A proof-of-concept study on a catalytic [NiFe] intermediate reveals structural and dynamical details of hydrogenases by ultrafast and two-dimensional infrared spectroscopies.
NAD+-reducing [NiFe] hydrogenases are valuable
biocatalysts
for H2-based energy conversion and the regeneration of
nucleotide cofactors. While most hydrogenases are sensitive toward
O2 and elevated temperatures, the soluble NAD+-reducing [NiFe] hydrogenase from Hydrogenophilus
thermoluteolus (HtSH) is O2-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 O2 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.
Ultrafast two-dimensional infrared (2D-IR) spectroscopy of Escherichia coli Hyd-1 (EcHyd-1) reveals the structural and dynamic influence of the protein scaffold on the Fe(CO)(CN)2unit of the active site. Measurements on...
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