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Hydrogenases and Oxygen View Article Online / Journal Homepage / Table of Contents for this issue Chemical Science Dynamic Article LinksC< Cite this: Chem. Sci., 2012, 3, 1739 www.rsc.org/chemicalscience PERSPECTIVE Hydrogenases and oxygen Martin Tillmann Stiebritz*a and Markus Reiher*b Received 23rd December 2011, Accepted 3rd February 2012 DOI: 10.1039/c2sc01112c Hydrogenases represent a heterogeneous group of enzymes consisting of three evolutionary unrelated classes, i.e. [NiFe], [FeFe] and [Fe] hydrogenases. They allow the uni-cellular organisms in which they are expressed to use hydrogen as energy source or to reduce protons as a sink for excess reduction equivalents. Because of this capability there is growing interest in exploiting these enzymes in the field of sustainable energy generation. However, most hydrogenases which are appealing in this context are reversibly or irreversibly inhibited by dioxygen. Here, we summarize the current picture of oxygen- induced inhibition of the different classes of hydrogenases and discuss possible avenues that might lead to tailored oxygen-robust enzyme variants. 1 Introduction discuss the current status of knowledge about the mechanisms of 1–5 O2 induced inhibition in order to clarify and evaluate strategies Hydrogenases catalyze the reversible oxidation of molecular for obtaining O -tolerant enzyme variants. + À 2 hydrogen, H2 # 2H +2e , and it is exactly this capability that makes them particularly interesting for sustainable energy production—either by directly exploiting their catalytic activity 2 Hydrogenase classes and their structural in electrochemical devices or by biomimetic complex-design characteristics inspired by their active sites. Considerable progress in the latter 6 2.1 Genetical and physiological classification field has recently been achieved with the prospect of becoming independent from enzyme based systems. However, despite the Downloaded by ETH-Zurich on 25 March 2013 Physiologically, hydrogenases are involved in anaerobic energy remarkable turnover rates observed, the over-potentials neces- conversion processes that are accompanied by H2 formation or sary to achieve catalysis are still significantly larger for synthetic consumption. Based on sequence similarity and active site Published on 23 February 2012 http://pubs.rsc.org | doi:10.1039/C2SC01112C 6 catalysts. On the other hand, the technical application of composition the three phylogenetically unrelated classes of hydrogenases is hampered by the fact that, to a variable extent, [FeFe], [NiFe] and [Fe] hydrogenases can be distinguished. these enzymes are rather sensitive towards molecular oxygen, Whereas enzymes that are catalytically active in H2 conversion which is presumably due to their evolutionary origin in anaerobic are distributed over unicellular members of the three domains living organisms. Any development exploiting their catalytic eubacteria, archaea and eukarya, hydrogenase-like genes are also capabilities has therefore to face and to cope with the oxygen- found in higher eukaryotes and even in humans, where their sensitivity problem of hydrogenases. protein products are presumably involved in the assembly of Recent years have seen significant progress in the mechanistic cytosolic iron–sulfur proteins.7–10 [NiFe] hydrogenases are het- understanding of how the active sites of different hydrogenases erodimeric proteins with the a subunit carrying the active site interact with molecular oxygen and reactive species formed whereas the b subunit contains Fe–S clusters involved in electron thereof. Hydrogenase chemistry has already been reviewed in the transport and facilitates protein complex formation and sub- literature, but this has not yet been done in view of the reactivity cellular localization. In terms of their physiological roles [NiFe] of all different classes of hydrogenases against reactive oxygen enzymes display the largest variability amongst the convergently species. Here, we pursue this goal in order to deduce general evolved hydrogenases. Based on homology they are grouped into reactivity principles that may eventually also be applied to other four classes as reviewed by Vignais et al.1–5 Group 1 comprises metallo-enzymes. After giving a brief overview of the various membrane-bound so-called uptake [NiFe] hydrogenases, that hydrogenase classes and the composition of their active sites, we allow cells to use H2 as an energy source by forming a proton motive force. They occur in bacteria and archaea. Based on sequence similarity the uptake hydrogenases of cyanobacteria aLaboratorium fur€ Physikalische Chemie, ETH Zurich, Wolfgang-Pauli- and the cytoplasmic hydrogen sensors that in bacteria regulate Strasse 10, 8093 Zurich,€ Switzerland. E-mail: [email protected]. hydrogenase