Carbon dioxide reduction to methane and coupling with acetylene to form propylene catalyzed by remodeled nitrogenase

Abstract

A doubly substituted form of the nitrogenase MoFe protein (α-70 Val→Ala , α-195 His→Gln ) has the capacity to catalyze the reduction of carbon dioxide (CO 2 ) to yield methane (CH 4 ). Under optimized conditions, 1 nmol of the substituted MoFe protein catalyzes the formation of 21 nmol of CH 4 within 20 min. The catalytic rate depends on the partial pressure of CO 2 (or concentration of HCO 3 − ) and the electron flux through nitrogenase. The doubly substituted MoFe protein also has the capacity to catalyze the unprecedented formation of propylene (H 2 C = CH-CH 3 ) through the reductive coupling of CO 2 and acetylene (HC≡CH). In light of these observations, we suggest that an emerging understanding of the mechanistic features of nitrogenase could be relevant to the design of synthetic catalysts for CO 2 sequestration and formation of olefins.is an abundant and stable form of carbon that is the product of respiration and burning of fossil fuels. As a result of these activities, the atmospheric concentration of CO 2 , a greenhouse gas, has been rising over the last century and contributing to global warming (1). There is strong interest in developing methods for sequestering CO 2 either by capturing it or by chemically converting it to valuable chemicals (2-5). Of particular interest are possible routes to reduction of CO 2 by multiple electrons to yield methanol (CH 3 OH) and methane (CH 4 ), which are renewable fuels (2). The reduction of CO 2 is difficult, with a limited number of reports of metal-based compounds able to catalyze these reactions (6-13). In biology, only a few enzymes are known to reduce CO 2 (14-18), and none of these can catalyze the eight electron reduction to CH 4 .The bacterial Mo-dependent nitrogenase enzyme catalyzes the multielectron/proton reduction of dinitrogen (N 2 ) to two ammonia (NH 3 ) at a metal cluster designated FeMo-cofactor [7Fe-9S-1Mo-1C-R-homocitrate] (Fig. 1) in a reaction that requires ATP hydrolysis and evolution of H 2 , with a minimal reaction stoichiometry shown in Eq. 1 (19)(20)(21)(22).Given that nitrogenase is effective at catalyzing the difficult multielectron reduction of N 2 , it was of interest to determine whether this enzyme might also catalyze the reduction of CO 2 to the level of CH 4 . Nitrogenase is known to have the capacity to reduce a variety of other small, relatively inert, doubly or triply bonded compounds, such as acetylene (HC≡CH) (19,23). It has been shown that an alternative form of nitrogenase, which contains V in place of Mo in the active site cofactor, has the remarkable capacity to reduce CO and couple multiple CO molecules, yielding short chain alkenes and alkanes such as ethylene (C 2 H 4 ), ethane (C 2 H 6 ), propylene (C 3 H 6 ), and propane (C 3 H 8 ) (24,25). In contrast, the Mo-nitrogenase is only able to reduce CO at exceedingly low rates (24). However, we have found that the MoFe protein can be remodeled by substitution of amino acid residues that provide the first shell of noncovalent interactions with the active site FeMo cofa...

Light-driven carbon dioxide reduction to methane by nitrogenase in a photosynthetic bacterium

“…A variant of the FeMo-containing nitrogenase protein, NifD, from Azotobacter vinelandii containing amino acid substitutions V70A and H195Q reduces CO 2 to CH 4 and also produces H 2 in vitro, but it is unable to reduce N 2 to NH 3 (3). Nucleotide substitutions to generate homologous substitutions of residues in R. palustris NifD (V75A and H201Q) were introduced into the nifD coding sequence by homologous recombination.…”

“…Electrons derived from the oxidation of either an organic carbon source (acetate) or an inorganic compound (thiosulfate) are used to reduce CO 2 to CH 4 . HCO 3 − acquired from outside the cell is converted to intracellular CO 2 by carbonic anhydrase (11). Some CO 2 is also generated from acetate metabolism.…”

“…R. palustris can also synthesize alternative V or Fe nitrogenase isozymes, but these isozymes are not expressed in the NifA* strain grown with ammonium (5, 9). The NifA* strain expressing the remodeled nitrogenase produced CH 4 when grown with acetate as a carbon source and HCO 3 − as a source of CO 2 ( Table 1). The growth rate of this strain was similar to that of its parent strain expressing WT nitrogenase.…”

Photosynthetic fuel for heterologous enzymes: the role of electron carrier proteins

“…Further, its ability to carry out reductive coupling of a wide variety of small alkenes, alkynes as well as N-, S-, and O-containing hydrocarbons makes this enzyme highly interesting for metabolic engineering (Seefeldt et al 2013). Yang and colleagues expanded the scope of nitrogenase reactions by introducing two amino acid substitutions in the substrate-binding pockets, conferring an ability to reduce CO 2 to methane or couple it with acetylene to form the polymer precursor propylene (Yang et al 2012). Recently, this engineered enzyme was shown to produce methane in a light-dependent manner upon expression in the purple non-sulfur bacterium Rhodopseudomonas palustris (Fixen et al 2016).…”

Recent Progress and Novel Applications in Enzymatic Conversion of Carbon Dioxide

“…It was found that the nitrogenase MoFe protein is able to catalyze the CO2 reduction to methane (CH4) [45]. In total, 21 nmol of CH4 can be formed using 1 nmol of this enzyme under optimized conditions within 20 min.…”