Lars Lauterbach

Re-Structuring of Marine Communities Exposed to Environmental Change: A Global Study on the Interactive Effects of Species and Functional Richness

Species richness is the most commonly used but controversial biodiversity metric in studies on aspects of community stability such as structural composition or productivity. The apparent ambiguity of theoretical and experimental findings may in part be due to experimental shortcomings and/or heterogeneity of scales and methods in earlier studies. This has led to an urgent call for improved and more realistic experiments. In a series of experiments replicated at a global scale we translocated several hundred marine hard bottom communities to new environments simulating a rapid but moderate environmental change. Subsequently, we measured their rate of compositional change (re-structuring) which in the great majority of cases represented a compositional convergence towards local communities. Re-structuring is driven by mortality of community components (original species) and establishment of new species in the changed environmental context. The rate of this re-structuring was then related to various system properties. We show that availability of free substratum relates negatively while taxon richness relates positively to structural persistence (i.e., no or slow re-structuring). Thus, when faced with environmental change, taxon-rich communities retain their original composition longer than taxon-poor communities. The effect of taxon richness, however, interacts with another aspect of diversity, functional richness. Indeed, taxon richness relates positively to persistence in functionally depauperate communities, but not in functionally diverse communities. The interaction between taxonomic and functional diversity with regard to the behaviour of communities exposed to environmental stress may help understand some of the seemingly contrasting findings of past research.

Probing the active site of an O2-tolerant NAD+-reducing [NiFe]-hydrogenase from Ralstonia eutropha H16 by in situ EPR and FTIR spectroscopy.

[NiFe]-hydrogenases catalyze the reversible cleavage of dihydrogen into two protons and two electrons. This process plays an important role in the energy metabolism of many microorganisms. For most [NiFe]-hydrogenases, the process of H2 cycling is extremely sensitive to molecular oxygen as O2 exhibits a high affinity to the active site. However, some organisms are capable of catalyzing H2 cycling even at ambient oxygen levels. Notably, the b-proteobacterium Ralstonia eutropha H16 (Re) harbors three different [NiFe]hydrogenases, all of which display a remarkable oxygentolerance. The underlying molecular mechanisms are not yet fully understood. For the regulatory hydrogenase (RH) of Re, a narrow gas tunnel is thought to restrict O2 access to the active site. The Re membrane-bound hydrogenase (MBH) has a high redox potential FeS cluster in close proximity to the active site, a property that might be related to the observation that O2-inhibited MBH re-activates rapidly at high potentials. The soluble hydrogenase (SH) of Re is a cytoplasmic NAD-reducing six-subunit enzyme that is closely related to cyanobacterial bidirectional [NiFe]-hydrogenases. 7] For purified SH, a modified catalytic site was proposed on the basis of numerous biochemical and spectroscopic studies. 8, 9] In contrast to “standard” [NiFe]-hydrogenases, in which the active site iron is kept in the low-spin iron(II) state by one carbonyl and two cyanide ligands, Fourier transform infrared (FTIR) spectroscopy and concomitant chemical analysis suggested one additional cyanide bound to each metal ion of the catalytic center. The nickelbound cyanide ligand has been proposed to prevent the formation of the so-called Niu-A state, which is the most oxidized, O2-inactivated state in [NiFe]-hydrogenases. [9]

NAD(H)-coupled hydrogen cycling – structure–function relationships of bidirectional [NiFe] hydrogenases

Hydrogenases catalyze the activation or production of molecular hydrogen. Due to their potential importance for future biotechnological applications, these enzymes have been in the focus of intense research for the past decades. Bidirectional [NiFe] hydrogenases are of particular interest as they couple the reversible cleavage of hydrogen to the redox conversion of NAD(H). In this account, we review the current state of knowledge about mechanistic aspects and structural determinants of these complex multi‐cofactor enzymes. Special emphasis is laid on the oxygen‐tolerant NAD(H)‐linked bidirectional [NiFe] hydrogenase from Ralstonia eutropha.

