Discovery of novel [FeFe]-hydrogenases for biocatalytic H₂-production

Abstract

A semi-synthetic screening method for mining the biodiversity of [FeFe]-hydrogenases, expanding the toolbox for biocatalytic H2-gas production.

Spectroscopic investigations under whole-cell conditions provide new insight into the metal hydride chemistry of [FeFe]-hydrogenase

“…To generate active hydrogenase in concentrations suitable for whole-cell spectroscopy, the HydA1 protein was heterologously expressed in BL21(DE3) E. coli cells, similar to previous reports. [44][45][46] The absence of the [FeFe]-hydrogenase specic maturation machinery in E. coli results in the synthesis of an inactive form of the enzyme containing the [4Fe-4S] H cluster but lacking the [2Fe] H subsite (apo-HydA1). 50 mL cell cultures were concentrated to 2 mL and depleted of O 2 at which point the synthetic cofactor mimic [2Fe] adt was added to the medium, and the H-cluster assembly monitored by EPR spectroscopy and H 2 production assays.…”

“…It is noteworthy that diverging reactivity was recently reported for two putative sensory [FeFe]-hydrogenases of different origin, which appear to stabilize in an H red -like state both in vitro and in whole cells. 46,50 This arguably reects the different physiological roles of sensory hydrogenases and the prototypical HydA1 enzyme. 51 In addition to the relatively stable catalytic intermediates H ox and H red , the study revealed complex metal-hydride chemistry occurring in living cells and two different hydride states were observed.…”

“…7,13,[41][42][43] We recently adopted this strategy for in vivo applications, enabling the preparation of fully functional, semisynthetic enzymes in both E. coli and cyanobacteria. 6,[44][45][46] Herein, we take advantage of this protocol to perform spectroscopic investigations of [FeFe]-hydrogenase in whole cells (Fig. 1).…”

“…In the next step, we used attenuated total reection Fouriertransform infrared (ATR FTIR) spectroscopy to probe the catalytic competence of hydrated [2Fe] adt -HydA1-containing E. coli lms. Following reported protocols, 46 E. coli cell suspensions were deposited on the silicon crystal of the ATR optic, dried under inert gas (i.e., N 2 or Ar), and rehydrated by running the gas through a wash bottle, forming a bacterial lm that can react to changes in atmospheric gas composition. When the [2Fe] adt -HydA1-containing E. coli lm was kept under 100 mbar deuterium gas (D 2 ) instead of N 2 , the appearance of a band at 2515 cm À1 suggests that deuterium ions (D + ) were released into bulk H 2 O (Fig.…”

New Approaches to an Old Problem: Original Techniques in [FeFe]-hydrogenase Research

“…[66][67][68] Arguably, the main impact of artificial maturation to-date has been the simplified preparation of the active enzyme 60,69 including site-selective isotopologues, [70][71][72] which also facilitates rapid screening protocols. 30 Another important aspect is the preparation of [FeFe]-hydrogenases in which the diiron site is synthetically modified in order to generate non-natural H-clusters and enzymes with new properties. [73][74][75][76] In the case of Group A [FeFe]-hydrogenase, the active site cavity has been extensively explored through site-directed mutagenesis (Chapter 2.1.2).…”