Chi Chung Lee

Vanadium Nitrogenase Reduces CO

An enzyme that reduces nitrogen to ammonia can also reduce carbon monoxide to hydrocarbons. Vanadium nitrogenase not only reduces dinitrogen to ammonia but also reduces carbon monoxide to ethylene, ethane, and propane. The parallelism between the two reactions suggests a potential link in mechanism and evolution between the carbon and nitrogen cycles on Earth.

Radical SAM-Dependent Carbon Insertion into the Nitrogenase M-Cluster

A Radical Mechanism The bacterial enzyme nitrogenase plays a key role in the global nitrogen cycle by catalyzing the reduction of N2 to ammonia. At the heart of the enzyme is a metal-sulfur cluster that contains an interstitial light atom recently identified as a carbide. The identification raised questions concerning the role of the carbide in the enzyme mechanism and how it is inserted into the metal cluster. Wiig et al. (p. 1672; see the Perspective by Boal and Rosenzweig) now show that the carbide derives from S-adenosylmethionine (SAM) and is inserted into the core by the radical SAM enzyme NifB. The carbon atom in the middle of a large metal cluster originates from the one-carbon donor S-adenosylmethionine. The active site of nitrogenase, the M-cluster, is a metal-sulfur cluster containing a carbide at its core. Using radiolabeling experiments, we show that this carbide originates from the methyl group of S-adenosylmethionine (SAM) and that it is inserted into the M-cluster by the assembly protein NifB. Our SAM cleavage and deuterium substitution analyses suggest a similarity between the mechanism of carbon insertion by NifB and the proposed mechanism of RNA methylation by the radical SAM enzymes RlmN and Cfr, which involves methyl transfer from one SAM equivalent, followed by hydrogen atom abstraction from the methyl group by a 5′-deoxyadenosyl radical generated from a second SAM equivalent. This work is an initial step toward unraveling the importance of the interstitial carbide and providing insights into the nitrogenase mechanism.

Extending the carbon chain: hydrocarbon formation catalyzed by vanadium/molybdenum nitrogenases.

The molybdenum nitrogenase enzyme can reduce carbon monoxide, albeit inefficiently, in addition to its native substrate, nitrogen. In a small-scale reaction, vanadium-dependent nitrogenase has previously been shown to catalyze reductive catenation of carbon monoxide (CO) to ethylene, ethane, propylene, and propane. Here, we report the identification of additional hydrocarbon products [α-butylene, n-butane, and methane (CH4)] in a scaled-up reaction featuring 20 milligrams of vanadium-iron protein, the catalytic component of vanadium nitrogenase. Additionally, we show that the more common molybdenum-dependent nitrogenase can generate the same hydrocarbons from CO, although CH4 was not detected. The identification of CO as a substrate for both molybdenum- and vanadium-nitrogenases strengthens the hypothesis that CO reduction is an evolutionary relic of the function of the nitrogenase family. Moreover, the comparison between the CO-reducing capacities of the two nitrogenases suggests that the identity of heterometal at the active cofactor site affects the efficiency and product distribution of this reaction.

Unique features of the nitrogenase VFe protein from Azotobacter vinelandii

Nitrogenase is an essential metalloenzyme that catalyzes the biological conversion of dinitrogen (N2) to ammonia (NH3). The vanadium (V)-nitrogenase is very similar to the “conventional” molybdenum (Mo)-nitrogenase, yet it holds unique properties of its own that may provide useful insights into the general mechanism of nitrogenase catalysis. So far, characterization of the vanadium iron (VFe) protein of Azotobacter vinelandii V-nitrogenase has been focused on 2 incomplete forms of this protein: αβ2 and α2β2, both of which contain the small δ-subunit in minor amounts. Although these studies provided important information about the V-dependent nitrogenase system, they were hampered by the heterogeneity of the protein samples. Here, we report the isolation and characterization of a homogeneous, His-tagged form of VFe protein from A. vinelandii. This VFe protein has a previously-unsuspected, α2β2δ4-heterooctameric composition. Further, it contains a P-cluster that is electronically and, perhaps, structurally different from the P-cluster of molybdenum iron (MoFe) protein. More importantly, it is catalytically distinct from the MoFe protein, particularly with regard to the mechanism of H2 evolution. A detailed EPR investigation of the origins and interplays of FeV cofactor- and P-cluster-associated signals is presented herein, which lays the foundation for future kinetic and structural analysis of the VFe protein.

