Stability of the H-cluster under whole-cell conditions-formation of an Htrans-like state and its reactivity towards oxygen.

Hydrogenases are metalloenzymes that catalyze the reversible oxidation of molecular hydrogen into protons and electrons. For this purpose, [FeFe]-hydrogenases utilize a hexanuclear iron cofactor, the H-cluster. This biologically unique cofactor provides the enzyme with outstanding catalytic activities, but it is also highly oxygen sensitive. Under in vitro conditions, oxygen stable forms of the H-cluster denoted Htrans and Hinact can be generated via treatment with sulfide under oxidizing conditions. Herein, we show that an Htrans-like species forms spontaneously under intracellular conditions on a time scale of hours, concurrent with the cells ceasing H2 production. Addition of cysteine or sulfide during the maturation promotes the formation of this H-cluster state. Moreover, it is found that formation of the observed Htrans-like species is influenced by both steric factors and proton transfer, underscoring the importance of outer coordination sphere effects on H-cluster reactivity.

Characterization of a putative sensory [FeFe]-hydrogenase provides new insight into the role of the active site architecture.

[FeFe]-hydrogenases are known for their high rates of hydrogen turnover, and are intensively studied in the context of biotechnological applications. Evolution has generated a plethora of different subclasses with widely different characteristics. The M2e subclass is phylogenetically distinct from previously characterized members of this enzyme family and its biological role is unknown. It features significant differences in domain- and active site architecture, and is most closely related to the putative sensory [FeFe]-hydrogenases. Here we report the first comprehensive biochemical and spectroscopical characterization of an M2e enzyme, derived from Thermoanaerobacter mathranii. As compared to other [FeFe]-hydrogenases characterized to-date, this enzyme displays an increased H2 affinity, higher activation enthalpies for H+/H2 interconversion, and unusual reactivity towards known hydrogenase inhibitors. These properties are related to differences in active site architecture between the M2e [FeFe]-hydrogenase and "prototypical" [FeFe]-hydrogenases. Thus, this study provides new insight into the role of this subclass in hydrogen metabolism and the influence of the active site pocket on the chemistry of the H-cluster.

Spectroscopic investigations under whole-cell conditions provide new insight into the metal hydride chemistry of [FeFe]-hydrogenase.

Hydrogenases are among the fastest H2 evolving catalysts known to date and have been extensively studied under in vitro conditions. Here, we report the first mechanistic investigation of an [FeFe]-hydrogenase under whole-cell conditions. Functional [FeFe]-hydrogenase from the green alga Chlamydomonas reinhardtii is generated in genetically modified Escherichia coli cells by addition of a synthetic cofactor to the growth medium. The assembly and reactivity of the resulting semi-synthetic enzyme was monitored using whole-cell electron paramagnetic resonance and Fourier-transform Infrared difference spectroscopy as well as scattering scanning near-field optical microscopy. Through a combination of gas treatments, pH titrations, and isotope editing we were able to corroborate the formation of a number of proposed catalytic intermediates in living cells, supporting their physiological relevance. Moreover, a previously incompletely characterized catalytic intermediate is reported herein, attributed to the formation of a protonated metal hydride species.

Discovery of novel [FeFe]-hydrogenases for biocatalytic H2-production.

