[FeFe]-hydrogenase maturation: H-cluster assembly intermediates tracked by electron paramagnetic resonance, infrared, and X-ray absorption spectroscopy.

Content Provider	Europe PMC
Author	Németh, Brigitta Senger, Moritz Redman, Holly J. Ceccaldi, Pierre Broderick, Joan Magnuson, Ann Stripp, Sven T. Haumann, Michael Berggren, Gustav
Abstract	Abstract[FeFe]-hydrogenase enzymes employ a unique organometallic cofactor for efficient and reversible hydrogen conversion. This so-called H-cluster consists of a [4Fe–4S] cubane cysteine linked to a diiron complex coordinated by carbon monoxide and cyanide ligands and an azadithiolate ligand (adt = NH(CH2S)2)·[FeFe]-hydrogenase apo-protein binding only the [4Fe–4S] sub-complex can be fully activated in vitro by the addition of a synthetic diiron site precursor complex ([2Fe]adt). Elucidation of the mechanism of cofactor assembly will aid in the design of improved hydrogen processing synthetic catalysts. We combined electron paramagnetic resonance, Fourier-transform infrared, and X-ray absorption spectroscopy to characterize intermediates of H-cluster assembly as initiated by mixing of the apo-protein (HydA1) from the green alga Chlamydomonas reinhardtii with [2Fe]adt. The three methods consistently show rapid formation of a complete H-cluster in the oxidized, CO-inhibited state (Hox-CO) already within seconds after the mixing. Moreover, FTIR spectroscopy support a model in which Hox-CO formation is preceded by a short-lived Hred′-CO-like intermediate. Accumulation of Hox-CO was followed by CO release resulting in the slower conversion to the catalytically active state (Hox) as well as formation of reduced states of the H-cluster.Graphic abstractElectronic supplementary materialThe online version of this article (10.1007/s00775-020-01799-8) contains supplementary material, which is available to authorized users.
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ISSN	09498257
Journal	Journal of Biological Inorganic Chemistry [J Biol Inorg Chem]
Volume Number	25
DOI	10.1007/s00775-020-01799-8
PubMed Central reference number	PMC7399679
Issue Number	5
PubMed reference number	32661785
e-ISSN	14321327
Language	English
Publisher	Springer Berlin Heidelberg
Publisher Date	2020-07-13
Publisher Place	Berlin/Heidelberg
Access Restriction	Open
Rights License	Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2020
Subject Keyword	Metalloenzymes [FeFe]-hydrogenase H-cluster assembly Time-resolved spectroscopy Maturation intermediates
Content Type	Text
Resource Type	Article
Subject	Biochemistry Inorganic Chemistry