Extending the Carbon Chain: Hydrocarbon Formation Catalyzed by Vanadium/Molybdenum Nitrogenases

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Science  05 Aug 2011:
Vol. 333, Issue 6043, pp. 753-755
DOI: 10.1126/science.1206883


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.

Nitrogenase enzymes catalyze the reduction of N2 to NH3, a key step in the global nitrogen cycle (1). The molybdenum (Mo)– and vanadium (V)–dependent nitrogenases are two homologous members of this enzyme family, distinguished primarily by the heterometals (Mo or V) at their respective cofactor centers (FeMoco or FeVco) (2). Both nitrogenases are composed of a specific reductase (nifH- or vnfH-encoded Fe protein) and a catalytic component (nifDK-encoded MoFe protein or vnfDGK-encoded VFe protein). Additionally, both enzymes presumably form a functional complex between the two component proteins during catalysis (3), in which electrons are transferred in an adenosine triphosphate (ATP)–dependent process from the reductase to the cofactor site of the catalytic component, where substrate reduction eventually occurs.

The homology between Mo and V nitrogenases renders their substrate profiles highly similar (4); yet, the two nitrogenases react differently with CO. In a small-scale reaction (5, 6), V nitrogenase catalyzes reductive catenation of CO to C2H4, C2H6, and C3H8; in contrast, these products have not been detected in the same reaction catalyzed by the wild-type Mo nitrogenase, although a very recent report has indicated that the Valα70-substituted Mo nitrogenase variants can catalyze the formation of low–molecular-weight hydrocarbon products from CO (79). The ability of V nitrogenase to catalyze hydrocarbon formation from CO is somewhat analogous to the capacities of late transition metal catalysts in the industrial Fischer-Tropsch process (10). However, the enzymatic reaction uses H+, rather than of H2, for hydrocarbon formation (11), and it requires the participation of both components of the enzyme, the hydrolysis of ATP, and the supplies of electron sources (7). Thus, V nitrogenase–based hydrocarbon formation probably involves the reductive protonation of CO, a process that parallels the nitrogenase-based NH3 formation pathway, which involves the reductive protonation of N2.

A fourth hydrocarbon product of V nitrogenase catalysis, C3H6, can be detected in the standard reaction upon the substitution of H2O by D2O (11). One possible explanation for this effect is a stalling of the reaction by D2O and the consequent accumulation of intermediates that are normally turned over quickly into other products. This observation leads to the speculation that other hydrocarbon products may have remained unidentified because of their minor yields. In this scenario, a sufficiently scaled-up reaction would allow us to identify these products either directly in H2O or after the substitution of H2O by D2O.

Indeed, when we scaled up the reaction 130-fold, C3H6—along with C2H4, C2H6, and C3H8—was readily detected in the H2O-based reactions using either 12CO (fig. S1, B and C, “1” traces) or 13CO (fig. S1, B and C, “2” traces) as the substrate (12). Moreover, we identified three additional products in the large-scale, H2O-based assays. Gas chromatography–mass spectrometry (GC-MS) analyses revealed the identities of these products as CH4, α-C4H8, and n-C4H10, which displayed mass/charge (m/z) ratios of 16.032, 56.064, and 58.080, respectively, in the reaction of 12CO (fig. S1, A and D, “1” traces) and the expected mass shifts of +1, +4, and +4, respectively, on substituting 13CO for 12CO (fig. S1, A and D, “2” traces). This complete set of products was also detected in the D2O-based reactions with the mass shifts expected for the D/H substitution (fig. S1, A to D, “3” and “4” traces).

Unexpectedly, when V nitrogenase was replaced by Mo nitrogenase in the scaled-up reaction, the same two- and three-carbon products were detected in the H2O-based reaction (fig. S2, B and C). Thus, like its V counterpart, Mo nitrogenase used CO and H+ as the carbon and hydrogen sources for hydrocarbon formation. Furthermore, the deuterium effect documented for C3H6 in the small-scale V nitrogenase reaction (11) was duplicated for α-C4H8 and n-C4H10 in the scaled-up Mo nitrogenase reaction. These four-carbon products, which were not detected in the H2O-based reaction (fig. S2D, 1 and 2 traces), could be clearly identified in the D2O-based reaction (fig. S2D, 3 and 4 traces). In contrast, the one-carbon product (CH4) remained unidentified, even with the substitution of H2O by D2O (fig. S2A, 3 and 4 traces), suggesting that CH4 (if any) was formed at an extremely low yield in the Mo nitrogenase-catalyzed reaction (13).

