Semisynthetic Hydrogenases Propel Biological Energy Research into a New Era  James A. Birrell, Olaf Rüdiger, Edward J. Reijerse, Wolfgang Lubitz  Joule  Volume 1, Issue 1, Pages 61-76 (September 2017) DOI: 10.1016/j.joule.2017.07.009 Copyright © 2017 Elsevier Inc. Terms and Conditions

Joule 2017 1, 61-76DOI: (10.1016/j.joule.2017.07.009) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 1 Active-Site Structure and Proposed Catalytic Mechanism of [FeFe] Hydrogenases (A) Structure of the H-cluster in the Hox state with the [2Fe] and [4Fe-4S] subclusters, as well as two cysteine side chains, one bridging the two clusters and one serving as the initial proton acceptor site. The site of H2 binding is indicated with an arrow. The figure was made in PyMOL using the PDB file PDB: 1HFE.11 (B) Proposed catalytic cycle for hydrogen conversion by the H-cluster in the [FeFe] hydrogenases. The dark orange diamond represents the [4Fe-4S] and the orange rectangle the [2Fe] subcluster; the oxidation states of the subclusters are given. The states Hox, Hred, HredH+, HsredH+, and Hhyd have been identified by IR spectroscopy under equilibrium or steady-state conditions.12,13 Joule 2017 1, 61-76DOI: (10.1016/j.joule.2017.07.009) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 2 Natural and Artificial Maturation of the [FeFe] Hydrogenases (A) Scheme describing the laboratory synthesis of [2Fe] precursor complexes for use in artificial maturation.25 (B) Scheme describing the processes of natural and artificial maturation. Left: the [2Fe] cofactor is synthesized by the concerted action of the maturases HydE and HydG, followed by insertion into the hydrogenase using HydF. Right: the [2Fe] precursor is chemically synthesized and inserted into the recombinantly produced pro-hydrogenase either directly or using HydF. (C) Artificial maturation of CrHydA1 followed by IR spectroscopy (modified from Berggren et al.26) and CO release.27 Joule 2017 1, 61-76DOI: (10.1016/j.joule.2017.07.009) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 3 Chemical Modifications of the [2Fe] Subcluster (A) Scheme indicating the modifications to the [2Fe] subcluster made so far. (B) IR spectra and properties associated with hydrogenases produced using the chemically modified precursors containing NH, CH2, or O in the bridging dithiolate ligand. (C) H2 production activity for CrHydA1 maturated with the indicated [2Fe] precursors; for details see Siebel et al.33 The error bars indicate the standard deviation based on triplicate measurements. Joule 2017 1, 61-76DOI: (10.1016/j.joule.2017.07.009) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 4 Isotopic Labeling of the [2Fe] Subcluster for Spectroscopic Studies (A) Scheme indicating the sites in the [2Fe] subcluster isotopically labeled so far. (B) Electron paramagnetic resonance and ESEEM (electron-spin echo envelope modulation) used to measure nuclear hyperfine interactions, e.g., of nitrogen ligands in the H-cluster. The excitation energy of an electron in an external magnetic field depends on the spin state of the magnetic nucleus, which is detected via modulation of the spin echo formed during a three-pulse ESEEM experiment. The two-dimensional form of this experiment (HYSCORE) increases the resolution; here pulse spacing times t1 and t2 are varied. An example for detection of the bridgehead nitrogen (15N) in CrHydA1-ADT is given at the bottom.36 (C) Nuclear resonance vibrational spectroscopy (NRVS) on 57Fe-labeled [2Fe] subcomplexes used to study the properties of an Fe-bound hydride. Synchrotron (X-ray) radiation excites the 57Fe nucleus and recoil-free (nuclear forward) scattering is predominantly observed (Mössbauer effect). In addition, nuclear inelastic scattering (NRVS) can be detected, associated with vibrations of nuclei bound to the iron. In CrHydA1-ODT (bottom panel) NRVS was used to observe vibrational modes indicative of the presence of a terminal hydride, confirmed by H/D exchange and supported by DFT calculations.13 Joule 2017 1, 61-76DOI: (10.1016/j.joule.2017.07.009) Copyright © 2017 Elsevier Inc. Terms and Conditions