Tracing the interstitial carbide of the nitrogenase cofactor during substrate turnover

Abstract

The fate of the interstitial atom of the nitrogenase cofactor during substrate turnover has remained a topic of interest since the discovery of this atom more than a decade ago. In this study, we labeled the interstitial carbide atom with (14)C and (13)C isotopes and traced the fate of the isotope under turnover conditions. Our results show that the interstitial carbide cannot be exchanged upon turnover, nor can it be used as a substrate and incorporated into the products. These observations point to a role of the interstitial carbide in stabilizing the cofactor structure, although a function of this atom in indirectly modulating the reactivity of the cofactor or directly interacting with the substrate cannot be excluded.

Figure 1

Structure of the FeMoco ([MoFe₇S₉Chomocitrate]). The cluster is shown as a ball-and-stick model, with the atoms colored as follows: Fe, orange; S, yellow; Mo, cyan; O, red; C, grey; and N, blue. PYMOL was used to generate this figure using PDB entry 3U7Q.

Figure 2

Intensities of ¹⁴C labels in MoFe protein samples without turnover (A) or upon turnover of C₂H₂ (B) and N₂ (C). Shown is the autoradiography detecting radiation of the ¹⁴C label in the MoFe protein-bound FeMoco. All MoFe protein samples contained an equivalent amount of ¹⁴C-labeled FeMoco.

Figure 3

GC-MS analysis of hydrocarbon products generated from the turnover of (A) ¹²CO by unlabeled FeMoco; (B) ¹³CO by unlabeled FeMoco; and (C) ¹²CO by ¹³C-labeled FeMoco. The relative intensity of each hydrocarbon product traced at a given mass is arbitrarily set at 100%.