The observation of CO reduction by Mo nitrogenase comes as a big surprise, as CO has never been shown to be a substrate of Mo nitrogenase; instead, it has long been regarded as a potent inhibitor for the reduction of all known substrates of Mo nitrogenase except H+ (1). The presence of CO suppresses the ability of Mo nitrogenase to turn over substrates other than H+; consequently, the electrons that are normally used for the reduction of these substrates are routed toward the concomitant reduction of H+. The fact that CO inhibits the V nitrogenase–catalyzed H+ reduction by ~76% suggests a rerouting of electrons from H+ reduction to CO reduction, an argument supported by the formation of hydrocarbon products from CO in this reaction (4, 7). In contrast, CO exerts a minimal impact on the H+ reduction by Mo nitrogenase, indicating that an almost untraceable quantity of electrons (14) are diverted from H+ reduction to CO reduction, thereby limiting the ability of Mo nitrogenase to generate hydrocarbons from CO.

Consistent with this hypothesis, quantitative GC analyses show that Mo nitrogenase (Fig. 1B) is considerably less efficient than its V counterpart (Fig. 1A) in hydrocarbon formation, and the total specific activity of product formation by the former is only ~0.1% and ~2%, respectively, of that by the latter in H2O- and D2O-based reactions (Fig. 1C). The disparate CO-reducing activities of V and Mo nitrogenase parallel the differential capacities of synthetic V and Mo compounds to reductively couple two CO moieties into functionalized acetylene ligands (15), suggesting that V (a group VB transition metal) is superior to Mo (a group VIB transition metal) in this particular type of reaction. The substitution of H2O by D2O, however, has a much more dramatic effect on the reaction catalyzed by Mo nitrogenase than that catalyzed by its V counterpart, leading to an increase in the specific activity of total product formation by ~21-fold in the case of the former (Fig. 1C, Mo, 2 versus 1) and by only ~12% in the case of the latter (Fig. 1C, V, 2 versus 1). In both cases, the increase in hydrocarbon formation is accompanied by a ~30% decrease in D2 evolution, suggesting a possible shift of electrons from D+ reduction toward CO reduction (16).

Fig. 1

Specific activities for individual product formation catalyzed by (A) V and (B) Mo nitrogenase and (C) comparison of total activities for hydrocarbon formation by the two nitrogenases in the presence of (1) H2O or (2) D2O. Numbers in circles: 1, CH4; 2, C2H4; 3, C2H6; 4, C3H6; 5, C3H8; 6, α-C4H8; and 7, n-C4H10. Expanded scales of α-C4H8 and n-C4H10 formation are shown as insets. Specific activities were determined by quantitative GC analyses and calculated on the basis of the formation of products (see fig. S3) in the first 10 min of reaction (linear range). Data are presented as mean ± SD (N = 5 repetitions of each experiment.).

A closer examination of the product distributions of V and Mo nitrogenases reveals additional features that set the reactions catalyzed by the two nitrogenases apart (Fig. 2 and table S1). Compared with V nitrogenase (Fig. 2A, 1), Mo nitrogenase (Fig. 2B, 1) displays a much lower C2H4/C2H6 ratio in its product profile. Moreover, Mo nitrogenase generates a lower percentage of two-carbon products and rather seems to shift the H2O-based reaction toward the formation of C3H6 and C3H8. In the presence of D2O, however, Mo nitrogenase shifts the product distribution back toward two-carbon products; additionally, there is an appreciable increase in the C2H4/C2H6 ratio of its product profile (Fig. 2B, 2). Contrary to Mo nitrogenase, V nitrogenase is less affected by the deuterium effect. Nevertheless, V nitrogenase shows a small decrease in the C2H4/C2H6 ratio of its product profile and a slight shift toward the formation of three- and four-carbon products in the D2O-based reaction, an opposite trend compared with its Mo counterpart (Fig. 2A, 2). Notably, these opposite changes in D2O render a closer resemblance between the product profiles of V and Mo nitrogenases (Fig. 2, A versus B, 2), pointing to possible similarities between the CO-reducing mechanisms of these two nitrogenases.

Fig. 2

Distributions of hydrocarbons formed by (A) V and (B) Mo nitrogenases in the presence of (1) H2O or (2) D2O. The total amounts of hydrocarbons detected in (A) V- and (B) Mo-based reactions were set as 100%, and the percentages of individual products were determined accordingly for each nitrogenase. The alkene/alkane ratios of V and Mo nitrogenases are summarized in table S1.

One common trait shared by the reactions catalyzed by V and Mo nitrogenases is the overwhelmingly predominant formation/release of C2H4 (Figs. 1 and 2). The fact that CH4—the one-carbon product that could result directly from the reductive protonation of CO—is observed in much lower abundance than C2H4 suggests that the formation of a C-C bond between CO derivatives is relatively rapid. The observation that C2H6—the two-carbon product that could originate from the reductive protonation of C2H4—drops sharply in quantity relative to C2H4 indicates that the release of C2H4 is favored over the further reduction of this product. Furthermore, the huge discrepancy between the amounts of two- and three-/four-carbon products points to a potential competition between the formation of two-carbon products (particularly C2H4) and the extension of the carbon chain from the same two-carbon intermediate. The preferred C2H4 formation catalyzed by nitrogenases probably reflects a dominant impact of kinetic factors that guide the overall product distribution, resulting in a drastically decreased yield of hydrocarbon formation beyond two-carbon length.

