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.