A new screening method for [FeFe]-hydrogenases is described, circumventing the need for specialized expression conditions as well as protein purification for initial characterization. [FeFe]-hydrogenases catalyze the formation and oxidation of molecular hydrogen at rates exceeding 103 s-1, making them highly promising for biotechnological applications. However, the discovery of novel [FeFe]-hydrogenases is slow due to their oxygen sensitivity and dependency on a structurally unique cofactor, complicating protein expression and purification. Consequently, only a very limited number have been characterized, hampering their implementation. With the purpose of increasing the throughput of [FeFe]-hydrogenase discovery, we have developed a screening method that allows for rapid identification of novel [FeFe]-hydrogenases as well as their characterization with regards to activity (activity assays and protein film electrochemistry) and spectroscopic properties (electron paramagnetic resonance and Fourier transform infrared spectroscopy). The method is based on in vivo artificial maturation of [FeFe]-hydrogenases in Escherichia coli and all procedures are performed on either whole cells or non-purified cell lysates, thereby circumventing extensive protein purification. The screening was applied on eight putative [FeFe]-hydrogenases originating from different structural sub-classes and resulted in the discovery of two new active [FeFe]-hydrogenases. The [FeFe]-hydrogenase from Solobacterium moorei shows high H2-gas production activity, while the enzyme from Thermoanaerobacter mathranii represents a hitherto uncharacterized [FeFe]-hydrogenase sub-class. This latter enzyme is a putative sensory hydrogenase and our in vivo spectroscopy study reveals distinct differences compared to the well established H2 producing HydA1 hydrogenase from Chlamydomonas reinhardtii.

In†Vivo EPR Characterization of Semi-Synthetic [FeFe] Hydrogenases.

EPR spectroscopy reveals the formation of two different semi-synthetic hydrogenases in vivo. [FeFe] hydrogenases are metalloenzymes that catalyze the interconversion of molecular hydrogen and protons. The reaction is catalyzed by the H-cluster, consisting of a canonical iron-sulfur cluster and an organometallic [2Fe] subsite. It was recently shown that the enzyme can be reconstituted with synthetic cofactors mimicking the composition of the [2Fe] subsite, resulting in semi-synthetic hydrogenases. Herein, we employ EPR spectroscopy to monitor the formation of two such semi-synthetic enzymes in whole cells. The study provides the first spectroscopic characterization of semi-synthetic hydrogenases in vivo, and the observation of two different oxidized states of the H-cluster under intracellular conditions. Moreover, these findings underscore how synthetic chemistry can be a powerful tool for manipulation and examination of the hydrogenase enzyme under in vivo conditions.

Photophysics of Coumarin and Carbostyril-Sensitized Luminescent Lanthanide Complexes: Implications for Complex Design in Multiplex Detection.

Luminescent lanthanide (Ln(III)) complexes with coumarin or carbostyril antennae were synthesized and their photophysical properties evaluated using steady-state and time-resolved UV-vis spectroscopy. Ligands bearing distant hydroxycoumarin-derived antennae attached through triazole linkers were modest sensitizers for Eu(III) and Tb(III), whereas ligands with 7-amidocarbostyrils directly linked to the coordination site could reach good quantum yields for multiple Ln(III), including the visible emitters Sm(III) and Dy(III), and the near-infrared emitters Nd(III) and Yb(III). The highest lanthanide-centered luminescence quantum yields were 35% (Tb), 7.9% (Eu), 0.67% (Dy), and 0.18% (Sm). Antennae providing similar luminescence intensities with 2-4 Ln-emitters were identified. Photoredox quenching of the carbostyril antenna excited states was observed for all Eu(III)-complexes and should be sensitizing in the case of Yb(III); the scope of the process extends to Ln(III) for which it has not been seen previously, specifically Dy(III) and Sm(III). The proposed process is supported by photophysical and electrochemical data. A FRET-type mechanism was identified in architectures with both distant and close antennae for all of the Lns. This mechanism seems to be the only sensitizing one at long distance and probably contributes to the sensitization at shorter distances along with the triplet pathway. The complexes were nontoxic to either bacterial or mammalian cells. Complexes of an ester-functionalized ligand were taken up by bacteria in a concentration-dependent manner. Our results suggest that the effects of FRET and photoredox quenching should be taken into consideration when designing luminescent Ln complexes. These results also establish these Ln(III)-complexes for multiplex detection beyond the available two-color systems.

Electron-transfer subunits of the NiFe hydrogenases in Thiocapsa roseopersicina BBS.

