Michael Haumann

Photosynthetic O2 Formation Tracked by Time-Resolved X-ray Experiments

Plants and cyanobacteria produce atmospheric dioxygen from water, powered by sunlight and catalyzed by a manganese complex in photosystem II. A classic S-cycle model for oxygen evolution involves five states, but only four have been identified. The missing S4 state is particularly important because it is directly involved in dioxygen formation. Now progress comes from an x-ray technique that can monitor redox and structural changes in metal centers in real time with 10-microsecond resolution. We show that in the O2-formation step, an intermediate is formed—the enigmatic S4 state. Its creation is identified with a deprotonation process rather than the expected electron-transfer mechanism. Subsequent electron transfer would give an additional S4′ state, thus extending the fundamental S-state cycle of dioxygen formation.

How oxygen attacks [FeFe] hydrogenases from photosynthetic organisms

Green algae such as Chlamydomonas reinhardtii synthesize an [FeFe] hydrogenase that is highly active in hydrogen evolution. However, the extreme sensitivity of [FeFe] hydrogenases to oxygen presents a major challenge for exploiting these organisms to achieve sustainable photosynthetic hydrogen production. In this study, the mechanism of oxygen inactivation of the [FeFe] hydrogenase CrHydA1 from C. reinhardtii has been investigated. X-ray absorption spectroscopy shows that reaction with oxygen results in destruction of the [4Fe-4S] domain of the active site H-cluster while leaving the di-iron domain (2FeH) essentially intact. By protein film electrochemistry we were able to determine the order of events leading up to this destruction. Carbon monoxide, a competitive inhibitor of CrHydA1 which binds to an Fe atom of the 2FeH domain and is otherwise not known to attack FeS clusters in proteins, reacts nearly two orders of magnitude faster than oxygen and protects the enzyme against oxygen damage. These results therefore show that destruction of the [4Fe-4S] cluster is initiated by binding and reduction of oxygen at the di-iron domain—a key step that is blocked by carbon monoxide. The relatively slow attack by oxygen compared to carbon monoxide suggests that a very high level of discrimination can be achieved by subtle factors such as electronic effects (specific orbital overlap requirements) and steric constraints at the active site.

Synthetic manganese–calcium oxides mimic the water-oxidizing complex of photosynthesis functionally and structurally

In the worldwide search for sustainable energy technologies, water oxidation by abundant low-cost materials is of key importance. In nature, this process is efficiently catalyzed by an intricate manganese–calcium (Mn4Ca) complex bound to the proteins of photosystem II (PSII). Recently synthetic manganese–calcium oxides were found to be active catalysts of water oxidation but at the atomic level their structure has remained elusive. To investigate these amorphous catalysts, extended-range X-ray absorption spectroscopy (XAS) at the K-edges of both manganese and calcium was performed. The XAS results reveal striking similarities between the synthetic material and the natural Mn4Ca complex. The oxidation state of manganese in the active oxides was found to be close to +4, but MnIII ions are present as well at a level of about 20%. Neighboring Mn ions are extensively interconnected by two bridging oxygens, a characteristic feature of layered manganese oxides. However, the oxides do not exhibit long-range order, as opposed to canonical, but catalytically inactive MnIII- or MnIV-oxides. Two different Ca-containing motifs were identified. One of them results in the formation of Mn3CaO4 cubes, as also proposed for the natural paragon in PSII. Other calcium ions likely interconnect oxide-layer fragments. We conclude that these readily synthesized manganese–calcium oxides are the closest structural and functional analogs to the native PSII catalyst found so far. Evolutionary implications are considered. From the differences to inactive manganese oxides, we infer structural features facilitating the catalysis of water oxidation in both the protein-bound Mn4Ca complex of PSII and in the synthetic oxides.

Rapid Loss of Structural Motifs in the Manganese Complex of Oxygenic Photosynthesis by X-ray Irradiation at 10–300 K

Structural changes upon photoreduction caused by x-ray irradiation of the water-oxidizing tetramanganese complex of photosystem II were investigated by x-ray absorption spectroscopy at the manganese K-edge. Photoreduction was directly proportional to the x-ray dose. It was faster in the higher oxidized S2 state than in S1; seemingly the oxidizing potential of the metal site governs the rate. X-ray irradiation of the S1 state at 15 K initially caused single-electron reduction to S0* accompanied by the conversion of one di-μ-oxo bridge between manganese atoms, previously separated by ∼2.7 Å, to a mono-μ-oxo motif. Thereafter, manganese photoreduction was 100 times slower, and the biphasic increase in its rate between 10 and 300 K with a breakpoint at ∼200 K suggests that protein dynamics is rate-limiting the radical chemistry. For photoreduction at similar x-ray doses as applied in protein crystallography, halfway to the final MnII4 state the complete loss of inter-manganese distances <3Å was observed, even at 10 K, because of the destruction of μ-oxo bridges between manganese ions. These results put into question some structural attributions from recent protein crystallography data on photosystem II. It is proposed to employ controlled x-ray photoreduction in metalloprotein research for: (i) population of distinct reduced states, (ii) estimating the redox potential of buried metal centers, and (iii) research on protein dynamics.

