X-ray emission spectroscopy evidences a central carbon in the nitrogenase iron-molybdenum cofactor

Abstract

Nitrogenase is a complex enzyme that catalyzes the reduction of dinitrogen to ammonia. Despite insight from structural and biochemical studies, its structure and mechanism await full characterization. An iron-molybdenum cofactor (FeMoco) is thought to be the site of dinitrogen reduction, but the identity of a central atom in this cofactor remains unknown. Fe Kβ x-ray emission spectroscopy (XES) of intact nitrogenase MoFe protein, isolated FeMoco, and the FeMoco-deficient nifB protein indicates that among the candidate atoms oxygen, nitrogen, and carbon, it is carbon that best fits the XES data. The experimental XES is supported by computational efforts, which show that oxidation and spin states do not affect the assignment of the central atom to C(4-). Identification of the central atom will drive further studies on its role in catalysis.

Figure 1

The FeMoco (a) and P-cluster (b) of nitrogenase (adapted from PDB: 1MIN). Orange = Fe; Yellow= S; Cyan = Mo; Black = C^4-, N^3-, or O^2-. For clarity, the homocitrate and histidine ligands to the Mo have been omitted.

Figure 2

(a) Normalized V2C XES spectra of isolated FeMoco (red) and a representative fit to the data (black dotted line). (b) Comparison of the normalized V2C XES data for FeMoco (red), the MoFe protein (gray), and the ∆nifB MoFe protein (black). Inset: V2C satellite region for Fe₂O₃ (red), Fe₃N (blue), and MoFe protein (gray).

Figure 3

(a) Comparison of the calculated V2C XES spectra of FeMoco with an interstitial C^4- (black), N^3- (blue) and O^2- (red) and of the spectra of the P-clusters (gray). (b) Calculated V2C XES spectra of FeMoco with an interstitial C^4- (black), the P-clusters (gray). (c) Experimental difference spectrum of FeMoco with the P-clusters (gray), as well as calculated difference spectra of the P-cluseters with FeMoco containing interstitial C^4- (black), N^3- (blue), and O^2- (red).