Catalytic properties of the isolated diaphorase fragment of the NAD-reducing [NiFe]-hydrogenase from Ralstonia eutropha.

The NAD+-reducing soluble hydrogenase (SH) from Ralstonia eutropha H16 catalyzes the H2-driven reduction of NAD+, as well as reverse electron transfer from NADH to H+, in the presence of O2. It comprises six subunits, HoxHYFUI2, and incorporates a [NiFe] H+/H2 cycling catalytic centre, two non-covalently bound flavin mononucleotide (FMN) groups and an iron-sulfur cluster relay for electron transfer. This study provides the first characterization of the diaphorase sub-complex made up of HoxF and HoxU. Sequence comparisons with the closely related peripheral subunits of Complex I in combination with UV/Vis spectroscopy and the quantification of the metal and FMN content revealed that HoxFU accommodates a [2Fe2S] cluster, FMN and a series of [4Fe4S] clusters. Protein film electrochemistry (PFE) experiments show clear electrocatalytic activity for both NAD+ reduction and NADH oxidation with minimal overpotential relative to the potential of the NAD+/NADH couple. Michaelis-Menten constants of 56 µM and 197 µM were determined for NADH and NAD+, respectively. Catalysis in both directions is product inhibited with K I values of around 0.2 mM. In PFE experiments, the electrocatalytic current was unaffected by O2, however in aerobic solution assays, a moderate superoxide production rate of 54 nmol per mg of protein was observed, meaning that the formation of reactive oxygen species (ROS) observed for the native SH can be attributed mainly to HoxFU. The results are discussed in terms of their implications for aerobic functioning of the SH and possible control mechanism for the direction of catalysis.

Systematic evaluation of the dihydrogen-oxidising and NAD+-reducing soluble [NiFe]-hydrogenase from Ralstonia eutropha H16 as a cofactor regeneration catalyst

Abstract The oxygen-tolerant NAD+-reducing soluble hydrogenase (SH) from Ralstonia eutropha H16 has been described as a promising catalyst for cofactor regeneration in biocatalysed reductions. In this study, the actual potential of this enzyme for application in technical synthesis was evaluated. An overproduced, purified version of the enzyme was coupled to the carbonyl reductase from Candida parapsilosis (CPCR), where it allowed an almost quantitative conversion of the model substrate; total turnover numbers (TTN: nproduct/nenzyme) of up to 143,666 were achieved. This was distinctly superior to the commonly used NADH regenerating enzyme formate dehydrogenase (FDH) from Candida boidinii. In a systematic quantitative approach, maximum activity for NAD+ reduction was observed at 35 °C and pH 8, which corresponds to that of native SH. The half-life of the enzyme under these conditions was 5.3 hours. In the presence of sodium salts, distinct inhibitory effects were observed while ammonium and potassium ions increased the enzyme stability. Overall, a high but not unusual sensitivity of SH for changes in temperature, pH and mechanical stress in a reactor was found. Technical application in chemical synthesis can therefore be considered a feasible goal.

Asymmetric Biocatalytic Amination of Ketones at the Expense of NH3 and Molecular Hydrogen.

A biocatalytic system is presented for the stereoselective amination of ketones at the expense of NH3 and molecular hydrogen. By using a NAD(+)-reducing hydrogenase, an alanine dehydrogenase, and a suitable ω-transaminase, the R- as well as the S-enantiomer of various amines could be prepared with up to >99% ee and 98% conversion.

Reversible active site sulfoxygenation can explain the oxygen tolerance of a NAD+-reducing [NiFe] hydrogenase and its unusual infrared spectroscopic properties.

