Combining a Nitrogenase Scaffold and a Synthetic Compound into an Artificial Enzyme

Abstract

Nitrogenase catalyzes substrate reduction at its cofactor center ([(Cit)MoFe 7 S 9 C] n− ; designated M-cluster). Here, we report the formation of an artificial, nitrogenase-mimicking enzyme upon insertion of a synthetic model complex ([Fe 6 S 9 (SEt) 2 ] 4− ; designated Fe 6 RHH ) into the catalytic component of nitrogenase (designated NifDK). Two Fe 6 RHH clusters were inserted into NifDK, rendering the resultant protein (designated NifDK Fe ) in a similar conformation to that upon insertion of native M-clusters. NifDK Fe could work together with the reductase component of nitrogenase to reduce C 2 H 2 in an ATP-dependent reaction. It could also act as an enzyme on its own in the presence of Eu(II) DTPA, displaying a strong activity in C 2 H 2 reduction while demonstrating an ability to reduce CN − to C1-C3 hydrocarbons in an ATP-independent manner.

Reconstruction of Nitrogenase Predecessors Suggests Origin from Maturase-Like Proteins

“…This is particularly important for assessing whether metal availability significantly guides protein evolution or whether other internal biophysical constraints lead to background-dependent, epistatic interactions ( Williams and da Silva 1996 ; Anbar and Knoll 2002 ; Moore et al 2017 ; Smethurst and Shcherbik 2021 ). Efforts to generate artificial nitrogenases and nitrogenase metalloclusters ( Tanifuji et al 2015 ; Sickerman et al 2017 ) may expand the suite of molecular structures capable of reducing N 2 , but biotic experiments integrating gene regulatory and protein–protein interaction constraints are needed to test different macroevolutionary hypotheses of nitrogenase emergence. A survey of such functional constraints on nitrogenase and maturase predecessors could reveal the sequence of biomolecular functions conducive for the evolution of nitrogen fixation, which could then be integrated into a more comprehensive accounting of internal selective forces, geochemical features, and planetary environments that can host similar evolutionary pathways ( Kacar et al 2021 ).…”

Reconstruction of nitrogenase predecessors suggests origin from maturase-like proteins

“…This is particularly important for assessing whether metal availability significantly guides protein evolution or whether other internal biophysical constraints lead to background-dependent, epistatic interactions (Williams and da Silva 1996; Anbar and Knoll 2002; Moore et al 2017; Smethurst and Shcherbik 2021). Efforts to generate artificial nitrogenases and nitrogenase metalloclusters (Tanifuji et al 2015; Sickerman et al 2017) may expand the suite of molecular structures capable of reducing N 2 , but biotic experiments integrating gene regulatory and protein-protein interaction constraints are needed to test different macroevolutionary hypotheses of nitrogenase emergence. A survey of such functional constraints on nitrogenase and maturase predecessors could reveal the sequence of biomolecular functions conducive for the evolution of nitrogen fixation, which could then be integrated into a more comprehensive accounting of internal selective forces, geochemical features and planetary environments that can host similar evolutionary pathways (Kacar et al 2020).…”

Semisynthetic Hydrogenases Propel Biological Energy Research into a New Era

“…66 More recently, a FeMoco deficient nitrogenase was combined with a synthetic mimic to generate an artificial enzyme with partial catalytic activity. 67 One can envision that ultimately, when effective strategies are available for synthesis of the true FeMoco or analogous compounds that give native-like catalytic activity when inserted into nitrogenase, the same advances in understanding of enzyme function can be made as discussed here for hydrogenase.…”