The enzyme hydrogenase reversibly converts dihydrogen to protons and electrons at a metal catalyst. The location of the abundant hydrogens is of key importance for understanding structure and ...function of the protein. However, in protein X-ray crystallography the detection of hydrogen atoms is one of the major problems, since they display only weak contributions to diffraction and the quality of the single crystals is often insufficient to obtain sub-ångström resolution. Here we report the crystal structure of a standard NiFe hydrogenase (∼91.3 kDa molecular mass) at 0.89 Å resolution. The strictly anoxically isolated hydrogenase has been obtained in a specific spectroscopic state, the active reduced Ni-R (subform Ni-R1) state. The high resolution, proper refinement strategy and careful modelling allow the positioning of a large part of the hydrogen atoms in the structure. This has led to the direct detection of the products of the heterolytic splitting of dihydrogen into a hydride (H(-)) bridging the Ni and Fe and a proton (H(+)) attached to the sulphur of a cysteine ligand. The Ni-H(-) and Fe-H(-) bond lengths are 1.58 Å and 1.78Å, respectively. Furthermore, we can assign the Fe-CO and Fe-CN(-) ligands at the active site, and can obtain the hydrogen-bond networks and the preferred proton transfer pathway in the hydrogenase. Our results demonstrate the precise comprehensive information available from ultra-high-resolution structures of proteins as an alternative to neutron diffraction and other methods such as NMR structural analysis.
The investigation of water oxidation in photosynthesis has remained a central topic in biochemical research for the last few decades due to the importance of this catalytic process for technological ...applications. Significant progress has been made following the 2011 report of a high-resolution X-ray crystallographic structure resolving the site of catalysis, a protein-bound Mn
4
CaO
x
complex, which passes through ≥5 intermediate states in the water-splitting cycle. Spectroscopic techniques complemented by quantum chemical calculations aided in understanding the electronic structure of the cofactor in all (detectable) states of the enzymatic process. Together with isotope labeling, these techniques also revealed the binding of the two substrate water molecules to the cluster. These results are described in the context of recent progress using X-ray crystallography with free-electron lasers on these intermediates. The data are instrumental for developing a model for the biological water oxidation cycle.
Hydrogenases catalyze the reversible conversion of molecular hydrogen to protons and electrons via a heterolytic splitting mechanism. The active sites of NiFe hydrogenases comprise a dinuclear Ni-Fe ...center carrying CO and CN
ligands. The catalytic activity of the standard (O
-sensitive) NiFe hydrogenases vanishes under aerobic conditions. The O
-tolerant NiFe hydrogenases can sustain H
oxidation activity under atmospheric conditions. These hydrogenases have very similar active site structures that change the ligand sphere during the activation/catalytic process. An important structural difference between these hydrogenases has been found for the proximal iron-sulphur cluster located in the vicinity of the active site. This unprecedented 4Fe-3S-6Cys cluster can supply two electrons, which lead to rapid recovery of the O
inactivation, to the NiFe active site.
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•FeFe hydrogenases use earth abundant metals for efficient hydrogen activation.•Understanding their catalytic mechanism may help synthesise new catalysts.•Their active site H-cluster ...contains two sites: a 4Fe-4S site and a 2Fe site.•Proton-coupled electron transfer and hydride formation are important steps.•We discuss the dominant models for the mechanism and future research directions.
Ever since the discovery of hydrogenases over 90 years ago (Stephenson and Stickland, 1931), their structure and mechanism of action have been intensively investigated. Of the three classes of hydrogenases, the FeFe hydrogenases show the highest activity and reversibility, and, thus, have garnered special interest. In this review, we tell the story of how structural, spectroscopic, functional and theoretical studies have all contributed substantially to our modern understanding of the catalytic cycle. We present a brief historical overview of the main events preceding the X-ray crystal structure determination, and then discuss how this defining moment over 20 years ago (Peters et al., 1998; Nicolet et al., 1999) revolutionised our understanding. We then detail the studies leading up to one model for the catalytic cycle in the simple enzyme from Chlamydomonas reinhardtii, containing only the active site H-cluster, as well as how the situation differs in enzymes containing additional iron-sulphur clusters. We then discuss the studies that led to a second model in the literature. Finally, we highlight the open questions and discuss how these could be answered.
