O₂-Tolerant H₂ Activation by an Isolated Large Subunit of a [NiFe] Hydrogenase

Abstract

The catalytic properties of hydrogenases are nature's answer to the seemingly simple reaction H ⇌ 2H + 2e. Members of the phylogenetically diverse subgroup of [NiFe] hydrogenases generally consist of at least two subunits, where the large subunit harbors the H-activating [NiFe] site and the small subunit contains iron-sulfur clusters mediating e transfer. Typically, [NiFe] hydrogenases are susceptible to inhibition by O. Here, we conducted system minimization by isolating and analyzing the large subunit of one of the rare members of the group of O-tolerant [NiFe] hydrogenases, namely the preHoxG protein of the membrane-bound hydrogenase from Ralstonia eutropha. Unlike previous assumptions, preHoxG was able to activate H as it clearly performed catalytic hydrogen/deuterium exchange. However, it did not execute the entire catalytic cycle described for [NiFe] hydrogenases. Remarkably, H activation was performed by preHoxG even in the presence of O, although the unique [4Fe-3S] cluster located in the small subunit and described to be crucial for tolerance toward O was absent. These findings challenge the current understanding of O tolerance of [NiFe] hydrogenases. The applicability of this minimal hydrogenase in basic and applied research is discussed.

Infrared Characterization of the Bidirectional Oxygen-Sensitive [NiFe]-Hydrogenase from E. coli

“…This is not the case. With respect to O 2 tolerance, the role of the proximal iron-sulfur cluster in the membrane-bound [NiFe] hydrogenase of Ralstonia eutropha has been questioned recently [56]. The example of HYD-2 from E. coli shows that the reaction with O 2 may involve additional check screws like proton-and electron transfer that remain to be evaluated.…”

A membrane‐bound [NiFe]‐hydrogenase large subunit precursor whose C‐terminal extension is not essential for cofactor incorporation but guarantees optimal maturation

“…Ralstonia eutropha strains HF649, HF1063, HP3, and HP9 were cultivated in FGN mod medium as described elsewhere (Hartmann et al, 2018; Lenz et al, 2018). Cells were harvested by centrifugation (11,500 g , 4°C, 12 min), and the resulting cell pellet was frozen in liquid nitrogen and stored at − 80°C.…”

“…Subsequently, the mature subunits form the HoxGK heterodimer, which is then translocated via the Tat pathway through the cytoplasmic membrane and attached to a membrane‐integral cytochrome b (Frielingsdorf, Schubert, Pohlmann, Lenz, & Friedrich, 2011; Schubert, Lenz, Krause, Volkmer, & Friedrich, 2007). Deletion of the gene encoding the MBH‐specific endopeptidase HoxM results in the accumulation of a HoxG preform still carrying the C‐terminal extension (Bernhard, Schwartz, Rietdorf, & Friedrich, 1996; Hartmann et al, 2018).…”

Insights Into the Redox Sensitivity of Chloroflexi Hup-Hydrogenase Derived From Studies in Escherichia coli: Merits and Pitfalls of Heterologous [NiFe]-Hydrogenase Synthesis

“…Because D. mccartyi species are not amenable to large-scale biochemical analysis, and are currently genetically intractable, using the E. coli Hyd- and Fdh-negative host strains developed here will provide a means of studying the biochemical mechanism(s) underlying the loss of HupSL activity in response to oxidizing redox conditions and whether this effect is linked to a particular iron-sulfur cluster, or clusters, in HupS, or whether the bimetallic cofactor in HupL is the target of irreversible inactivation. A recent study by Hartmann et al (2018) indicates that, at least for certain [NiFe]-hydrogenases, the NiFe(CN) 2 CO cofactor is not sensitive to oxidative conditions, suggesting that it might indeed be the electron-transfer pathway that is disrupted by non-reducing redox potentials.…”