Characterization of Isolated Nitrogenase FeVco

The cofactors of the Mo- and V-nitrogenases (i.e., FeMoco and FeVco) are homologous metal centers with distinct catalytic properties. So far, there has been only one report on the isolation of FeVco from Azotobacter chroococcum. However, this isolated FeVco species did not carry the full substrate-reducing capacity, as it is unable to restore the N(2)-reducing ability of the cofactor-deficient MoFe protein. Here, we report the isolation and characterization of a fully active species of FeVco from A. vinelandii. Our metal and activity analyses show that FeVco has been extracted intact, carrying with it the characteristic capacity to reduce C(2)H(2) to C(2)H(6) and, perhaps even more importantly, the ability to reduce N(2) to NH(3). Moreover, our EPR and XAS/EXAFS investigations indicate that FeVco is similar to, yet distinct from FeMoco in electronic properties and structural topology, which could account for the differences in the reactivity of the two cofactors. The outcome of this study not only permits the proposal of the first EXAFS-based structural model of the isolated FeVco but also lays a foundation for future catalytic and structural investigations of this unique metallocluster.

P-cluster maturation on nitrogenase MoFe protein.

Biosynthesis of nitrogenase P-cluster has attracted considerable attention because it is biologically important and chemically unprecedented. Previous studies suggest that P-cluster is formed from a precursor consisting of paired [4Fe–4S]-like clusters and that P-cluster is assembled stepwise on MoFe protein, i.e., one cluster is assembled before the other. Here, we specifically tackle the assembly of the second P-cluster by combined biochemical and spectroscopic approaches. By using a P-cluster maturation assay that is based on purified components, we show that the maturation of the second P-cluster requires the concerted action of NifZ, Fe protein, and MgATP and that the action of NifZ is required before that of Fe protein/MgATP, suggesting that NifZ may act as a chaperone that facilitates the subsequent action of Fe protein/MgATP. Furthermore, we provide spectroscopic evidence that the [4Fe–4S] cluster-like fragments can be converted to P-clusters, thereby firmly establishing the physiological relevance of the previously identified P-cluster precursor.

ATP‐Independent Formation of Hydrocarbons Catalyzed by Isolated Nitrogenase Cofactors