Thiocapsa roseopersicina BBS contains at least three different active NiFe hydrogenases: two membrane-bound enzymes and one apparently localized in the cytoplasm. In addition to the small and large structural subunits, additional proteins are usually associated with the NiFe hydrogenases, connecting their activity to other redox processes in the cells. The operon of the membrane-associated hydrogenase, HynSL, has an unusual gene arrangement: between the genes coding for the large and small subunits, there are two open reading frames, namely isp1 and isp2. Isp1 is a b-type haem-containing transmembrane protein, whereas Isp2 displays marked sequence similarity to the heterodisulfide reductases. The other membrane-bound (Hup) NiFe hydrogenase contains the hupC gene, which codes for a cytochrome b-type protein that probably plays a role in electron transport. The operon of the NAD(+)-reducing Hox hydrogenase contains a hoxE gene. In addition to the hydrogenase and diaphorase parts of the complex, the fifth HoxE subunit may serve as a third redox gate of this enzyme. The physiological functions of these putative electron-mediating subunits were studied by disruption of their genes. The deletion of some accessory proteins dramatically reduced the in vivo activities of the hydrogenases, although they were fully active in vitro. The absence of HupC resulted in a decrease in HupSL activity in the membrane, but removal of the Isp1 and Isp2 proteins did not have any significant effect on the location of HynSL activity. Through the use of a tagged HoxE protein, the whole Hox hydrogenase pentamer could be purified as an intact complex.

Hydrogen independent expression of hupSL genes in Thiocapsa roseopersicina BBS.

The expression of many membrane bound [NiFe] hydrogenases is regulated by their substrate molecule, hydrogen. The HupSL hydrogenase, encoded in the hupSLCDHIR operon, probably plays a role in hydrogen recycling in the phototrophic purple bacterium, Thiocapsa roseopersicina BBS. RpoN, coding for sigma factor 54, was shown to be important for expression, suggesting a regulated biosynthsis from the hup gene cluster. The response regulator gene, hupR, has been identified in the hup operon and expression of hupSL was reduced in a chromosomal hupR mutant, which indicated that HupR was implicated in the activation process. The hupT and hupUV genes were isolated, and show similarity to the histidine kinase element of the H2-driven signal transduction system and to the regulatory hydrogenases of Ralstonia eutropha and Rhodobacter capsulatus, respectively. Although the genes of the entire H2 sensing and regulation system were present, the expression of the hupSL genes was not affected by the presence or absence of H2. Using reverse transcription PCR, we could not detect any mRNA specific to the hupTUV genes in cells grown under diverse conditions. The hupT and hupUV mutant strains had the same phenotype as the wild-type strains. The hupT gene product, expressed from a plasmid, repressed HupSL synthesis as expected while introduction of actively expressed hupTUV genes together derepressed the HupSL activity in T. roseopersicina. The gene product of hupUV behaves similarly to other regulatory hydrogenases and shows H-D exchange activity.

Modular broad-host-range expression vectors for single-protein and protein complex purification.

A set of modular broad-host-range expression vectors with various affinity tags (six-His-tag, FLAG-tag, Strep-tag II, T7-tag) was created. The complete nucleotide sequences of the vectors are known, and these small vectors can be mobilized by conjugation. They are useful in the purification of proteins and protein complexes from gram-negative bacterial species. The plasmids were easily customized for Thiocapsa roseopersicina, Rhodobacter capsulatus, and Methylococcus capsulatus by inserting an appropriate promoter. These examples demonstrate the versatility and flexibility of the vectors. The constructs harbor the T7 promoter for easy overproduction of the desired protein in an appropriate Escherichia coli host. The vectors were useful in purifying different proteins from T. roseopersicina. The FLAG-tag-Strep-tag II combination was utilized for isolation of the HynL-HypC2 protein complex involved in hydrogenase maturation. These tools should be useful for protein purification and for studying protein-protein interactions in a range of bacterial species.