In Vivo EPR Characterization of Semi‐Synthetic [FeFe] Hydrogenases

Abstract

EPR spectroscopy reveals the formation of two different semi‐synthetic hydrogenases in vivo. [FeFe] hydrogenases are metalloenzymes that catalyze the interconversion of molecular hydrogen and protons. The reaction is catalyzed by the H‐cluster, consisting of a canonical iron–sulfur cluster and an organometallic [2Fe] subsite. It was recently shown that the enzyme can be reconstituted with synthetic cofactors mimicking the composition of the [2Fe] subsite, resulting in semi‐synthetic hydrogenases. Herein, we employ EPR spectroscopy to monitor the formation of two such semi‐synthetic enzymes in whole cells. The study provides the first spectroscopic characterization of semi‐synthetic hydrogenases in vivo, and the observation of two different oxidized states of the H‐cluster under intracellular conditions. Moreover, these findings underscore how synthetic chemistry can be a powerful tool for manipulation and examination of the hydrogenase enzyme under in vivo conditions.

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.…”

“…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).…”

“…The preparation of the semi-synthetic hydrogenase was performed following a literature protocol with minor modications. 44,45 The apo-HydA1 protein was expressed in 50 mL E. coli cultures as described in the "Overexpression of the apo-HydA1 hydrogenase" section. Aer the 16-18 h expression period the cells were harvested, deaerated and transferred to the glove-box.…”

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

“…[19][20][21][22][23] The well-studied [FeFe]hydrogenase from C. reinhardtii (Cr-HydA1) belonging to subclass M1 (sub-class nomenclature is derived from Meyer 20 and Calusinska et al 21 ) was included as a positive control as it has previously been shown to work under the presented in vivo conditions. 24,26 M1 is the structurally simplest known [FeFe]hydrogenase sub-class consisting only of the H-domain ( Fig. 1).…”

“…24,25 Moreover, we have reported how the cofactor of the resulting semi-synthetic enzyme can be monitored in vivo by electron paramagnetic resonance (EPR). 26 Herein we present how the combination of articial maturation and biophysical characterization under in vivo conditions can be turned into a tool for efficient screening of novel [FeFe]hydrogenases. The method is applicable to a range of E. coli expression and growth conditions, and allows for basic characterization without the need for time-consuming protein purication.…”