In vitro synthesis of the iron-molybdenum cofactor of nitrogenase from iron, sulfur, molybdenum, and homocitrate using purified proteins

Abstract

Biological nitrogen fixation, the conversion of atmospheric N2 to NH3, is an essential process in the global biogeochemical cycle of nitrogen that supports life on Earth. Most of the biological nitrogen fixation is catalyzed by the molybdenum nitrogenase, which contains at its active site one of the most complex metal cofactors known to date, the iron-molybdenum cofactor (FeMo-co). FeMo-co is composed of 7Fe, 9S, Mo, R-homocitrate, and one unidentified light atom. Here we demonstrate the complete in vitro synthesis of FeMo-co from Fe(2+), S(2-), MoO4(2-), and R-homocitrate using only purified Nif proteins. This synthesis provides direct biochemical support to the current model of FeMo-co biosynthesis. A minimal in vitro system, containing NifB, NifEN, and NifH proteins, together with Fe(2+), S(2-), MoO4(2-), R-homocitrate, S-adenosyl methionine, and Mg-ATP, is sufficient for the synthesis of FeMo-co and the activation of apo-dinitrogenase under anaerobic-reducing conditions. This in vitro system also provides a biochemical approach to further study the function of accessory proteins involved in nitrogenase maturation (as shown here for NifX and NafY). The significance of these findings in the understanding of the complete FeMo-co biosynthetic pathway and in the study of other complex Fe-S cluster biosyntheses is discussed.

Fig. 1.

Current model for FeMo-co biosynthesis and insertion into apo-NifDK. The model shows in dark blue the core components of the FeMo-co biosynthesis pathway: NifB, NifEN, and NifH (this work). Simple [Fe-S] clusters (most likely [2Fe-2S] and [4Fe-4S]), which can be chemically reconstituted on NifB, are converted into NifB-co precursor in a reaction that requires SAM and reducing conditions. Presumably, NifS and NifU would be responsible for the synthesis of those “NifB-co precursors” on NifB in vivo (8, 9). NifB-co is a common precursor for the biosynthesis of FeMo-co and the cofactors for the alternative nitrogenases, iron–vanadium cofactor and iron-only cofactor. NifEN binds NifB-co and converts it into the VK cluster. Incorporation of Mo and R-homocitrate into the VK cluster and final assembly of FeMo-co require NifH and MgATP. The resulting FeMo-co is inserted into apo-NifDK to activate it. Analogous reactions are hypothesized to occur during the maturation of alternative nitrogenases. The model shows in light blue the other Nif/Naf proteins known to participate in FeMo-co biosynthesis and insertion into apo-NifDK. NifS, NifU, NifQ, and NifV provide physiologically relevant forms of Fe, S, Mo, and R-homocitrate. NifX and NafY may assist NifB, NifEN, and NifDK on FeMo-co precursor trafficking among them and may serve as a reservoir of precursors giving some buffering capacity to the pathway.

Fig. 2.

Purification of A. vinelandii Nif/Naf proteins and in vitro activation of apo-NifDK with purified Nif proteins. (A) SDS/PAGE analysis of purified NifB, NifEN, NifH, and apo-NifDK proteins on a 12% polyacrylamide gel. (B) SDS/PAGE analysis of purified NifX and NafY on a 14% polyacrylamide gel. (C) NifB-dependent in vitro activation of apo-NifDK. The complete reaction (NifB-dependent assay) contained NifB, NifEN, NifH, NifX, NafY, apo-NifDK, FeSO₄, Na₂S, SAM, Na₂MoO₄, R-homocitrate, Mg-ATP, and DTH. In the NifB-co-dependent assay, purified NifB-co substitutes for NifB, FeSO₄, Na₂S, and SAM. Data are the mean and standard deviation of two to four independent assays.

Fig. 3.

Effect of apo-NifDK concentration on the specific activity of reconstituted NifDK. Reactions were set essentially as those in Fig. 2C, but the concentration of apo-NifDK present in the assay varied from 0 to 2.4 μM. ▲, NifB-dependent reactions; ■, NifB-co-dependent reactions. (A) Plot of C₂H₂ reduction activity versus apo-NifDK concentration in the assay. (B) Specific activity of in vitro activated NifDK versus apo-NifDK concentration in the assay, as calculated from data in A. The percentage of apo-NifDK activated in the assay is indicated on the right axis. The specific activity of purified NifDK determined under the same reaction conditions (1,234 nmol C₂H₄ formed per min⁻¹·mg⁻¹) represents 100% activation.

Fig. 4.

NifB, NifEN, and NifH are essential for in vitro activation of apo-NifDK. Shown is the effect of NifB, NifEN, or NifH concentration on in vitro apo-NifDK activation. The NifB-dependent assays were conducted in the absence of NifX and NafY (see Experimental Procedures) with the following modifications: in A the concentration of NifB in the assay was increased from 0 to 11 μM, in B the concentration of NifEN in the assay was increased from 0 to 4.5 μM, and in C the concentration of NifH in the assay was increased from 0 to 7.5 μM. In C, apo-NifDK activation was terminated by addition of 0.3 mM (NH₄)₂MoS₄ before the addition of excess NifH for the determination of NifDK activity. The scale of activity axes in B and C are the same. Representative experiments are shown.

Fig. 5.

Effect of NifX and NafY on the in vitro activation of apo-NifDK. Shown is the effect of NifX (A and B) or NafY (C and D) in the NifB-co-dependent (A and C) or in the NifB-dependent (B and D) reactions. The NifB-co-dependent or NifB-dependent assays were supplemented with 0 to 30 μM NifX (A and B) or 0 to 30 μM NafY (C and D). A representative experiment is shown for each titration experiment.

Fig. 6.

Substrate requirements for in vitro activation of apo-NifDK. FeMo-co synthesis and apo-NifDK activation reactions (containing NifB, NifEN, NifH, and apo-NifDK as protein components) were conducted in the absence of one or more substrates at a time. A reaction containing all substrates (complete reaction) and a NifB-co-dependent reaction for FeMo-co synthesis and apo-NifDK activation were carried out as controls. Acid-washed vials were used to show the Mo dependency of the reaction. Data are the average and standard deviation of two to four independent determinations.