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Title (Primary) An in vitro reconstitution system to monitor iron transfer to the active site during the maturation of [NiFe]-hydrogenase
Author Soboh, B.; Adrian, L.; Stripp, S.T.
Journal Journal of Biological Chemistry
Year 2022
Department UBT
Volume 298
Issue 9
Page From art. 102291
Language englisch
Topic T7 Bioeconomy
Supplements https://ars.els-cdn.com/content/image/1-s2.0-S0021925822007335-mmc1.docx
Keywords biosynthesis; cofactor assembly; FTIR spectroscopy; metalloproteins; protein-protein interactions
Abstract NiFe]-hydrogenases comprise a small and a large subunit. The latter harbors the biologically unique [NiFe](CN)2CO active site cofactor. The maturation process includes the assembly of the [Fe](CN)2CO cofactor precursor, nickel binding, endoproteolytic cleavage of the large subunit, and dimerization with the small subunit to yield active enzyme. The biosynthesis of the [Fe](CN)2CO moiety of [NiFe]-Hydrogenase 1 (Hyd-1) and Hyd-2 occurs on the scaffold complex HybG-HypD (GD), whereas the HypC-HypD complex (CD) is specific for the assembly of Hyd-3. The metabolic source and the route for delivering iron to the active site remain unclear. To investigate the maturation process of O2-tolerant Hyd-1 from Escherichia coli, we developed an enzymatic in vitro reconstitution system that allows for the synthesis of Hyd-1 using only purified components. Together with this in vitro reconstitution system, we employed biochemical analyses, infrared spectroscopy (ATR FTIR), mass spectrometry, and microscale thermophoresis (MST) to monitor the iron transfer during the maturation process and to understand how the [Fe](CN)2CO cofactor precursor is ultimately incorporated into the large subunit. We demonstrate the direct transfer of iron from 57Fe-labeled GD complex to the large subunit of Hyd-1. Our data reveal that the GD complex exclusively interacts with the large subunit of Hyd-1 and Hyd-2 but not with the large subunit of Hyd-3. Furthermore, we show that the presence of iron in the active site is a prerequisite for nickel insertion. Taken together, these findings reveal how the [Fe](CN)2CO cofactor precursor is transferred and incorporated into the active site of [NiFe]-hydrogenase.
Persistent UFZ Identifier https://www.ufz.de/index.php?en=20939&ufzPublicationIdentifier=26402
Soboh, B., Adrian, L., Stripp, S.T. (2022):
An in vitro reconstitution system to monitor iron transfer to the active site during the maturation of [NiFe]-hydrogenase
J. Biol. Chem. 298 (9), art. 102291