ATP-independent substrate reduction by nitrogenase P-cluster variant

Abstract

The P-cluster of nitrogenase is largely known for its function to mediate electron transfer to the active cofactor site during catalysis. Here, we show that a P-cluster variant (designated P*-cluster), which consists of paired [Fe 4 S 4 ]-like clusters, can catalyze ATP-independent substrate reduction in the presence of a strong reductant, europium (II) diethylenetriaminepentaacetate [Eu(II)-DTPA]. The observation of a decrease of activity in the rank ΔnifH, ΔnifBΔnifZ, and ΔnifB MoFe protein, which corresponds to a decrease of the amount of P*-clusters in these cofactor-deficient proteins, firmly establishes P*-cluster as a catalytically active metal center in Eu(II)-DTPA-driven reactions. More excitingly, the fact that P*-cluster is not only capable of catalyzing the two-electron reduction of proton, acetylene, ethylene, and hydrazine, but also capable of reducing cyanide, carbon monoxide, and carbon dioxide to alkanes and alkenes, points to a possibility of developing biomimetic catalysts for hydrocarbon production under ambient conditions.N itrogenases are a family of metalloenzymes that catalyze the nucleotide-dependent reduction of dinitrogen to ammonia under ambient conditions. The best-characterized member of this enzyme family is the molybdenum (Mo)-nitrogenase of Azotobacter vinelandii, which consists of two redox-active component proteins (1). One of the proteins, termed iron (Fe) protein (encoded by nifH), is a γ 2 -dimer that contains a [Fe 4 S 4 ] cluster between the two subunits and a MgATP binding site within each subunit; the other, termed molybdenum-iron (MoFe) protein (encoded by nifDK), is an α 2 β 2 -heterotetramer that houses two complex metal clusters per αβ-dimer: the P-cluster ([Fe 8 S 7 ]), which is located at each α/β-subunit interface; and the iron-molybdenum (FeMo) cofactor, or FeMoco ([MoFe 7 S 9 C-homocitrate]), which is buried within each α-subunit (2-4). Catalysis by nitrogenase presumably involves repeated association and dissociation between the two component proteins and ATP-dependent electron transfer from the [Fe 4 S 4 ] cluster of the Fe protein, through the P-cluster, to the FeMoco of MoFe protein, where substrate reduction occurs (5).The P-cluster has long been regarded as a "capacitor" that mediates the electron transfer between the [Fe 4 S 4 ] cluster of Fe protein and the cofactor site of MoFe protein during catalysis (1). Although the question of whether the P-cluster can directly reduce substrates was raised previously, it has remained a topic of debate because of the difficulty of distinguishing the contribution of the P-cluster from that of the cofactor to catalysis. As such, MoFe protein variants carrying the P-cluster species alone need to be generated to address the catalytic capacity of P-cluster. Recent studies of the biosynthesis of nitrogenase have led to the identification of three cofactor-deficient forms of MoFe protein (Fig. 1). One form, designated ΔnifB MoFe protein, contains two intact, [Fe 8 S 7 ] P-clusters (6); another, designated ΔnifH MoFe pro...

ATP-dependent substrate reduction at an [Fe 8 S 9 ] double-cubane cluster

“…The FeMoCo as well as the P-cluster of nitrogenase are thought to be formed from two closely spaced, paired [Fe 4 S 4 ]-clusters, resembling DCCs (34). The P-cluster is produced on the nitrogenase scaffold by action of NifH from its precursor P*-cluster (35). Excitingly, in contrast to the mature P-cluster, the P*-cluster is catalytically active and catalyzes the reduction of acetylene and hydrazine, as well as the reduction of CO and CN to alkanes and alkenes (35).…”

Uncoupling binding of substrate CO from turnover by vanadium nitrogenase

“…Stock solutions of Eu II -EGTA and Eu II -DTPA were prepared as described previously (20,21). The dithionite-reduced VFe protein was passed through a G25 column in a buffer containing 25 mM Tris·HCl (pH 8.0) and 500 mM NaCl to remove excess dithionite before exposure to 100% CO.…”

Activation of CO2 by Vanadium Nitrogenase

“…Interestingly,t race amounts of 13 C 2 H 4 , 13 C 2 H 6 , 13 C 3 H 6 ,a nd 13 C 3 H 8 have been detected in the reaction of ac ofactor-deficient NifDK variant with 13 CO 2 and Eu II DTPA. [73] This particular protein variant cannote xpress NifH, which is essential for maturationo fb otht he M-cluster and P-cluster; [23] thus, the socalled DnifHN ifDK lacks M-cluster and possesses aP -cluster precursor called the P*-cluster,w hich is believed to be similar in compositiona nd structure to the P*-clustero fV nfDGK in Vnitrogenase. [26,[74][75][76] Detectiono fh ydrocarbonsf rom the Eu II DTPA-driven reduction of CO 2 with DnifHN ifDK raises the possibility that the P*-cluster may exhibit marginal catalytic properties of its own.…”

“…[26,[74][75][76] Detectiono fh ydrocarbonsf rom the Eu II DTPA-driven reduction of CO 2 with DnifHN ifDK raises the possibility that the P*-cluster may exhibit marginal catalytic properties of its own. [73] Both VnfDGK and DnifHN ifDK are thought to have roughly analogous P*-clusters, i.e.,apair of Fe 4 S 4 -like clusters that displays imilar spectroscopics ignals. [26] As such, the site of ATP-independent CO 2 reduction for VnfDGK in the presence of Eu II DTPAm ay occur at either P*cluster,V -cluster,o rs ome combination of both;m ore work is necessary to understand the exact natureo ft his reaction.…”