On the structure of the manganese complex of photosystem II: extended-range EXAFS data and specific atomic-resolution models for four S-states.

The water-oxidizing manganese complex bound to the proteins of photosystem II (PSII) was studied by X-ray absorption spectroscopy on PSII membrane particles. An extended range for collection of extended X-ray absorption fine-structure (EXAFS) data was used (up to 16.6 Å−1). The EXAFS suggests the presence of two Mn–Mn distances close to 2.7 Å (per Mn4Ca complex); the existence of a third Mn–Mn distance below 2.9 Å is at least uncertain. Interestingly, a distance of 3.7 Å is clearly resolved in the extended-range data and tentatively assigned to a Mn–Mn distance. Taking into account the above EXAFS results (inter alia), we present a model for the structure of the PSII manganese complex, which differs from previous atomic-resolution models. Emphasizing the hypothetical character, we propose for all semi-stable S-states: (i) a structure of the Mn4Ca(μ-O)n core, (ii) a model of the amino acid environment, and (iii) assignments of distinct Mn oxidation states to all the individual Mn ions. This specific working model may permit discussion, verification and invalidation of its various features in comparison with experimental and theoretical findings.

Recent developments in research on water oxidation by photosystem II.

Photosynthetic water oxidation chemistry at the unique manganese-calcium complex of photosystem II (PSII) is of fundamental importance and serves as a paragon in the development of efficient synthetic catalysts. A recent crystal structure of PSII shows the atoms of the water-oxidizing complex; its Mn4CaO5 core resembles inorganic manganese-calcium oxides. Merging of crystallographic and spectroscopic information reverses radiation-induced modifications at the Mn-complex in silico and facilitates discussion of the O-O bond chemistry. Coordinated proton movements are promoted by a water network connecting the Mn4CaO5 core with the oxidant, a tyrosine radical and one possibly mobile chloride ion. A basic reaction-cycle model predicts an alternating proton and electron removal from the catalytic site, which facilitates energetically efficient water oxidation.

Alternating electron and proton transfer steps in photosynthetic water oxidation

Water oxidation by cyanobacteria, algae, and plants is pivotal in oxygenic photosynthesis, the process that powers life on Earth, and is the paradigm for engineering solar fuel–production systems. Each complete reaction cycle of photosynthetic water oxidation requires the removal of four electrons and four protons from the catalytic site, a manganese–calcium complex and its protein environment in photosystem II. In time-resolved photothermal beam deflection experiments, we monitored apparent volume changes of the photosystem II protein associated with charge creation by light-induced electron transfer (contraction) and charge-compensating proton relocation (expansion). Two previously invisible proton removal steps were detected, thereby filling two gaps in the basic reaction-cycle model of photosynthetic water oxidation. In the S2 → S3 transition of the classical S-state cycle, an intermediate is formed by deprotonation clearly before electron transfer to the oxidant (). The rate-determining elementary step (τ, approximately 30 µs at 20 °C) in the long-distance proton relocation toward the protein–water interface is characterized by a high activation energy (Ea = 0.46 ± 0.05 eV) and strong H/D kinetic isotope effect (approximately 6). The characteristics of a proton transfer step during the S0 → S1 transition are similar (τ, approximately 100 µs; Ea = 0.34 ± 0.08 eV; kinetic isotope effect, approximately 3); however, the proton removal from the Mn complex proceeds after electron transfer to . By discovery of the transient formation of two further intermediate states in the reaction cycle of photosynthetic water oxidation, a temporal sequence of strictly alternating removal of electrons and protons from the catalytic site is established.

