Uncoupling binding of substrate CO from turnover by vanadium nitrogenase

Abstract

Biocatalysis by nitrogenase, particularly the reduction of N 2 and CO by this enzyme, has tremendous significance in environmentand energy-related areas. Elucidation of the detailed mechanism of nitrogenase has been hampered by the inability to trap substrates or intermediates in a well-defined state. Here, we report the capture of substrate CO on the resting-state vanadium-nitrogenase in a catalytically competent conformation. The close resemblance of this active CO-bound conformation to the recently described structure of CO-inhibited molybdenum-nitrogenase points to the mechanistic relevance of sulfur displacement to the activation of iron sites in the cofactor for CO binding. Moreover, the ability of vanadium-nitrogenase to bind substrate in the resting-state uncouples substrate binding from subsequent turnover, providing a platform for generation of defined intermediate(s) of both CO and N 2 reduction.nitrogenase | vanadium | carbon monoxide | turnover | substrate binding N itrogenases are complex metalloenzymes that catalyze the reduction of a variety of substrates under ambient conditions (1-3). Among them, two reactions bear significant relevance to environment-and energy-related areas: (i) the reduction of dinitrogen (N 2 ), a key element of nitrogen cycle in our biosphere, to the bio-accessible form of ammonia (NH 3 ); and (ii) the reduction of carbon monoxide (CO), a waste product from car and factory exhausts, to useful hydrocarbon products. The molybdenum (Mo)-and vanadium (V)-nitrogenases are two homologous members of this enzyme family. Both enzymes consist of a reductase component (nifH-or vnfH-encoded Fe protein) and a catalytic component (nifDK-encoded MoFe protein or vnfDGK-encoded VFe protein). Substrate turnover by both nitrogenases involves the formation of a functional complex between the two component proteins (1-3), which enables adenosine triphosphate (ATP)-dependent, interprotein transfer of electrons from the reductase component to the cofactor site of the catalytic component for the subsequent reduction of substrates.Designated the M and V cluster, respectively, the cofactors of Mo-and V-nitrogenases closely resemble each other in geometry (3-7). Previous Fe K-edge X-ray absorption spectroscopy (XAS)/ extended X-ray absorption fine structure (EXAFS) analysis revealed that the two cofactors had nearly indistinguishable metalsulfur core structures, each comprising MFe 3 S 3 (M = Mo or V) and Fe 4 S 3 subclusters bridged by three μ 2 -coordinated, belt-sulfur (S) atoms ( Fig. 1 A and B) (3, 6, 8). Recently, we performed a K-valence X-ray emission spectroscopy (XES) study of both protein-bound and solvent-extracted V clusters (Fig. 1C), which identified a carbide (C 4-)-specific XES feature of the V cluster that was observed earlier in the case of the M cluster (9). Thus, the two cofactors not only share an overall homology in structure, but also have the same inner strengths that originate from the μ 6 -coordinated interstitial carbide. Surprisingly, despite the significant homology bet...

Nitrogenases—A Tale of Carbon Atom(s)

“…The CO-bound M-and V-clusters displayed EPR signals with strikingly similar line shapes (Figure 8 c,d), [52] thus suggesting a close resemblance between the two CO-bound conformations (Figure 8 a,b). [52] However, while the "inhibited" state of the CO-bound M-cluster was hardly capable of turnover, the CO-bound V-cluster was catalytically competent and, thus, represented a substratebound state of nitrogenase. GC-MS analysis demonstrated that 13 CO could be preloaded onto the V-cluster and subsequently turned over into hydrocarbon products.…”

“…. PDB entry 4TKV, [54] XAS/EXAFS, [51] and XES [52] data were used to generate these models. Atoms are colored as described in Figure 1 b.…”

“…[52] The role of Mo or V in nitrogenase catalysis is unclear, although it has been proposed that the heterometal could function in modulating the redox properties of the cofactor.…”

“…In the absence of extra CO, turnover of the V-cluster-bound 13 CO resulted in the formation of 13 CH 4 (Figure 8 e, 1 ) and 13 CD 4 ( Figure 8 e, 2 ) in H 2 O and D 2 O, respectively. [52] When 1 % extra 12 CO was supplied, however, the V-cluster-bound 13 CO could also undergo reductive C À C coupling, thereby generating 13 (Figure 8 e, 4 ) as additional turnover products in H 2 O-based reactions. [52] The displacement of a belt-S atom by a CO moietya binding mode that is likely adopted by the catalytically competent conformation of the CO-bound V-cluster-points to a nitrogenase-based mechanism of CO activation that relies on a substantial rearrangement of the belt-S atom to uncover and activate the two Fe sites for the initial binding of CO and, possibly, the subsequent attachment of additional CO moieties for CÀC coupling.…”

Cleaving the N,N Triple Bond: The Transformation of Dinitrogen to Ammonia by Nitrogenases

“…In addition, there is a high degree of sequence identity between the MoFe protein and the VFe protein, which centers around the residues that are either ligated to or in the surroundings of the P-cluster and the cofactor [5,22]. The P-cluster of the VFe protein has long been thought to closely resemble the P-cluster of the MoFe protein in structure, but the recent characterization of the A. vinelandii VFe protein suggested otherwise [22,92]. The EPR data of the dithionite-reduced and IDS-oxidized forms of the VFe protein strongly indicate that the electronic properties of the P-cluster in the VFe protein are quite different than those of its counterpart in the MoFe protein [92].…”