Journal
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
Volume -, Issue -, Pages -Publisher
AMER CHEMICAL SOC
DOI: 10.1021/jacs.2c06400
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Funding
- Deutsche Forschungsgemein-schaft (DFG, German Research Foundation) [10043725 (ZE 510/2-1), 10043724 (LE 2934/1-1), 311062227 (ZE 510/2-2), 311062227 (LE 2934/1-2), 2008-390540038]
- EU's Horizon 2020 Research and Innovation Programme [810856]
- Leverhulme Trust [RPG-2018-188]
- Einstein Foundation Berlin
- MEXT KAKENHI [18H05516]
- JSPS KAKENHI [19H00984]
- STFC [20130007]
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The NAD(+)-reducing [NiFe] hydrogenase from Hydrogenophilus thermoluteolus shows promising potential for biotechnological applications due to its O2 tolerance and thermostability. Through studying the active-site structure and catalytic activity of native HtSH and its variants, it was revealed that oxidized HtSH features unusual active-site states with unique responses to O2, potentially serving as a mechanism to protect the enzyme from oxidative damage.
NAD(+)-reducing [NiFe] hydrogenases are valuable biocatalysts for H-2-based energy conversion and the regeneration of nucleotide cofactors. While most hydrogenases are sensitive toward O-2 and elevated temperatures, the soluble NAD(+)-reducing [NiFe] hydrogenase from Hydrogenophilus thermoluteolus (HtSH) is O-2 tolerant and thermostable. Thus, it represents a promising candidate for biotechnological applications. Here, we have investigated the catalytic activity and active-site structure of native HtSH and variants in which a glutamate residue in the active-site cavity was replaced by glutamine, alanine, and aspartate. Our biochemical, spectroscopic, and theoretical studies reveal that at least two active-site states of oxidized HtSH feature an unusual architecture in which the glutamate acts as a terminal ligand of the active-site nickel. This observation demonstrates that crystallographically observed glutamate coordination represents a native feature of the enzyme. One of these states is diamagnetic and characterized by a very high stretching frequency of an iron-bound active-site CO ligand. Supported by density-functional-theory calculations, we identify this state as a high-valent species with a biologically unprecedented formal Ni(IV) ground state. Detailed insights into its structure and dynamics were obtained by ultrafast and two-dimensional infrared spectroscopy, demonstrating that it represents a conformationally strained state with unusual bond properties. Our data further show that this state is selectively and reversibly formed under oxic conditions, especially upon rapid exposure to high O-2 levels. We conclude that the kinetically controlled formation of this six-coordinate high-valent state represents a specific and precisely orchestrated stereoelectronic response toward O2 that could protect the enzyme from oxidative damage.
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