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Issue 31, 2009
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14NHYSCORE investigation of the H-cluster of [FeFe] hydrogenase: evidence for a nitrogen in the dithiol bridge

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Abstract

Hydrogenases are enzymes catalyzing the reversible heterolytic splitting of molecular hydrogen. Despite extensive investigations of this class of enzymes its catalytic mechanism is not yet well understood. In this paper spectroscopic investigations of the active site of [FeFe] hydrogenase are presented. The so-called H-cluster consists of a bi-nuclear catalytically active subcluster connected to a [4Fe4S] ferredoxin-like unit via a Cys–thiol bridge. An important feature of the H-cluster is that both irons in the bi-nuclear subcluster are coordinated by CN and CO ligands. The bi-nuclear site also carries a dithiol bridge, whose central atom has not yet been identified. Nitrogen and oxygen are the most probable candidates from a mechanistic point of view. Here we present a study of the 14N nuclear quadrupole and hyperfine interactions of the active oxidized state of the H-cluster using advanced EPR methods. In total three 14N nuclei with quadrupole couplings of 0.95 MHz, 0.35 MHz and 1.23 MHz were detected using hyperfine sublevel correlation spectroscopy (HYSCORE). The assignment of the signals is based on their 14N quadrupole couplings in combination with DFT calculations. One signal is assigned to the CN ligand of the distal iron, one to a Lys side chain nitrogen and one to the putative nitrogen of the dithiol bridge. Hence, these results provide the first experimental evidence for a di-(thiomethyl)amine ligand (–S–CH2–NH–CH2–S–) in the bi-nuclear subcluster. This finding is important for understanding the mechanism of [FeFe] hydrogenases, since the nitrogen is likely to act as an internal base facilitating the heterolytic splitting/formation of H2.

Graphical abstract: 14N HYSCORE investigation of the H-cluster of [FeFe] hydrogenase: evidence for a nitrogen in the dithiol bridge

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Publication details

The article was received on 24 Mar 2009, accepted on 01 May 2009 and first published on 09 Jun 2009


Article type: Paper
DOI: 10.1039/B905841A
Phys. Chem. Chem. Phys., 2009,11, 6592-6599

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    14 N HYSCORE investigation of the H-cluster of [FeFe] hydrogenase: evidence for a nitrogen in the dithiol bridge

    A. Silakov, B. Wenk, E. Reijerse and W. Lubitz, Phys. Chem. Chem. Phys., 2009, 11, 6592
    DOI: 10.1039/B905841A

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