A second conserved feature between the reactions catalyzed by the two nitrogenases is the formation of products in the following rank: CH2=CH2 >> CH3–CH3 > CH3–CH2–CH3 > CH2=CH2–CH3 > CH3–CH2–CH2–CH3 > CH2=CH2–CH2–CH3 (Figs. 1 and 2). As such, except for the two-carbon products, both nitrogenases display a preference toward the formation of saturated three- or four-carbon alkanes rather than their corresponding unsaturated alkenes, suggesting a possible mechanistic switch after the formation of the first C-C bond. One plausible account for the reaction beyond the two-carbon stage involves the formation of alkene before a fast protonation step that converts alkene to alkane. Such a hypothesis is supported by two lines of evidence. First, Mo nitrogenase displays a product profile of higher three- and four-carbon alkene/alkane ratios than its V counterpart (table S1), which could be explained by its limited access to electrons for CO reduction that slows down the coupled electron/proton addition to the unsaturated C=C bonds of alkenes. Second, both V and Mo nitrogenases show increased three- and four-carbon alkene/alkane ratios in the presence of D2O (table S1), which may reflect a stalling effect of deuterium on the protonation step (16).

The discrepancy between the CO-reducing capacities of Mo and V nitrogenases suggests a prevailing impact of heterometal chemistry on the efficiency of the reaction; at the same time, the merging of the product profiles of the two nitrogenases in D2O points to a critical role of coupled electron/proton tunneling in the mechanism of this particular reaction. The discovery that Mo nitrogenase is capable of using CO as a substrate, albeit at a considerably lower rate than its V counterpart, more broadly supports the hypothesis that CO reduction is an evolutionary relic of the function of this enzyme family. The parallelism between the nitrogenase-catalyzed CO and N2 reduction—demonstrated further by the ability of V nitrogenase to form CH4, an analogous product of NH3—strengthens the theory that the ancestral nitrogenase may represent an evolutionary link between carbon and nitrogen cycles on earth (7). In a practical vein, the industrial Fischer-Tropsch process uses H2, a costly syngas component, for CO reduction and shows a tendency toward excessive formation of CH4, a low-value product (17), whereas the nitrogenase-based reaction uses H+ for hydrocarbon formation and favors the formation of C-C bond over the cleavage of C-O bond. These features make nitrogenase an attractive template for cost-efficient hydrocarbon formation that is directed toward C-C coupling and carbon-chain extension. Borrowing a trick or two from this ancient enzyme family, perhaps an efficient strategy could be developed in the future for controlled fuel production from CO? Only time will tell.

Supporting Online Material

Materials and Methods

Figs. S1 to S4

Table S1

References (24, 25)

References and Notes

  1. A small-scale nitrogenase reaction typically features 0.15 mg VFe or MoFe protein, the catalytic component of V or Mo nitrogenase. Such an assay condition was established empirically some 30 years ago (6) and has since been used as the conventional scale of in vitro nitrogenase assays in the field.
  2. Based on the retention time on a gas chromatograph column, the low–molecular-weight hydrocarbon products formed by the Valα70-substituted Mo nitrogenase variants have been assigned as methane, ethylene, ethane, propylene, and propane (9).
  3. Materials and methods are available as supporting material on Science Online.
  4. Given the detection threshold of CH4 (0.0007 nmol per nmol protein per min) and the activity of V nitrogenase in CH4 production (0.12 nmol per nmol protein per min), the Mo nitrogenase is at least 170-fold less active than its V counterpart in catalyzing the formation of CH4 from CO.
  5. Only 0.04% of electrons can be traced in the hydrocarbon products formed in the Mo nitrogenase–catalyzed reaction.
  6. The effect of deuterium on nitrogenase-catalyzed CO reduction is probably multifaceted. Apart from the inverse kinetic isotope effects (i.e., kH/kD < 1, where kH and kD are the rate constants of H2O- and D2O-based reactions, respectively) of D2O that favor the formation of deuterated products (18, 19), other solvent effects of D2O—such as those affecting the protein conformation, the interaction between proteins, and the network of hydrogen bonds—have been observed (2023).
  7. Acknowledgments: We thank D. C. Rees and N. Dalleska of the California Institute of Technology (Pasadena, CA) for help with the GC-MS analysis. This work was supported by Herman Frasch Foundation grant 617-HF07 (M.W.R.) and NIH grant GM 67626 (M.W.R.).
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