Iron-sulfur Centers Research Articles

Acetylene hydratase: a non-redox enzyme with tungsten and iron-sulfur centers at the active site.

In living systems, tungsten is exclusively found in microbial enzymes coordinated by the pyranopterin cofactor, with additional metal coordination provided by oxygen and/or sulfur, and/or selenium atoms in diverse arrangements. Prominent examples are formate dehydrogenase, formylmethanofuran dehydrogenase, and aldehyde oxidoreductase all of which catalyze redox reactions. The bacterial enzyme acetylene hydratase (AH) stands out of its class as it catalyzes the conversion of acetylene to acetaldehyde, clearly a non-redox reaction and a reaction distinct from the reduction of acetylene to ethylene by nitrogenase. AH harbors two pyranopterins bound to W, and a [4Fe-4S] cluster. W is coordinated by four dithiolene sulfur atoms, one cysteine sulfur, and one oxygen ligand. AH activity requires a strong reductant suggesting W(IV) as the active oxidation state. Two different types of reaction pathways have been proposed. The 1.26Å structure reveals a water molecule coordinated to W which could gain a partially positive net charge by the adjacent protonated Asp-13, enabling a direct attack of C2H2. To access the W-Asp site, a substrate channel was evolved distant from where it is found in other members of the DMSOR family. Computational studies of this second shell mechanism led to unrealistically high energy barriers, and alternative pathways were proposed where C2H2 binds directly to W. The architecture of the catalytic cavity, the specificity for C2H2 and the results from site-directed mutagenesis do not support this first shell mechanism. More investigations including structural information on the binding of C2H2 are needed to present a conclusive answer.

Structural principles for computational and de novo design of 4Fe–4S metalloproteins

Iron-sulfur centers in metalloproteins can access multiple oxidation states over a broad range of potentials, allowing them to participate in a variety of electron transfer reactions and serving as catalysts for high-energy redox processes. The nitrogenase FeMoCO cluster converts di-nitrogen to ammonia in an eight-electron transfer step. The 2(Fe4S4) containing bacterial ferredoxin is an evolutionarily ancient metalloprotein fold and is thought to be a primordial progenitor of extant oxidoreductases. Controlling chemical transformations mediated by iron-sulfur centers such as nitrogen fixation, hydrogen production as well as electron transfer reactions involved in photosynthesis are of tremendous importance for sustainable chemistry and energy production initiatives. As such, there is significant interest in the design of iron-sulfur proteins as minimal models to gain fundamental understanding of complex natural systems and as lead-molecules for industrial and energy applications. Herein, we discuss salient structural characteristics of natural iron-sulfur proteins and how they guide principles for design. Model structures of past designs are analyzed in the context of these principles and potential directions for enhanced designs are presented, and new areas of iron-sulfur protein design are proposed. This article is part of a Special issue entitled Biodesign for Bioenergetics — the design and engineering of electronic transfer cofactors, protein networks, edited by Ronald L. Koder and J.L Ross Anderson.

Isolation and characterization of a new [FeFe]-hydrogenase from Clostridium perfringens.

This paper reports the first characterization of an [FeFe]-hydrogenase from a Clostridium perfringens strain previously isolated in our laboratory from a pilot-scale bio-hydrogen plant that efficiently produces H2 from waste biomasses. On the basis of sequence analysis, the enzyme is a monomer formed by four domains hosting various iron-sulfur centres involved in electron transfer and the catalytic center H-cluster. After recombinant expression in Escherichia coli, the purified protein catalyzes H2 evolution at high rate of 1645±16s(-1) . The optimal conditions for catalysis are in the pH range 6.5-8.0 and at the temperature of 50°C. EPR spectroscopy showed that the H-cluster of the oxidized enzyme displays a spectrum coherent with the Hox state, whereas the CO-inhibited enzyme has a spectrum coherent with the Hox -CO state. FTIR spectroscopy showed that the purified enzyme is composed of a mixture of redox states, with a prevalence of the Hox ; upon reduction with H2 , vibrational modes assigned to the Hred state were more abundant, whereas binding of exogenous CO resulted in a spectrum assigned to the Hox -CO state. The spectroscopic features observed are similar to those of the [FeFe]-hydrogenases class, but relevant differences were observed given the different protein environment hosting the H-cluster.