The photosynthetic protein complex photosystem II oxidizes water to molecular oxygen at an embedded tetramanganese-calcium cluster. Resolving the geometric and electronic structure of this cluster in ...its highest metastable catalytic state (designated S₃) is a prerequisite for understanding the mechanism of O-O bond formation. Here, multifrequency, multidimensional magnetic resonance spectroscopy reveals that all four manganese ions of the catalyst are structurally and electronically similar immediately before the final oxygen evolution step; they all exhibit a 4+ formal oxidation state and octahedral local geometry. Only one structural model derived from quantum chemical modeling is consistent with all magnetic resonance data; its formation requires the binding of an additional water molecule. O-O bond formation would then proceed by the coupling of two proximal manganese-bound oxygens in the transition state of the cofactor.
The understanding of light‐induced biological water oxidation in oxygenic photosynthesis is of great importance both for biology and (bio)technological applications. The chemically difficult ...multistep reaction takes place at a unique protein‐bound tetra‐manganese/calcium cluster in photosystem II whose structure has been elucidated by X‐ray crystallography (Umena et al. Nature 2011, 473, 55). The cluster moves through several intermediate states in the catalytic cycle. A detailed understanding of these intermediates requires information about the spatial and electronic structure of the Mn4Ca complex; the latter is only available from spectroscopic techniques. Here, the important role of Electron Paramagnetic Resonance (EPR) and related double resonance techniques (ENDOR, EDNMR), complemented by quantum chemical calculations, is described. This has led to the elucidation of the cluster's redox and protonation states, the valence and spin states of the manganese ions and the interactions between them, and contributed substantially to the understanding of the role of the protein surrounding, as well as the binding and processing of the substrate water molecules, the O‐O bond formation and dioxygen release. Based on these data, models for the water oxidation cycle are developed.
Light‐induced water oxidation and dioxygen release in photosynthesis is catalyzed by a paramagnetic μ‐oxo‐bridged Mn4Ca cofactor. It passes through five metastable states (S0 – S4) whose structure is described, focusing on the essential electronic structure obtained from spectroscopy, especially EPR techniques, supported by quantum chemistry. A catalytic cycle is presented, which describes structural isomers of key S‐state intermediates facilitating substrate binding and cofactor activation.
A central question in biological water splitting concerns the oxidation states of the manganese ions that comprise the oxygen-evolving complex of photosystem II. Understanding the nature and order of ...oxidation events that occur during the catalytic cycle of five S
states (
= 0-4) is of fundamental importance both for the natural system and for artificial water oxidation catalysts. Despite the widespread adoption of the so-called "high-valent scheme"-where, for example, the Mn oxidation states in the S
state are assigned as III, IV, IV, IV-the competing "low-valent scheme" that differs by a total of two metal unpaired electrons (
III, III, III, IV in the S
state) is favored by several recent studies for the biological catalyst. The question of the correct oxidation state assignment is addressed here by a detailed computational comparison of the two schemes using a common structural platform and theoretical approach. Models based on crystallographic constraints were constructed for all conceivable oxidation state assignments in the four (semi)stable S states of the oxygen evolving complex, sampling various protonation levels and patterns to ensure comprehensive coverage. The models are evaluated with respect to their geometric, energetic, electronic, and spectroscopic properties against available experimental EXAFS, XFEL-XRD, EPR, ENDOR and Mn K pre-edge XANES data. New 2.5 K
Mn ENDOR data of the S
state are also reported. Our results conclusively show that the entire S state phenomenology can only be accommodated within the high-valent scheme by adopting a single motif and protonation pattern that progresses smoothly from S
(III, III, III, IV) to S
(IV, IV, IV, IV), satisfying all experimental constraints and reproducing all observables. By contrast, it was impossible to construct a consistent cycle based on the low-valent scheme for all S states. Instead, the low-valent models developed here may provide new insight into the over-reduced S states and the states involved in the assembly of the catalytically active water oxidizing cluster.