Introduction of Methionines in the Gas Channel Makes [NiFe] Hydrogenase Aero-Tolerant

Hydrogenases catalyze the conversion between 2H(+) + 2e(-) and H(2)(1). Most of these enzymes are inhibited by O(2), which represents a major drawback for their use in biotechnological applications. Improving hydrogenase O(2) tolerance is therefore a major contemporary challenge to allow the implementation of a sustainable hydrogen economy. We succeeded in improving O(2) tolerance, which we define here as the ability of the enzyme to resist for several minutes to O(2) exposure, by substituting with methionines small hydrophobic residues strongly conserved in the gas channel. Remarkably, the mutated enzymes remained active in the presence of an O(2) concentration close to that found in aerobic solutions in equilibrium with air, while the wild type enzyme is inhibited in a few seconds. Crystallographic and spectroscopic studies showed that the structure and the chemistry at the active site are not affected by the mutations. Kinetic studies demonstrated that the inactivation is slower and reactivation faster in these mutants. We propose that in addition to restricting O(2) diffusion to the active site of the enzyme, methionine may also interact with bound peroxide and provide an assisted escape route for H(2)O(2) toward the gas channel. These results show for the first time that it is possible to improve O(2)-tolerance of [NiFe] hydrogenases, making possible the development of biohydrogen production systems.

Bacterial formate hydrogenlyase complex

Significance The isolation of an active formate hydrogenlyase is a breakthrough in understanding the molecular basis of bacterial hydrogen production. For over 100 years, Escherichia coli has been known to evolve H2 when cultured under fermentative conditions. Glucose is metabolized to formate, which is then oxidized to CO2 with the concomitant reduction of protons to H2 by a single complex called formate hydrogenlyase, which had been genetically, but never biochemically, characterized. In this study, innovative molecular biology and electrochemical experiments reveal a hydrogenase enzyme with the unique ability to sustain H2 production even under high partial pressures of H2. Harnessing bacterial H2 production offers the prospect of a source of fully renewable H2 energy, freed from any dependence on fossil fuel. Under anaerobic conditions, Escherichia coli can carry out a mixed-acid fermentation that ultimately produces molecular hydrogen. The enzyme directly responsible for hydrogen production is the membrane-bound formate hydrogenlyase (FHL) complex, which links formate oxidation to proton reduction and has evolutionary links to Complex I, the NADH:quinone oxidoreductase. Although the genetics, maturation, and some biochemistry of FHL are understood, the protein complex has never been isolated in an intact form to allow biochemical analysis. In this work, genetic tools are reported that allow the facile isolation of FHL in a single chromatographic step. The core complex is shown to comprise HycE (a [NiFe] hydrogenase component termed Hyd-3), FdhF (the molybdenum-dependent formate dehydrogenase-H), and three iron-sulfur proteins: HycB, HycF, and HycG. A proportion of this core complex remains associated with HycC and HycD, which are polytopic integral membrane proteins believed to anchor the core complex to the cytoplasmic side of the membrane. As isolated, the FHL complex retains formate hydrogenlyase activity in vitro. Protein film electrochemistry experiments on Hyd-3 demonstrate that it has a unique ability among [NiFe] hydrogenases to catalyze production of H2 even at high partial pressures of H2. Understanding and harnessing the activity of the FHL complex is critical to advancing future biohydrogen research efforts.

The structure of the manganese complex of Photosystem II in its dark-stable S1-state-EXAFS results in relation to recent crystallographic data

Determination of the nuclear geometry of the Mn4Ca complex of photosystem II (PSII) may facilitate an in-depth discussion of the mechanism of photosynthetic water oxidation. The first step of structural analysis by EXAFS (extended X-ray absorption fine-structure) spectroscopy and protein crystallography is the determination of the structure of the dark-stable S1-state of the Mn complex. Approaches for normalization and simulation of EXAFS spectra are critically evaluated, in particular with respect to the accuracy of the determined coordination numbers. It is shown that the number of Mn–Mn interactions with about 2.7 A length is likely to be two in the S1-state. The presence and orientation of the 2.7 A Mn–Mn vectors as well as of the Mn–Ca distances of ∼3.3 A length is predicted by EXAFS analysis and seems to be compatible with the available crystallographic data (Zouni, Witt, J. Kern, P. Fromme, N. Kraus, W. Saenger and P. Orth, Nature, 2001, 409, Kamiya and Shen, Proc. Natl. Acad. Sci. USA, 2003, 100, 98; Ferreira, Iverson, Maghlaoui, Barber and Iwata, Science, 2004, 303, 1831). In the light of XAS results on radiation-induced modifications of the Mn4Ca complex, however, we conclude that in the course of the crystallographic data collection the Mn4Ca complex may be significantly affected by X-ray photoreduction so that the obtained electron density maps do not represent the intact complex in its S1-state. Thus, any detailed structural modelling by combination of EXAFS results and crystallographic data seems to be premature. Two motifs for the connection of Mn ions in the complex, namely either by (μ2-O)2 or (μ2-O, μ3-O) bridges, may account for Mn–Mn distances close to 2.7 A. Three basic possibilities to arrange the Mn ions of the Mn4Ca complex in its S1-state are discussed.