Menaquinone as the Secondary Electron Acceptor in the Type I Homodimeric Photosynthetic Reaction Center of Heliobacterium modesticaldum.

The type I photosynthetic reaction center (RC) of heliobacteria (hRC) is a homodimer containing cofactors almost analogous to those in the plant photosystem I (PS I). However, its three-dimensional structure is not yet clear. PS I uses phylloquinone (PhyQ) as a secondary electron acceptor (A1), while the available evidence has suggested that menaquinone (MQ) in hRC has no function as A1. The present study identified a new transient electron spin-polarized electron paramagnetic resonance (ESP-EPR) signal, arising from the radical pair of the oxidized electron donor and the reduced electron acceptor (P800(+)MQ(-)), in the hRC core complex and membranes from Heliobacterium modesticaldum. The ESP signal could be detected at 5-20 K upon flash excitation only after prereduction of the iron-sulfur center, F(X), and was selectively lost by extraction of MQ with diethyl ether. MQ was suggested to be located closer to F(X) than PhyQ in PS I based on the simulation of the unique A/E (A, absorption; E, emission) ESP pattern, the reduction/oxidation rates of MQ, and the power saturation property of the static MQ(-) signal. The result revealed the quinone usage as the secondary electron acceptor in hRC, as in the case of PS I.

Directionality of Electron Transfer in Type I Reaction Center Proteins: High-Frequency EPR Study of PS I with Removed Iron-Sulfur Centers.

A key step of photosynthetic solar energy conversion involves rapid light-induced sequential electron-transfer steps that result in the formation of a stabilized charge-separated state. These primary reactions take place in large integral membrane reaction center (RC) proteins, wherein a series of donor/acceptor cofactors are specifically positioned for efficient electron transfer. RCs can be divided in two classes, Type I and Type II and examples of both types, photosystem I (PS I) and photosystem II (PS II), are involved in the oxygenic photosynthesis of higher plants, cyanobacteria, and algae. High-resolution X-ray crystal structures reveal that PS I and PS II contain two nearly symmetric branches of redox cofactors, termed the A and B branches. While unidirectional ET along the A branch in Type II RCs is well established, there is still a debate of whether primary photochemistry in Type I RCs is unidirectional along the A branch or bidirectional proceeding down both of the A and B branches. Light-induced electron transfer through the B branch has been observed in genetically modified PS I and in native PS I pretreated with strong reducing conditions to reduce three [4Fe-4S] clusters, the terminal electron acceptors of PS I; however, the extent of asymmetry of ET along both cofactor branches remains an open question. To prove that bidirectional ET in PS I is not simply an artifact of a reducing environment or genetic modification and to determine the degree of PS I ET asymmetry, we have examined biochemically modified Synechococcus leopoliensis PS I RCs, wherein the [4Fe-4S] clusters FX, FA, and FB have been removed to prevent secondary ET from phylloquinones (A1A/A1B) to FX. For these Fe-removed proteins, we observe that ET along both the A and B branches occurs with a ratio close to 1. Together with previously reported data, the concomitant structural and kinetic information obtained with HF EPR unambiguously proves the bidirectional nature of ET in PS I over a broad temperature range.

An Iron Reservoir to the Catalytic Metal: THE RUBREDOXIN IRON IN AN EXTRADIOL DIOXYGENASE

The rubredoxin motif is present in over 74,000 protein sequences and 2,000 structures, but few have known functions. A secondary, non-catalytic, rubredoxin-like iron site is conserved in 3-hydroxyanthranilate 3,4-dioxygenase (HAO), from single cellular sources but not multicellular sources. Through the population of the two metal binding sites with various metals in bacterial HAO, the structural and functional relationship of the rubredoxin-like site was investigated using kinetic, spectroscopic, crystallographic, and computational approaches. It is shown that the first metal presented preferentially binds to the catalytic site rather than the rubredoxin-like site, which selectively binds iron when the catalytic site is occupied. Furthermore, an iron ion bound to the rubredoxin-like site is readily delivered to an empty catalytic site of metal-free HAO via an intermolecular transfer mechanism. Through the use of metal analysis and catalytic activity measurements, we show that a downstream metabolic intermediate can selectively remove the catalytic iron. As the prokaryotic HAO is often crucial for cell survival, there is a need for ensuring its activity. These results suggest that the rubredoxin-like site is a possible auxiliary iron source to the catalytic center when it is lost during catalysis in a pathway with metabolic intermediates of metal-chelating properties. A spare tire concept is proposed based on this biochemical study, and this concept opens up a potentially new functional paradigm for iron-sulfur centers in iron-dependent enzymes as transient iron binding and shuttling sites to ensure full metal loading of the catalytic site.

