1. Stepwise assembly of the active site of [NiFe]-hydrogenase
- Author
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Caserta, Giorgio, Hartmann, Sven, Van Stappen, Casey, Karafoulidi-Retsou, Chara, Lorent, Christian, Yelin, Stefan, Keck, Matthias, Schoknecht, Janna, Sergueev, Ilya, Yoda, Yoshitaka, Hildebrandt, Peter, Limberg, Christian, DeBeer, Serena, Zebger, Ingo, Frielingsdorf, Stefan, and Lenz, Oliver
- Abstract
[NiFe]-hydrogenases are biotechnologically relevant enzymes catalyzing the reversible splitting of H2into 2e−and 2H+under ambient conditions. Catalysis takes place at the heterobimetallic NiFe(CN)2(CO) center, whose multistep biosynthesis involves careful handling of two transition metals as well as potentially harmful CO and CN−molecules. Here, we investigated the sequential assembly of the [NiFe] cofactor, previously based on primarily indirect evidence, using four different purified maturation intermediates of the catalytic subunit, HoxG, of the O2-tolerant membrane-bound hydrogenase from Cupriavidus necator. These included the cofactor-free apo-HoxG, a nickel-free version carrying only the Fe(CN)2(CO) fragment, a precursor that contained all cofactor components but remained redox inactive and the fully mature HoxG. Through biochemical analyses combined with comprehensive spectroscopic investigation using infrared, electronic paramagnetic resonance, Mössbauer, X-ray absorption and nuclear resonance vibrational spectroscopies, we obtained detailed insight into the sophisticated maturation process of [NiFe]-hydrogenase.
- Published
- 2023
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