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Postbiosynthetic modification of a precursor to the nitrogenase iron-molybdenum cofactor.

ABSTRACT: Nitrogenases utilize Fe-S clusters to reduce N₂ to NH₃ The large number of Fe sites in their catalytic cofactors has hampered spectroscopic investigations into their electronic structures, mechanisms, and biosyntheses. To facilitate their spectroscopic analysis, we are developing methods for incorporating ⁵⁷Fe into specific sites of nitrogenase cofactors, and we report herein site-selective ⁵⁷Fe labeling of the L-cluster-a carbide-containing, [Fe₈S₉C] precursor to the Mo nitrogenase catalytic cofactor. Treatment of the isolated L-cluster with the chelator ethylenediaminetetraacetate followed by reconstitution with ⁵⁷Fe²⁺ results in ⁵⁷Fe labeling of the terminal Fe sites in high yield and with high selectivity. This protocol enables the generation of L-cluster samples in which either the two terminal or the six belt Fe sites are selectively labeled with ⁵⁷Fe. Mössbauer spectroscopic analysis of these samples bound to the nitrogenase maturase Azotobacter vinelandii NifX reveals differences in the primary coordination sphere of the terminal Fe sites and that one of the terminal sites of the L-cluster binds to H35 of Av NifX. This work provides molecular-level insights into the electronic structure and biosynthesis of the L-cluster and introduces postbiosynthetic modification as a promising strategy for studies of nitrogenase cofactors.

SUBMITTER: Srisantitham S

PROVIDER: S-EPMC7980433 | biostudies-literature |

REPOSITORIES: biostudies-literature

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