A structural model for FOXRED1, an FAD-dependent oxidoreductase necessary for NADH: Ubiquinone oxidoreductase (complex I) assembly

The biogenesis of mitochondrial respiratory chain components is complex. Mammalian complex I (NADH:ubiquinone oxidoreductase) contains 44 different subunits, an FMN and seven iron–sulfur centers. Its assembly involves at least twelve additional proteins, called assembly factors. One of these is FOXRED1, a 486-amino acid FAD-dependent oxidoreductase. FOXRED1 is a member of the d-amino acid oxidase (DAO) family. A structural model of FOXRED1 reveals a large substrate-binding cavity and a putative oxygen-binding site. These features strongly suggest that FOXRED1 is catalytically active as an oxidoreductase. A metabolic role for FOXRED1 in the biogenesis of complex I should be considered.

Electron tunneling rates in respiratory complex I are tuned for efficient energy conversion.

Respiratory complex I converts the free energy of ubiquinone reduction by NADH into a proton motive force, a redox reaction catalyzed by flavin mononucleotide(FMN) and a chain of seven iron–sulfur centers. Electron transfer rates between the centers were determined by ultrafast freeze-quenching and analysis by EPR and UV/Vis spectroscopy. The complex rapidly oxidizes three NADH molecules. The electron-tunneling rate between the most distant centers in the middle of the chain depends on the redox state of center N2 at the end of the chain, and is sixfold slower when N2 is reduced. The conformational changes that accompany reduction of N2 decrease the electronic coupling of the longest electron-tunneling step. The chain of iron–sulfur centers is not just a simple electron-conducting wire; it regulates the electron-tunneling rate synchronizing it with conformation-mediated proton pumping, enabling efficient energy conversion. Synchronization of rates is a principle means of enhancing the specificity of enzymatic reactions.

Iron-Sulfur Cluster-dependent Catalysis of Chlorophyllide a Oxidoreductase from Roseobacter denitrificans

Bacteriochlorophyll a biosynthesis requires the stereo- and regiospecific two electron reduction of the C7-C8 double bond of chlorophyllide a by the nitrogenase-like multisubunit metalloenzyme, chlorophyllide a oxidoreductase (COR). ATP-dependent COR catalysis requires interaction of the protein subcomplex (BchX)2 with the catalytic (BchY/BchZ)2 protein to facilitate substrate reduction via two redox active iron-sulfur centers. The ternary COR enzyme holocomplex comprising subunits BchX, BchY, and BchZ from the purple bacterium Roseobacter denitrificans was trapped in the presence of the ATP transition state analog ADP·AlF4(-). Electron paramagnetic resonance experiments revealed a [4Fe-4S] cluster of subcomplex (BchX)2. A second [4Fe-4S] cluster was identified on (BchY/BchZ)2. Mutagenesis experiments indicated that the latter is ligated by four cysteines, which is in contrast to the three cysteine/one aspartate ligation pattern of the closely related dark-operative protochlorophyllide a oxidoreductase (DPOR). In subsequent mutagenesis experiments a DPOR-like aspartate ligation pattern was implemented for the catalytic [4Fe-4S] cluster of COR. Artificial cluster formation for this inactive COR variant was demonstrated spectroscopically. A series of chemically modified substrate molecules with altered substituents on the individual pyrrole rings and the isocyclic ring were tested as COR substrates. The COR enzyme was still able to reduce the B ring of substrates carrying modified substituents on ring systems A, C, and E. However, substrates with a modification of the distantly located propionate side chain were not accepted. A tentative substrate binding mode was concluded in analogy to the related DPOR system.