Among the four photo-driven transitions of the water-oxidizing tetramanganese-calcium cofactor of biological photosynthesis, the second-last step of the catalytic cycle, that is the S
to S
state ...transition, is the crucial step that poises the catalyst for the final O-O bond formation. This transition, whose intermediates are not yet fully understood, is a multi-step process that involves the redox-active tyrosine residue and includes oxidation and deprotonation of the catalytic cluster, as well as the binding of a water molecule. Spectroscopic data has the potential to shed light on the sequence of events that comprise this catalytic step, which still lacks a structural interpretation. In this work the S
-S
state transition is studied and a key intermediate species is characterized: it contains a Mn
O
Ca cubane subunit linked to a five-coordinate Mn(iv) ion that adopts an approximately trigonal bipyramidal ligand field. It is shown using high-level density functional and multireference wave function calculations that this species accounts for the near-infrared absorption and electron paramagnetic resonance observations on metastable S
-S
intermediates. The results confirm that deprotonation and Mn oxidation of the cofactor must precede the coordination of a water molecule, and lead to identification of a novel low-energy water binding mode that has important implications for the identity of the substrates in the mechanism of biological water oxidation.
Protonation states of water ligands and oxo bridges are intimately involved in tuning the electronic structures and oxidation potentials of the oxygen evolving complex (OEC) in Photosystem II, ...steering the mechanistic pathway, which involves at least five redox state intermediates S(n) (n = 0-4) resulting in the oxidation of water to molecular oxygen. Although protons are practically invisible in protein crystallography, their effects on the electronic structure and magnetic properties of metal active sites can be probed using spectroscopy. With the twin purpose of aiding the interpretation of the complex electron paramagnetic resonance (EPR) spectroscopic data of the OEC and of improving the view of the cluster at the atomic level, a complete set of protonation configurations for the S(2) state of the OEC were investigated, and their distinctive effects on magnetic properties of the cluster were evaluated. The most recent X-ray structure of Photosystem II at 1.9 Å resolution was used and refined to obtain the optimum structure for the Mn(4)O(5)Ca core within the protein pocket. Employing this model, a set of 26 structures was constructed that tested various protonation scenarios of the water ligands and oxo bridges. Our results suggest that one of the two water molecules that are proposed to coordinate the outer Mn ion (Mn(A)) of the cluster is deprotonated in the S(2) state, as this leads to optimal experimental agreement, reproducing the correct ground state spin multiplicity (S = 1/2), spin expectation values, and EXAFS-derived metal-metal distances. Deprotonation of Ca(2+)-bound water molecules is strongly disfavored in the S(2) state, but dissociation of one of the two water ligands appears to be facile. The computed isotropic hyperfine couplings presented here allow distinctions between models to be made and call into question the assumption that the largest coupling is always attributable to Mn(III). The present results impose limits for the total charge and the proton configuration of the OEC in the S(2) state, with implications for the cascade of events in the Kok cycle and for the water splitting mechanism.
Hydrogenases catalyze the formation of hydrogen. The cofactor ('H-cluster') of FeFe-hydrogenases consists of a 4Fe-4S cluster bridged to a unique 2Fe subcluster whose biosynthesis in vivo requires ...hydrogenase-specific maturases. Here we show that a chemical mimic of the 2Fe subcluster can reconstitute apo-hydrogenase to full activity, independent of helper proteins. The assembled H-cluster is virtually indistinguishable from the native cofactor. This procedure will be a powerful tool for developing new artificial H₂-producing catalysts.
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