Reversible Glutamate Coordination to High-Valent Nickel Protects the Active Site of a [NiFe] Hydrogenase from Oxygen

Abstract

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.

The Fully Oxidized State of the Glutamate Coordinated O2-Tolerant [NiFe]-Hydrogenase Shows a Ni(III)/Fe(III) Open-Shell Singlet Ground State

“…Comparison of experimental and calculated vibrational spectra for the oxidized state of the [NiFe]-hydrogenase HtSH . a Experimental and Ni(IV)/Fe(II) calculated spectra are taken from ref . Closed-shell (CS), triplet state (TS), and broken-symmetry (BS) spectra are from this work.…”

“…31,32 Recently, the effect of such a reversible glutamate coordination was investigated in order to link crystallographic and spectral features. 1 Ultrafast and two-dimensional infrared spectroscopy were used to give details into the structure and dynamics of the formation of the conformationally strained oxidized structure. The analysis and interpretation of experimental data was supported by density functional theory (DFT) calculations.…”

A Hitchhiker’s Guide to Supplying Enzymatic Reducing Power into Synthetic Cells

“… 75 Importantly, unless anaerobic conditions are ensured thorough the whole isolation procedure, the purified SH requires a short reducing pretreatment (with more than 5 μM NADH) to be active in both the conformations of tetramer or hexamer, as the enzyme is inactivated over time when in contact with the air dioxygen. 76 A recent insight on the active site of SH of H. thermoluteolus suggested a protective role of Glu32 from oxidative damage, 112 preventing the access of dioxygen to the embedded and cysteine-coordinated nickel ion; further exploration on the mechanism of oxygen tolerance is needed to promote a broader biotechnological use of SHs. In-depth studies on the cofactor specificity have not been conducted so far on SHs, but a first insight was provided by the work of Preissler and colleagues: 113 they relaxed the strict NAD + -dependence of SH isolated from C. necator via a double mutation E341A/S342R that allowed to recognize also NADP + as a substrate with similar affinity to NAD + , although with a four times lower turnover number.…”

“…75 Importantly, unless anaerobic conditions are ensured thorough the whole isolation procedure, the purified SH requires a short reducing pretreatment (with more than 5 μM NADH) to be active in both the conformations of tetramer or hexamer, as the enzyme is inactivated over time when in contact with the air dioxygen. 76 A recent insight on the active site of SH of H. thermoluteolus suggested a protective role of Glu32 from oxidative damage, 112 preventing the access of dioxygen to the embedded and cysteine-coordinated nickel ion; further exploration on the mechanism of oxygen tolerance is needed to promote a broader biotechnological use of SHs.…”

Trendbericht Anorganische Chemie 2023: Nebengruppen, Bioanorganik und Koordinationschemie

“…39) Zuletzt zur Detektion hochvalenten Nickels in einer speziellen [Ni-Fe]-Hydrogenase (Abbildung 13c). 42) Das Nickelatom stabilisieren dabei eine sechsfache Koordination und eine reversibel bindende Glutamatfunktion. Diese zusätzliche Koordination von Glutamat W…”