Structure of L-serine dehydratase from Legionella pneumophila: novel use of the C-terminal cysteine as an intrinsic competitive inhibitor.

Here we report the first complete structure of a bacterial Fe–S l-serine dehydratase determined to 2.25 Å resolution. The structure is of the type 2 l-serine dehydratase from Legionella pneumophila that consists of a single polypeptide chain containing a catalytic α domain and a β domain that is structurally homologous to the “allosteric substrate binding” or ASB domain of d-3-phosphoglycerate dehydrogenase from Mycobacterium tuberculosis. The enzyme exists as a dimer of identical subunits, with each subunit exhibiting a bilobal architecture. The [4Fe-4S]2+ cluster is bound by residues from the C-terminal α domain and is situated between this domain and the N-terminal β domain. Remarkably, the model reveals that the C-terminal cysteine residue (Cys 458), which is conserved among the type 2 l-serine dehydratases, functions as a fourth ligand to the iron–sulfur cluster producing a “tail in mouth” configuration. The interaction of the sulfhydryl group of Cys 458 with the fourth iron of the cluster appears to mimic the position that the substrate would adopt prior to catalysis. A number of highly conserved or invariant residues found in the β domain are clustered around the iron–sulfur center. Ser 16, Ser 17, Ser 18, and Thr 290 form hydrogen bonds with the carboxylate group of Cys 458 and the carbonyl oxygen of Glu 457, whereas His 19 and His 61 are poised to potentially act as the catalytic base required for proton extraction. Mutation of His 61 produces an inactive enzyme, whereas the H19A protein variant retains substantial activity, suggesting that His 61 serves as the catalytic base. His 124 and Asn 126, found in an HXN sequence, point toward the Fe–S cluster. Mutational studies are consistent with these residues either binding a serine molecule that serves as an activator or functioning as a potential trap for Cys 458 as it moves out of the active site prior to catalysis.

Selective induction and subcellular distribution of ACONITASE 3 reveal the importance of cytosolic citrate metabolism during lipid mobilization in Arabidopsis.

Arabidopsis thaliana has three genes that encode distinct aconitases (ACO), but little is known about the function of each isoenzyme during plant development. In newly emerged seedlings of Arabidopsis, transcript and protein levels for ACO3 were selectively induced to yield more than 80% of total aconitase activity. Characterization of knockout mutants for each of the three ACOs suggests a major role for only ACO3in citrate metabolism. The aco3 mutant showed delayed early seedling growth, altered assimilation of [14C]acetate feeding and elevated citrate levels, which were nearly 4-fold greater than in wild-type, aco1 or aco2. However, both ACO1 and ACO2 are active in seedlings as shown by inhibition of aco3 growth by the toxin monofluoroacetate, and altered [14C]acetate assimilation and metabolite levels in aco1 and aco2. Relative levels of fumarate and malate differed between aco2 and aco3, indicating metabolically isolated pools of these metabolites in seedlings. Our inability to enrich ACO protein through mitochondria isolation, and the reduced cytosolic ACO activity of the iron-sulfur centre assembly mutant atm3-1, indicated a cytosolic localization of ACO3in 3-day-old seedlings. Subsequently, we determined that more than 90% of ACO3 was cytosolic. We conclude that ACO3 is cytosolic in young seedlings and functions in citrate catabolism consistent with the operation of the classic glyoxylate and not direct catabolism of citrate within mitochondria.

Role of oxygen and the OxyR protein in the response to iron limitation in Rhodobacter sphaeroides.

