‐range of 2 to 14 Å⁻¹.