Oxygen-tolerant [NiFe] hydrogenases are metalloenzymes that represent valuable model systems for sustainable H2 oxidation and production. The soluble NAD(+)-reducing [NiFe] hydrogenase (SH) from Ralstonia eutropha couples the reversible cleavage of H2 with the reduction of NAD(+) and displays a unique O2 tolerance. Here we performed IR spectroscopic investigations on purified SH in various redox states in combination with density functional theory to provide structural insights into the catalytic [NiFe] center. These studies revealed a standard-like coordination of the active site with diatomic CO and cyanide ligands. The long-lasting discrepancy between spectroscopic data obtained in vitro and in vivo could be solved on the basis of reversible cysteine oxygenation in the fully oxidized state of the [NiFe] site. The data are consistent with a model in which the SH detoxifies O2 catalytically by means of an NADH-dependent (per)oxidase reaction involving the intermediary formation of stable cysteine sulfenates. The occurrence of two catalytic activities, hydrogen conversion and oxygen reduction, at the same cofactor may inspire the design of novel biomimetic catalysts performing H2-conversion even in the presence of O2.

Rapid invasion and ecological interactions of Diplosoma listerianum in the North Sea, UK

This paper documents the arrival of Diplosoma listerianum into a habitat with no previously known history of the species. Once established, D. listerianum exploited rapid growth rates relative to the other fouling species present, to quickly become the dominant species in a local fouling assemblage. Most resident macrofoulers were out-competed for space and overgrown, although some resistance to overgrowth was demonstrated by the bryozoan Umbonula littoralis and the tunicate Ascidiella aspersa. In this instance, traits traditionally considered to be relevant for community resistance towards invasion, such as diversity, richness, dominant species identity and open space were not important in controlling the spread of D. listerianum

Impact of the iron-sulfur cluster proximal to the active site on the catalytic function of an O2-tolerant NAD(+)-reducing [NiFe]-hydrogenase.

The soluble NAD(+)-reducing hydrogenase (SH) from Ralstonia eutropha H16 belongs to the O2-tolerant subtype of pyridine nucleotide-dependent [NiFe]-hydrogenases. To identify molecular determinants for the O2 tolerance of this enzyme, we introduced single amino acids exchanges in the SH small hydrogenase subunit. The resulting mutant strains and proteins were investigated with respect to their physiological, biochemical, and spectroscopic properties. Replacement of the four invariant conserved cysteine residues, Cys41, Cys44, Cys113, and Cys179, led to unstable protein, strongly supporting their involvement in the coordination of the iron-sulfur cluster proximal to the catalytic [NiFe] center. The Cys41Ser exchange, however, resulted in an SH variant that displayed up to 10% of wild-type activity, suggesting that the coordinating role of Cys41 might be partly substituted by the nearby Cys39 residue, which is present only in O2-tolerant pyridine nucleotide-dependent [NiFe]-hydrogenases. Indeed, SH variants carrying glycine, alanine, or serine in place of Cys39 showed increased O2 sensitivity compared to that of the wild-type enzyme. Substitution of further amino acids typical for O2-tolerant SH representatives did not greatly affect the H2-oxidizing activity in the presence of O2. Remarkably, all mutant enzymes investigated by electron paramagnetic resonance spectroscopy did not reveal significant spectral changes in relation to wild-type SH, showing that the proximal iron-sulfur cluster does not contribute to the wild-type spectrum. Interestingly, exchange of Trp42 by serine resulted in a completely redox-inactive [NiFe] site, as revealed by infrared spectroscopy and H2/D(+) exchange experiments. The possible role of this residue in electron and/or proton transfer is discussed.

Active Site of the NAD(+)-Reducing Hydrogenase from Ralstonia eutropha Studied by EPR Spectroscopy.

Pulsed ENDOR and HYSCORE measurements were carried out to characterize the active site of the oxygen-tolerant NAD(+)-reducing hydrogenase of Ralstonia eutropha. The catalytically active Nia-C state exhibits a bridging hydride between iron and nickel in the active site, which is photodissociated upon illumination. Its hyperfine coupling is comparable to that of standard hydrogenases. In addition, a histidine residue could be identified, which shows hyperfine and nuclear quadrupole parameters in significant variance from comparable histidine residues that are conserved in standard [NiFe] hydrogenases, and might be related to the O2 tolerance of the enzyme.