BackgroundHigh intracellular levels of unbound iron can contribute to the production of reactive oxygen species (ROS) via the Fenton reaction, while depletion of iron limits the availability of iron-containing proteins, some of which have important functions in defence against oxidative stress. Vice versa increased ROS levels lead to the damage of proteins with iron sulphur centres. Thus, organisms have to coordinate and balance their responses to oxidative stress and iron availability. Our knowledge of the molecular mechanisms underlying the co-regulation of these responses remains limited. To discriminate between a direct cellular response to iron limitation and indirect responses, which are the consequence of increased levels of ROS, we compared the response of the α-proteobacterium Rhodobacter sphaeroides to iron limitation in the presence or absence of oxygen.ResultsOne third of all genes with altered expression under iron limitation showed a response that was independent of oxygen availability. The other iron-regulated genes showed different responses in oxic or anoxic conditions and were grouped into six clusters based on the different expression profiles. For two of these clusters, induction in response to iron limitation under oxic conditions was dependent on the OxyR regulatory protein. An OxyR mutant showed increased ROS production and impaired growth under iron limitation.ConclusionSome R. sphaeroides genes respond to iron limitation irrespective of oxygen availability. These genes therefore reflect a “core iron response” that is independent of potential ROS production under oxic, iron-limiting conditions. However, the regulation of most of the iron-responsive genes was biased by oxygen availability. Most strikingly, the OxyR-dependent activation of a subset of genes upon iron limitation under oxic conditions, including many genes with a role in iron metabolism, revealed that elevated ROS levels were an important trigger for this response. OxyR thus provides a regulatory link between the responses to oxidative stress and to iron limitation in R. sphaeroides.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-794) contains supplementary material, which is available to authorized users.

Diverse Bacterial Microcompartment Organelles

Bacterial microcompartments (MCPs) are sophisticated protein-based organelles used to optimize metabolic pathways. They consist of metabolic enzymes encapsulated within a protein shell, which creates an ideal environment for catalysis and facilitates the channeling of toxic/volatile intermediates to downstream enzymes. The metabolic processes that require MCPs are diverse and widely distributed and play important roles in global carbon fixation and bacterial pathogenesis. The protein shells of MCPs are thought to selectively control the movement of enzyme cofactors, substrates, and products (including toxic or volatile intermediates) between the MCP interior and the cytoplasm of the cell using both passive electrostatic/steric and dynamic gated mechanisms. Evidence suggests that specialized shell proteins conduct electrons between the cytoplasm and the lumen of the MCP and/or help rebuild damaged iron-sulfur centers in the encapsulated enzymes. The MCP shell is elaborated through a family of small proteins whose structural core is known as a bacterial microcompartment (BMC) domain. BMC domain proteins oligomerize into flat, hexagonally shaped tiles, which assemble into extended protein sheets that form the facets of the shell. Shape complementarity along the edges allows different types of BMC domain proteins to form mixed sheets, while sequence variation provides functional diversification. Recent studies have also revealed targeting sequences that mediate protein encapsulation within MCPs, scaffolding proteins that organize lumen enzymes and the use of private cofactor pools (NAD/H and coenzyme A [HS-CoA]) to facilitate cofactor homeostasis. Although much remains to be learned, our growing understanding of MCPs is providing a basis for bioengineering of protein-based containers for the production of chemicals/pharmaceuticals and for use as molecular delivery vehicles.

Decoding the s-nitrosoproteomic atlas in individualized human colorectal cancer tissues using a label-free quantitation strategy.

The abnormal S-nitrosylation induced by the overexpression and activation of inducible nitric oxide synthase (iNOS) modulates many human diseases, such as inflammation and cancer. To delineate the pathophysiological S-nitrosoproteome in cancer patients, we report an individualized S-nitrosoproteomic strategy with a label-free method for the site-specific quantification of S-nitrosylation in paired tumor and adjacent normal tissues from 11 patients with colorectal cancer (CRC). This study provides not only the first endogenous human S-nitrosoproteomic atlas but also the first individualized human tissue analysis, identifying 174 S-nitrosylation sites in 94 proteins. Fourteen novel S-nitrosylation sites with a high frequency of elevated levels in 11 individual patients were identified. An individualized S-nitrosylation quantitation analysis revealed that the detected changes in S-nitrosylation were regulated by both the expression level and the more dramatic post-translational S-nitrosylation of the targeted proteins, such as thioredoxin, annexin A4, and peroxiredoxin-4. These endogenous S-nitrosylated proteins illustrate the network of inflammation/cancer-related and redox reactions mediated by various S-nitrosylation sources, including iNOS, transnitrosylase, or iron-sulfur centers. Given the demonstrated sensitivity of individualized tissue analysis, this label-free approach may facilitate the study of the vastly under-represented S-nitrosoproteome and enable a better understanding of the effect of endogenous S-nitrosylation in cancer.

