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Nitrogen Fixation and Hydrogen Evolution by Sterically Encumbered Mo-Nitrogenase

[Image: see text] The substrate-reducing proteins of all nitrogenases (MoFe, VFe, and FeFe) are organized as α(2)ß(2)(γ(2)) multimers with two functional halves. While their dimeric organization could afford improved structural stability of nitrogenases in vivo, previous research has proposed both n...

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Autores principales: Cadoux, Cécile, Ratcliff, Daniel, Maslać, Nevena, Gu, Wenyu, Tsakoumagkos, Ioannis, Hoogendoorn, Sascha, Wagner, Tristan, Milton, Ross D.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Chemical Society 2023
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10207099/
https://www.ncbi.nlm.nih.gov/pubmed/37234119
http://dx.doi.org/10.1021/jacsau.3c00165
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author Cadoux, Cécile
Ratcliff, Daniel
Maslać, Nevena
Gu, Wenyu
Tsakoumagkos, Ioannis
Hoogendoorn, Sascha
Wagner, Tristan
Milton, Ross D.
author_facet Cadoux, Cécile
Ratcliff, Daniel
Maslać, Nevena
Gu, Wenyu
Tsakoumagkos, Ioannis
Hoogendoorn, Sascha
Wagner, Tristan
Milton, Ross D.
author_sort Cadoux, Cécile
collection PubMed
description [Image: see text] The substrate-reducing proteins of all nitrogenases (MoFe, VFe, and FeFe) are organized as α(2)ß(2)(γ(2)) multimers with two functional halves. While their dimeric organization could afford improved structural stability of nitrogenases in vivo, previous research has proposed both negative and positive cooperativity contributions with respect to enzymatic activity. Here, a 1.4 kDa peptide was covalently introduced in the proximity of the P cluster, corresponding to the Fe protein docking position. The Strep-tag carried by the added peptide simultaneously sterically inhibits electron delivery to the MoFe protein and allows the isolation of partially inhibited MoFe proteins (where the half-inhibited MoFe protein was targeted). We confirm that the partially functional MoFe protein retains its ability to reduce N(2) to NH(3), with no significant difference in selectivity over obligatory/parasitic H(2) formation. Our experiment concludes that wild-type nitrogenase exhibits negative cooperativity during the steady state regarding H(2) and NH(3) formation (under Ar or N(2)), with one-half of the MoFe protein inhibiting turnover in the second half. This emphasizes the presence and importance of long-range (>95 Å) protein–protein communication in biological N(2) fixation in Azotobacter vinelandii.
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spelling pubmed-102070992023-05-25 Nitrogen Fixation and Hydrogen Evolution by Sterically Encumbered Mo-Nitrogenase Cadoux, Cécile Ratcliff, Daniel Maslać, Nevena Gu, Wenyu Tsakoumagkos, Ioannis Hoogendoorn, Sascha Wagner, Tristan Milton, Ross D. JACS Au [Image: see text] The substrate-reducing proteins of all nitrogenases (MoFe, VFe, and FeFe) are organized as α(2)ß(2)(γ(2)) multimers with two functional halves. While their dimeric organization could afford improved structural stability of nitrogenases in vivo, previous research has proposed both negative and positive cooperativity contributions with respect to enzymatic activity. Here, a 1.4 kDa peptide was covalently introduced in the proximity of the P cluster, corresponding to the Fe protein docking position. The Strep-tag carried by the added peptide simultaneously sterically inhibits electron delivery to the MoFe protein and allows the isolation of partially inhibited MoFe proteins (where the half-inhibited MoFe protein was targeted). We confirm that the partially functional MoFe protein retains its ability to reduce N(2) to NH(3), with no significant difference in selectivity over obligatory/parasitic H(2) formation. Our experiment concludes that wild-type nitrogenase exhibits negative cooperativity during the steady state regarding H(2) and NH(3) formation (under Ar or N(2)), with one-half of the MoFe protein inhibiting turnover in the second half. This emphasizes the presence and importance of long-range (>95 Å) protein–protein communication in biological N(2) fixation in Azotobacter vinelandii. American Chemical Society 2023-05-09 /pmc/articles/PMC10207099/ /pubmed/37234119 http://dx.doi.org/10.1021/jacsau.3c00165 Text en © 2023 The Authors. Published by American Chemical Society https://creativecommons.org/licenses/by/4.0/Permits the broadest form of re-use including for commercial purposes, provided that author attribution and integrity are maintained (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Cadoux, Cécile
Ratcliff, Daniel
Maslać, Nevena
Gu, Wenyu
Tsakoumagkos, Ioannis
Hoogendoorn, Sascha
Wagner, Tristan
Milton, Ross D.
Nitrogen Fixation and Hydrogen Evolution by Sterically Encumbered Mo-Nitrogenase
title Nitrogen Fixation and Hydrogen Evolution by Sterically Encumbered Mo-Nitrogenase
title_full Nitrogen Fixation and Hydrogen Evolution by Sterically Encumbered Mo-Nitrogenase
title_fullStr Nitrogen Fixation and Hydrogen Evolution by Sterically Encumbered Mo-Nitrogenase
title_full_unstemmed Nitrogen Fixation and Hydrogen Evolution by Sterically Encumbered Mo-Nitrogenase
title_short Nitrogen Fixation and Hydrogen Evolution by Sterically Encumbered Mo-Nitrogenase
title_sort nitrogen fixation and hydrogen evolution by sterically encumbered mo-nitrogenase
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10207099/
https://www.ncbi.nlm.nih.gov/pubmed/37234119
http://dx.doi.org/10.1021/jacsau.3c00165
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