Formation of a homocitrate-free iron-molybdenum cluster on NifEN: implications for the role of homocitrate in nitrogenase assembly

Abstract

Molybdenum (Mo)-dependent nitrogenase is a complex metalloprotein that catalyzes the biological reduction of dinitrogen (N(2)) to ammonia (NH(3)) at the molybdenum-iron cofactor (FeMoco) site of its molybdenum-iron (MoFe) protein component. Here we report the formation of a homocitrate-free, iron-molybdenum ("FeMo") cluster on the biosynthetic scaffold of FeMoco, NifEN. Such a NifEN-associated "FeMo" cluster exhibits EPR features similar to those of the NifEN-associated, fully-complemented "FeMoco", which originate from the presence of Mo in both cluster species; however, "FeMo" cluster and "FeMoco" display different temperature-dependent changes in the line shape and the signal intensity of their respective EPR features, which reflect the impact of homocitrate on the redox properties of these clusters. XAS/EXAFS analysis reveals that the Mo centers in both "FeMo" cluster and "FeMoco" are present in a similar coordination environment, although Mo in "FeMo" cluster is more loosely coordinated as compared to that in "FeMoco" with respect to the Mo-O distances in the cluster, likely due to the absence of homocitrate that normally serves as an additional ligand for the Mo in the cluster. Subsequent biochemical investigation of the "FeMo" cluster not only facilitates the determination of the sequence of events in the mobilization of Mo and homocitrate during FeMoco maturation, but also permits the examination of the role of homocitrate in the transfer of FeMoco between NifEN and MoFe protein. Combined outcome of these studies establishes a platform for future structural analysis of the interactions between NifEN and MoFe protein, which will provide useful insights into the mechanism of cluster transfer between the two proteins.

Fig. 1

Native PAGE of protein samples. Lane 1, ΔnifB MoFe protein; lane 2, NifEN^Precursor; lane 3, NifEN^“FeMo”; lane 4, NifEN^“FeMoco”; lane 5, NifEN^Precursor plus ΔnifB MoFe protein; lane 6, NifEN^“FeMo” plus ΔnifB MoFe protein; lane 7, NifEN^“FeMoco” plus ΔnifB MoFe protein.

Fig. 2

Temperature-dependency of EPR spectra of NifEN^“FeMo” (A) and NifEN^“FeMoco” (B) in dithionite-reduced states. Spectra were measured at 4, 6, 10, 15, 20 and 30 K, respectively. The S = 3/2 features of NifEN^“FeMo” and NifEN^“FeMoco” are enlarged, and the g values of the S = 3/2 and S = 1/2 signals are indicated.

Fig. 3

Temperature-dependency of EPR spectra of NifEN^“FeMo” (A) and NifEN^“FeMoco” (B) in indigodisulfonate (IDS)-oxidized states. Spectra were measured at 4, 6, 10, 15, 20 and 30 K, respectively. The features of NifEN^“FeMo” and NifEN^“FeMoco” at g = 3.96 and 2.03 are enlarged.

Fig. 4

Mo K-edge x-ray absorption spectra (A) and smoothed second derivatives (B) of NifEN^“FeMo” (red) and NifEN^“FeMoco” (black); Mo K-edge EXAFS (C) and Fourier transforms (D) of data (pink) and fits (red) for NifEN^“FeMo”, and data (gray) and fits (black) for NifEN^“FeMoco”. The dashed line is centered at ~2.90 Å (D).

Fig. 5

(A) Reconstitution of purified ΔnifB MoFe protein by various NifEN species: NifEN^Precursor (➀); NifEN^Precursor plus MoO₄²⁻, homocitrate, Fe protein and MgATP (➁); NifEN^“FeMo” (➂); NifEN^“FeMo” plus homocitrate, Fe protein and MgATP (➃); NifEN^“FeMo” plus MoO₄²⁻, homocitrate, Fe protein and MgATP (➄); NifEN^HC (➅; HC: homocitrate); NifEN^HC plus MoO₄²⁻, Fe protein and MgATP (➆); NifEN^HC plus MoO₄²⁻, homocitrate, Fe protein and MgATP (➇). (B) Percentage activities of the crude extract of UW45 (a nifB-deletion strain) before (➀) and after (➁) incubation with NifEN^“FeMo” and upon removal of NifEN^“FeMo” (➂). (C) Percentage activities of the crude extract of UW45 before (➀) and after (➁) incubation with NifEN^“FeMoco” and upon removal of NifEN^“FeMoco” (➂). Consistent with our earlier report (18), the ΔnifB MoFe protein preparation used in this study can be activated to a comparable level by either NifEN^“FeMoco” (725±72 nmol C₂H₄ formation/mg protein/min) or isolated FeMoco (745±51 nmol C₂H₄ formation/mg protein/min), suggesting that NifEN^“FeMoco” is as proficient a FeMoco source as the isolated FeMoco. The maximum (100%) activities of acetylene reduction are set for 725±72, 1.6±0.3 and 22.8±3.8 nmol C₂H₄ formation/mg protein/min, respectively, in A, B and C.

Fig. 6

Plausible models depicting the interactions between ΔnifB MoFe protein and NifEN^“FeMoco” (A) and those between ΔnifB MoFe protein and NifEN^“FeMo” (B). For the purpose of simplicity, the interactions between one cofactor assembly site in NifEN and one cofactor binding site in ΔnifB MoFe protein are shown for NifEN^“FeMoco” (A) and NifEN^“FeMo” (B), respectively. The presence of homocitrate in NifEN-associated “FeMoco” and its contribution to the overall negative charge of the cluster facilitate the subsequent insertion of this cluster into the positively charged insertion funnel in ΔnifB MoFe protein, leading to the formation of a holo-MoFe protein that is fully proficient in catalysis (A). In contrast, the absence of homocitrate from the NifEN-associated “FeMo” cluster results in the attempted (and unsuccessful) delivery of “FeMo” cluster from NifEN to ΔnifB MoFe protein and the generation of a complex between the two proteins (B). A “chimeric” electron transfer chain could be formed in this complex, which utilizes the P-cluster on ΔnifB MoFe protein and the “FeMo” cluster on NifEN for catalysis (B). The “FeMo” cluster that is “stuck” between NifEN and ΔnifB MoFe protein likely “falls out” when the two proteins are pulled apart, which would be consistent with the absence of cluster from both NifEN and ΔnifB MoFe protein upon separation (B).