Defining the Far-red Limit of Photosystem I: The Primary Charge Separation is Functional to 840 nm

The far-red limit of photosystem I (PS I) photochemistry was studied by EPR spectroscopy using laser flashes between 730 and 850 nm. In manganese-depleted spinach thylakoid membranes, the primary donor in PS I, P700, was oxidized simultaneously with tyrosine Z, the secondary donor in PS II. It was found that at 295 K PS I photochemistry, observed as P700 (+) formation, was functional up to 840 nm. This is 30 nm further to the red region than was reported for PS II photochemistry (Thapper, A., Mamedov, F., Mokvist, F., Hammarström, L., and Styring, S. (2009) Plant Cell 21, 2391-2401). The same far-red limit for the P700 (+) formation was observed in a PS I reaction center core preparation from Nostoc punctiforme. The reduction of the acceptor side of PS I, observed as reduction of the iron-sulfur centers FA and FB by low temperature EPR measurements, was also functional at 15 K with light up to >830 nm. Taken together, these results, obtained from both plants and cyanobacteria, most likely rule out involvement of the red-absorbing antenna chlorophylls in this reaction. Instead we propose the existence of weak charge transfer bands absorbing in the far-red region in the ensemble of excitonically coupled chlorophyll a molecules around P700 similar to what has been found in the reaction center of PS II. These charge transfer bands could be responsible for the far-red light absorption leading to PS I photochemistry at wavelengths up to 840 nm.

Escherichia coli RIC participates in the formation of iron-sulfur centres through iron donation

Escherichia coli RIC participates in the formation of iron-sulfur centres through iron donation

Characterisation of Desulfovibrio vulgaris haem b synthase, a radical SAM family member

An alternative route for haem b biosynthesis is operative in sulfate-reducing bacteria of the Desulfovibrio genus and in methanogenic Archaea. This pathway diverges from the canonical one at the level of uroporphyrinogen III and progresses via a distinct branch, where sirohaem acts as an intermediate precursor being converted into haem b by a set of novel enzymes, named the alternative haem biosynthetic proteins (Ahb). In this work, we report the biochemical characterisation of the Desulfovibrio vulgaris AhbD enzyme that catalyses the last step of the pathway. Mass spectrometry analysis showed that AhbD promotes the cleavage of S-adenosylmethionine (SAM) and converts iron-coproporphyrin III via two oxidative decarboxylations to yield haem b, methionine and the 5′-deoxyadenosyl radical. Electron paramagnetic resonance spectroscopy studies demonstrated that AhbD contains two [4Fe–4S]2+/1+ centres and that binding of the substrates S-adenosylmethionine and iron-coproporphyrin III induces conformational modifications in both centres. Amino acid sequence comparisons indicated that D. vulgaris AhbD belongs to the radical SAM protein superfamily, with a GGE-like motif and two cysteine-rich sequences typical for ligation of SAM molecules and iron-sulfur clusters, respectively. A structural model of D. vulgaris AhbD with putative binding pockets for the iron-sulfur centres and the substrates SAM and iron-coproporphyrin III is discussed.

Single Molecule Force Spectroscopy Reveals the Molecular Mechanical Anisotropy of the FeS4 Metal Center in Rubredoxin

Mechanical anisotropy is an important feature of materials. Depending on the pulling direction, a material can exhibit very different mechanical properties. Mechanical anisotropy on the microscopic scale has also been observed for individual elastomeric proteins. Depending upon the pulling direction, a protein can unfold via different mechanical unfolding pathways and exhibit different mechanical stability. However, it remains to be demonstrated if the concept of mechanical anisotropy can be extended to molecular scale for small molecular objects containing only a few chemical bonds. Here, we choose the iron-sulfur center FeS4 in rubredoxin as a model system to demonstrate the molecular level mechanical anisotropy. We used single molecular force spectroscopy to investigate the mechanical rupture of the FeS4 center along different pulling directions. The FeS4 cluster is a simple molecular object with defined three-dimensional structure, where a ferric ion and four coordinating cysteinyl ligands are arranged into a distorted tetrahedral geometry. Mutating two specific residues in rubredoxin to cysteines provides anchoring points that enable us to stretch and rupture the FeS4 center along five distinct and precisely controlled directions. Our results showed that mechanical stability as well as the rupture mechanism and kinetics of the FeS4 center are strongly dependent upon the direction along which it is stretched, suggesting that the very small FeS4 center exhibits considerable mechanical anisotropy. It is likely that structural asymmetry in the FeS4 cluster and the modulation of the local environment due to partial unfolding of rubredoxin are responsible for the observed mechanical anisotropy. Our results suggest that mechanical anisotropy is a universal feature for any asymmetrical three-dimensional structure, even down to a molecular scale, and such mechanical anisotropy can be potentially utilized to control the mechanochemical reactivity of molecular objects.

