Discriminating changes in intracellular NADH/NAD+ levels due to anoxicity and H2 supply in R. eutropha cells using the Frex fluorescence sensor.

The hydrogen-oxidizing "Knallgas" bacterium Ralstonia eutropha can thrive in aerobic and anaerobic environments and readily switches between heterotrophic and autotrophic metabolism, making it an attractive host for biotechnological applications including the sustainable H2-driven production of hydrocarbons. The soluble hydrogenase (SH), one out of four different [NiFe]-hydrogenases in R. eutropha, mediates H2 oxidation even in the presence of O2, thus providing an ideal model system for biological hydrogen production and utilization. The SH reversibly couples H2 oxidation with the reduction of NAD+ to NADH, thereby enabling the sustainable regeneration of this biotechnologically important nicotinamide cofactor. Thus, understanding the interaction of the SH with the cellular NADH/NAD+ pool is of high interest. Here, we applied the fluorescent biosensor Frex to measure changes in cytoplasmic [NADH] in R. eutropha cells under different gas supply conditions. The results show that Frex is well-suited to distinguish SH-mediated changes in the cytoplasmic redox status from effects of general anaerobiosis of the respiratory chain. Upon H2 supply, the Frex reporter reveals a robust fluorescence response and allows for monitoring rapid changes in cellular [NADH]. Compared to the Peredox fluorescence reporter, Frex displays a diminished NADH affinity, which prevents the saturation of the sensor under typical bacterial [NADH] levels. Thus, Frex is a valuable reporter for on-line monitoring of the [NADH]/[NAD+] redox state in living cells of R. eutropha and other proteobacteria. Based on these results, strategies for a rational optimization of fluorescent NADH sensors are discussed.

Vibrational spectroscopy reveals the initial steps of biological hydrogen evolution.

[FeFe] hydrogenases are biocatalytic model systems for the exploitation and investigation of catalytic hydrogen evolution. Here, we used vibrational spectroscopic techniques to characterize, in detail, redox transformations of the [FeFe] and [4Fe4S] sub-sites of the catalytic centre (H-cluster) in a monomeric [FeFe] hydrogenase. Through the application of low-temperature resonance Raman spectroscopy, we discovered a novel metastable intermediate that is characterized by an oxidized [FeIFeII] centre and a reduced [4Fe4S]1+ cluster. Based on this unusual configuration, this species is assigned to the first, deprotonated H-cluster intermediate of the [FeFe] hydrogenase catalytic cycle. Providing insights into the sequence of initial reaction steps, the identification of this species represents a key finding towards the mechanistic understanding of biological hydrogen evolution.

Concepts in bio-molecular spectroscopy: vibrational case studies on metalloenzymes.

Spectroscopic techniques play a major role in the elucidation of structure-function relationships of biological macromolecules. Here we describe an integrated approach for bio-molecular spectroscopy that takes into account the special characteristics of such compounds. The underlying fundamental concepts will be exemplarily illustrated by means of selected case studies on biocatalysts, namely hydrogenase and superoxide reductase. The treatise will be concluded with an overview of challenges and future prospects, laying emphasis on functional dynamics, in vivo studies, and computational spectroscopy.

Reductive activation and structural rearrangement in superoxide reductase: a combined infrared spectroscopic and computational study.

Superoxide reductases (SOR) are a family of non-heme iron enzymes that limit oxidative stress by catalysing the reduction of superoxide to hydrogen peroxide and, thus, represent model systems for the detoxification of reactive oxygen species. In several enzymes of this type, reductive activation of the active site involves the reversible dissociation of a glutamate from the proposed substrate binding site at the iron. In this study we have employed IR spectroscopic and theoretical methods to gain insights into redox-linked structural changes of 1Fe-type superoxide reductases, focusing on the enzyme from the archaeon Ignicoccus hospitalis. Guided by crystal structure data and complemented by spectra calculation for an active site model, the main IR difference signals could be assigned. These signals reflect redox-induced structural changes in the first coordination sphere of the iron centre, adjacent loop and helical regions, and more remote ß-sheets. By comparison with the spectra obtained for the E23A mutant of Ignicoccus hospitalis SOR, it is shown that glutamate E23 dissociates reversibly from the ferrous iron during reductive activation of the wild type enzyme. Moreover, this process is found to trigger a global conformational transition of the protein that is strictly dependent on the presence of E23. Similar concerted structural changes can be inferred from the IR spectra of related SORs such as that from Archaeoglobus fulgidus, indicating a widespread mechanism. A possible functional role of this process in terms of synergistic effects during reductive activation of the homotetrameric enzyme is proposed.

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.