Nitrogenase-catalyzed acrylonitrile reductions

Abstract

Reduction of acrylonitrile is catalyzed by Azotobacter vinelandii nitrogenase (N 2ase); products are propylene, propane, and ammonia but not amines or imines; H 2 evolution occurs concurrently. ATP, Mg ++, and reductant (sodium dithionite) are required. The apparent K m for acrylonitrile is 10–50 mM. Acrylonitrile is a better N 2ase substrate than its saturated analogs propionitrile and acetonitrile. Acrylonitrile reduction is inhibited by N 2 and CO but not by H 2; substrate inhibition occurs at high acrylonitrile concentrations. The partitioning of electron flow through N 2ase between acrylonitrile reduction and H 2 production from H 3O + was directly measured by simultaneous gas chromatographic analyses of hydrocarbons and H 2. The effect of pH, Γ/2, and Na 2S 2O 4 concentrations on partitioning suggests the need for a more specific structure of or additional site on N 2ase for acrylonitrile reduction than for H 2 evolution. D 2O promotes acrylonitrile reduction by increasing the proportion of the total electron flow which reduces acrylonitrile. Neither the binding of acrylonitrile to N 2ase nor the propylene/propane product ratio is significantly affected by D 2O. The isotope effect on acrylonitrile reduction is explained in terms of relative activation energies of fast steps involved in the transfer of electrons to acrylonitrile and to H 2O + or D 3O +. The acrylic double bond shifts during reduction of acrylonitrile to propylene and reduction of the acrylic double bond to propane occurs in ca. 15% of the product. The propylene/propane product ratio shows major sensitivity to pH and ionic strength. Propane formation is suggested to arise from reduction of an allyl-enzyme intermediate or from reduction of acrylonitrile to enzyme-bound propionitrile and subsequent reduction of enzyme-bound propionitrile. Nitrogenase also catalyzes acrylonitrile reduction in vivo. Product ratios are similar in vivo and in vitro and are the best indication of similarity between isolated N 2ase and in vivo N 2ase. Compared with normal molybdenum-containing N 2ase, N 2ase from A. vinelandii cells grown on vanadium in place of molybdenum exhibits a larger apparent K m for acrylonitrile, a less favorable partitioning of electron flow toward acrylonitrile reduction, and a lower propylene/propane product ratio in vitro and in vivo. Molybdenum- and vanadium-containing N 2ases show the same D 2O enhancement of acrylonitrile reduction. In vivo and in vitro results imply a substrate site containing at least molybdenum and a close competition between acrylonitrile and H 3O + for reducing equivalents, and are consistent with a substrate site containing both iron and molybdenum.