The Functional Complexity of [NiFe] Hydrogenases in Sulfate Reducing Bacteria (Genus; Desulforvibrio spp)

Sulfate-reducing bacteria are categories of bacteria and archaea that can obtain energy by oxidizing organic compounds or molecular hydrogen (H2) while reducing sulfate (SO42−) to hydrogen sulfide (H2S). By analysis, these organisms "respire" sulfate rather than oxygen, a form of anaerobic respiration, the oxidation of hydrogen by the primary genus of Sulfate Reducing Bacteria (Desulfovibrio, Desulfovibrio desulfuricans) is catalyzed by enzymes called Hydrogenases. Three basic types of hydrogenases have been widely isolated from the primary genus of sulfate-reducing bacteria Desulfobibrio which differ in their structural subunits, metal compositions, physico-chemical characteristics, amino acid sequences, immunological activities, structural gene configuration and their catalytic properties. Broadly, hydrogenases can be considered as ‘iron containing hydrogenases and nickel-containing hydrogenases. The iron-sulfur-containing hydrogenase enzyme contains two ferredoxin-type (4Fe-4S) clusters and typical iron-sulfur center believed to be involved in the activation of H2 yet it is the most sensitive domain to CO and NO2−.eventhough it is not featured in all species of genus Desulfovibrio. The nickel-(iron-sulfur)-containing hydrogenases, [NiFe] hydrogenase posses two 4Fe-4S centers and one 3Fe-xS cluster in addition to nickel and have been found in all species of Desulfovibrio with strong resistance to CO and NO2- so far investigated. The genes encoding the large and small subunits of a periplasmic and membrane-bound species of the [NiFe] hydrogenase have been cloned in Escherichia coli and sequenced, however the functional complexity of the hydrogenase system remained unexplored as a result of the metabolic diversity in Desulfovibrio spp. The [NiFe] hydrogenase plays an important role in the energy metabolism of Desulfovibrio spp. Thus, the expression of the encoded structural genes would be an excellent marker for the metabolic functionalities under specific inducible environment.

Identification of crucial amino acids in mouse aldehyde oxidase 3 that determine substrate specificity.

In order to elucidate factors that determine substrate specificity and activity of mammalian molybdo-flavoproteins we performed site directed mutagenesis of mouse aldehyde oxidase 3 (mAOX3). The sequence alignment of different aldehyde oxidase (AOX) isoforms identified variations in the active site of mAOX3 in comparison to other AOX proteins and xanthine oxidoreductases (XOR). Based on the structural alignment of mAOX3 and bovine XOR, differences in amino acid residues involved in substrate binding in XORs in comparison to AOXs were identified. We exchanged several residues in the active site to the ones found in other AOX homologues in mouse or to residues present in bovine XOR in order to examine their influence on substrate selectivity and catalytic activity. Additionally we analyzed the influence of the [2Fe-2S] domains of mAOX3 on its kinetic properties and cofactor saturation. We applied UV-VIS and EPR monitored redox-titrations to determine the redox potentials of wild type mAOX3 and mAOX3 variants containing the iron-sulfur centers of mAOX1. In addition, a combination of molecular docking and molecular dynamic simulations (MD) was used to investigate factors that modulate the substrate specificity and activity of wild type and AOX variants. The successful conversion of an AOX enzyme to an XOR enzyme was achieved exchanging eight residues in the active site of mAOX3. It was observed that the absence of the K889H exchange substantially decreased the activity of the enzyme towards all substrates analyzed, revealing that this residue has an important role in catalysis.