gene expression in response to environmental H2 ethz.ch; Fax: +41 44 633 15 94; Tel: +41 44 633 38 66 concentration form group 2. Group 3 contains bidirectional het- bLaboratorium fur€ Physikalische Chemie, ETH Zurich, Wolfgang-Pauli- Strasse 10, 8093 Zurich,€ Switzerland. E-mail: [email protected]. eromultimeric cytoplasmic [NiFe] hydrogenases from bacteria ethz.ch; Fax: +41 44 633 15 94; Tel: +41 44 633 43 08 and archaea. Here, the basic hydrogenase dimer is associated This journal is ª The Royal Society of Chemistry 2012 Chem. Sci., 2012, 3, 1739–1751 | 1739 View Article Online with other subunits specific for soluble cofactors such as NAD— composition the common feature is the presence of Fe and/or Ni because these enzymes operate in a reversible fashion the as central ions coordinated by strong ligands, which due to their cofactors can be re-oxidized under anaerobiosis. The fourth toxicity do normally not occur in biological systems—namely group consists of energy-conserving, membrane-associated CO and CNÀ. hydrogenases which are found in bacteria and archaea. Structures of [NiFe] hydrogenases have mainly been solved for [FeFe] hydrogenases show the highest catalytic activity in the enzymes from sulfate-reducing bacteria of the Desulfovibrio hydrogen producing reaction regime, unlike [NiFe] hydrogenases genus15,17–22 and recently for enzymes from the purple sulfur 3 23 which are generally more biased toward H2 oxidation. This bacterium Allochromatium vinosum, the Knallgas bacterium makes them particularly interesting for sustainable energy Ralstonia eutropha24 and the hydrogen oxidizer Hydrogenovibrio production. With exceptions, hydrogen formation appears to be marinus.25 Therefore, all structures available belong to group 1 of the major physiological role of this class of enzymes which the class of [NiFe] hydrogenases. mainly occur as monomeric proteins in anaerobic prokaryotes The active site of [NiFe] hydrogenases, which is located in the (Clostridium spec., Desulfovibrio spec.), in the chloroplasts of large subunit of the enzyme, consists of a binuclear metal cluster unicellular algae (e.g. Chlamydomonas spec.) and in hydro- formed by Ni and Fe (see Fig. 1, middle). The Ni center is genosomes of anaerobic protozoa and certain fungi.2 Beside the coordinated by the sulfur groups of four cysteine residues, from active site, [FeFe] hydrogenases can contain varying numbers of which it shares two with the neighboring Fe atom, that addi- iron-sulfur (Fe–S) clusters, presumably involved in electron tionally carries two CNÀ and one CO ligands. In a special transport. More detailed information can be found in a number variant, the [NiFeSe] enzyme (see below) from Desulfomicrobium of excellent reviews about hydrogenases.1–4 [Fe] hydrogenase—or baculatum, one of the Ni-coordinating cysteine residues is Hmd (5,10-methylenetetrahydromethanopterin dehydroge- replaced by selenocysteine. nase)—is only found in the group of methanogenic arch- The active site is separated by a distance of approx. 6 A from aebacteria and catalyzes the heterolytic cleavage of H2 upon a regular [Fe4S4] cluster that resides in the small subunit and which a hydride ion is transferred to the cofactor 5,10-meth- which is most probably involved in electron transfer. This enyltetrahydromethanopterin.11 This pathway provides reduc- proximal Fe–S cluster is complemented by two further Fe–S tion equivalents for converting carbon dioxide into methane.12,13 clusters. In oxygen-sensitive [NiFe] hydrogenases, the medial Interestingly, the expression of this enzyme is tightly regulated cluster is a one-Fe ion deprived [Fe3S4] cluster and the distal one and only occurs when Ni, which is needed to build the active sites a regular [Fe4S4] cubane, in which, however, one Fe is coordi- of [NiFe] hydrogenases, is scarce.12 nated by a histidine instead of a cysteine residue. Crystal structures of [FeFe] hydrogenases have so far only been solved for the enzymes from Clostridium pasteur- 2.2 Protein structures and active sites ianum,14,26,27 Desulfovibrio desulfuricans28 and for the enzyme Fig. 1 gives an overview over the active sites of the three classes of from the unicellular green alga Chlamydomonas reinhardtii.29 The Downloaded by ETH-Zurich on 25 March 2013 hydrogenases and the protein structures of typical representa- latter however, though properly folded, does not contain the tives for which crystal structures have been solved. A comparison catalytically active subsite, because the protein was isolated from of the overall reactions catalyzed by the enzymes and their a strain deficient in maturation factors involved in active site Published on 23 February 2012 http://pubs.rsc.org | doi:10.1039/C2SC01112C 29 inhibition by O2 can be found in Fig. 2. In terms of
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