Such a task can be accomplished by combining NMF-extracted cofactors[6-8] with a strong reductant, europium (II) diethylenetriaminepentaacetate [Eu(II)-DPTA],[9] in an ATP-free buffer system. The isolated cofactors remain sufficiently stable in this buffer system, keeping >90% integrity within the first hour (Figure S1), thereby permitting the determination of activity over this time period. Driven by Eu(II)-DPTA (E0’ = -1.14 V at pH 8), both FeMoco and FeVco can reduce CO—under ambient conditions—to methane (CH4), ethene (C2H4), ethane (C2H6), propene (C3H6), propane (C3H8), 1-butene (C4H8), n-butane (C4H10) and 1-pentene (C5H10) (Figure 1; Figure S2). When CN- is used as a substrate, the same set of products—together with ammonia (NH3)—can be generated in both FeMoco- and FeVco-based reactions; only in this case, n-pentane (C5H12), 1-hexene (C6H12), n-hexane (C6H14) and n-heptane (C7H16) can be detected as additional products in the reaction catalyzed by FeMoco (Figure 1; Figure S2). The product profiles of CN-- and CO-reduction are similar, consistent with the earlier proposal of a common C-C coupling pathway utilized by the CN- isomer and CO.[10] However, the rates of product formation from CN- are considerably higher than those from CO, likely due to a previously observed, stabilizing effect of CN--binding on isolated cofactors.[11] Gas chromatograph-mass spectrometry (GC-MS) analysis further confirms that CO and CN- are the carbon sources for the hydrocarbons generated in these reactions, as all these products display the expected mass shifts when 12CO and 12CN- are replaced by 13CO and 13CN-, respectively, in the reaction (Figure 2). Figure 1 GC-MS analysis of hydrocarbons generated from the reduction of 12CO and 12CN- by isolated cofactors. Specific activities of hydrocarbon formation are expressed in nmol/μmol cofactor/hr above the corresponding traces and presented as means ± ... Figure 2 GC-M S analysis of hydrocarbons generated by isolated cofactors from the reduction of 13CO and 13CN-. There are interesting discrepancies in how cofactors react with the two carbonaceous substrates in the solvent-extracted/Eu(II)-DPTA-driven and protein-bound/ATP-driven states. Both isolated cofactors are less active than their protein-bound counterparts in CO reduction; however, the total amounts of hydrocarbon formation by the isolated FeMoco and FeVco are 67.9% and 0.05%, respectively, of those by the protein-bound FeMoco and FeVco.[3,4,12] Such a disparate decrease in CO-reducing efficiency renders FeMoco—which is only 0.1% as active as FeVco within the protein—comparably active with FeVco in the isolated state (Figure 1). With regard to CN-, the protein-bound cofactors normally reduce this substrate to CH4 and NH3[1]. This is not the case when CN- is reduced by isolated cofactors, as CH4 is no longer the major carbonaceous product, and alkenes/alkanes of two- to seven-carbon-length are detected as additional products in this reaction (Figure 1). The differences between the isolated- and protein-bound-cofactors in hydrocarbon formation highlight the significant impact of protein environment on the reactivity of nitrogenase cofactors.[13] Nevertheless, the ability of isolated cofactors to catalyze the ATP-independent formation of long-chain, liquid-phase hydrocarbons suggests the possibility of developing novel electrocatalysts for fuel production under ambient conditions. Understanding how nitrogenases catalyze the formation of hydrocarbons is crucial for achieving this goal, as these enzymes not only provide a prototype for such an electrocatalyst, but also serve as a biological blueprint for a synthetic matrix that immobilizes the catalyst and mimics the protein machinery for enhanced, ATP-independent electron transfer.

Spectroscopic Characterization of the Isolated Iron–Molybdenum Cofactor (FeMoco) Precursor from the Protein NifEN

The FeMoco of Mo-nitrogenase provides the active site for substrate reduction. Previously, a FeMoco precursor was captured on NifEN, a scaffold protein for FeMoco biosynthesis. Here, we report the isolation of FeMoco precursor from NifEN. The integrity of the precursor is reflected by the restoration of the precursor-specific EPR signal, as well as the full proficiency of the precursor in biosynthesis and catalysis upon its incorporation into the precursor-deficient NifEN. Moreover, XAS/EXAFS analysis supports the eight-iron model of the precursor, suggesting that the insertion of heterometal into the precursor involves exchanging one terminal iron atom for a molybdenum atom.

Stepwise formation of P-cluster in nitrogenase MoFe protein

The P-cluster of nitrogenase is one of the most complex biological metallocenters known to date. Despite the recent advances in the chemical synthesis of P-cluster topologs, the biosynthetic mechanism of P-cluster has not been well defined. Here, we present a combined biochemical, electron paramagnetic resonance, and X-ray absorption spectroscopy/extended X-ray absorption fine-structure investigation of the maturation process of P-clusters in ΔnifH molybdenum-iron (MoFe) protein. Our data indicate that the previously identified, [Fe4S4]-like cluster pairs in ΔnifH MoFe protein are indeed the precursors to P-clusters, which can be reductively coupled into the mature [Fe8S7] structures in the presence of Fe protein, MgATP, and dithionite. Moreover, our observation of a biphasic maturation pattern of P-clusters in ΔnifH MoFe protein provides dynamic proof for the previously hypothesized, stepwise assembly mechanism of the two P-clusters in the α2β2-tetrameric MoFe protein, i.e., one P-cluster is formed in one αβ dimer before the other in the second αβ dimer.

Tracing the interstitial carbide of the nitrogenase cofactor during substrate turnover.

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.