Javier Seravalli

Xenon in and at the End of the Tunnel of Bifunctional Carbon Monoxide Dehydrogenase/Acetyl-CoA Synthase,

A fascinating feature of some bifunctional enzymes is the presence of an internal channel or tunnel to connect the multiple active sites. A channel can allow for a reaction intermediate generated at one active site to be used as a substrate at a second active site, without the need for the intermediate to leave the safety of the protein matrix. One such bifunctional enzyme is carbon monoxide dehydrogenase/acetyl-CoA synthase from Moorella thermoacetica (mtCODH/ACS). A key player in the global carbon cycle, CODH/ACS uses a Ni-Fe-S center called the C-cluster to reduce carbon dioxide to carbon monoxide and uses a second Ni-Fe-S center, called the A-cluster, to assemble acetyl-CoA from a methyl group, coenzyme A, and C-cluster-generated CO. mtCODH/ACS has been proposed to contain one of the longest enzyme channels (138 A long) to allow for intermolecular CO transport. Here, we report a 2.5 A resolution structure of xenon-pressurized mtCODH/ACS and examine the nature of gaseous cavities within this enzyme. We find that the cavity calculation program CAVENV accurately predicts the channels connecting the C- and A-clusters, with 17 of 19 xenon binding sites within the predicted regions. Using this X-ray data, we analyze the amino acid composition surrounding the 19 Xe sites and consider how the protein fold is utilized to carve out such an impressive interior passageway. Finally, structural comparisons of Xe-pressurized mtCODH/ACS with related enzyme structures allow us to study channel design principles, as well as consider the conformational flexibility of an enzyme that contains a cavity through its center.

Biosynthesis and Reactivity of Cysteine Persulfides in Signaling

Hydrogen sulfide (H2S) elicits pleiotropic physiological effects ranging from modulation of cardiovascular to CNS functions. A dominant method for transmission of sulfide-based signals is via posttranslational modification of reactive cysteine thiols to persulfides. However, the source of the persulfide donor and whether its relationship to H2S is as a product or precursor is controversial. The transsulfuration pathway enzymes can synthesize cysteine persulfide (Cys-SSH) from cystine and H2S from cysteine and/or homocysteine. Recently, Cys-SSH was proposed as the primary product of the transsulfuration pathway with H2S representing a decomposition product of Cys-SSH. Our detailed kinetic analyses demonstrate a robust capacity for Cys-SSH production by the human transsulfuration pathway enzymes, cystathionine beta-synthase and γ-cystathionase (CSE) and for homocysteine persulfide synthesis from homocystine by CSE only. However, in the reducing cytoplasmic milieu where the concentration of reduced thiols is significantly higher than of disulfides, substrate level regulation favors the synthesis of H2S over persulfides. Mathematical modeling at physiologically relevant hepatic substrate concentrations predicts that H2S rather than Cys-SSH is the primary product of the transsulfuration enzymes with CSE being the dominant producer. The half-life of the metastable Cys-SSH product is short and decomposition leads to a mixture of polysulfides (Cys-S-(S)n-S-Cys). These in vitro data, together with the intrinsic reactivity of Cys-SSH for cysteinyl versus sulfur transfer, are consistent with the absence of an observable increase in protein persulfidation in cells in response to exogenous cystine and evidence for the formation of polysulfides under these conditions.

Functional copper at the acetyl-CoA synthase active site

The bifunctional CO dehydrogenase/acetyl-CoA synthase (CODH/ACS) plays a central role in the Wood–Ljungdahl pathway of autotrophic CO2 fixation. A recent structure of the Moorella thermoacetica enzyme revealed that the ACS active site contains a [4Fe-4S] cluster bridged to a binuclear Cu-Ni site. Here, biochemical and x-ray absorption spectroscopic (XAS) evidence is presented that the copper ion at the M. thermoacetica ACS active site is essential. Depletion of copper correlates with reduction in ACS activity and in intensity of the “NiFeC” EPR signal without affecting either the activity or the EPR spectroscopic properties associated with CODH. In contrast, Zn content is negatively correlated with ACS activity without any apparent relationship to CODH activity. Cu is also found in the methanogenic CODH/ACS from Methanosarcina thermophila. XAS studies are consistent with a distorted Cu(I)–S3 site in the fully active enzyme in solution. Cu extended x-ray absorption fine structure analysis indicates an average Cu–S bond length of 2.25 Å and a metal neighbor at 2.65 Å, consistent with the Cu–Ni distance observed in the crystal structure. XAS experiments in the presence of seleno-CoA reveal a Cu–S3Se environment with a 2.4-Å Se–Cu bond, strongly implicating a Cu–SCoA intermediate in the mechanism of acetyl-CoA synthesis. These results indicate an essential and functional role for copper in the CODH/ACS from acetogenic and methanogenic organisms.

Dietary selenium affects host selenoproteome expression by influencing the gut microbiota.

Colonization of the gastrointestinal tract and composition of the microbiota may be influenced by components of the diet, including trace elements. To understand how selenium regulates the intestinal microflora, we used high‐throughput sequencing to examine the composition of gut microbiota of mice maintained on selenium‐deficient, selenium‐sufficient, and selenium‐enriched diets. The microbiota diversity increased as a result of selenium in the diet. Specific phylotypes showed differential effects of selenium, even within a genus, implying that selenium had unique effects across microbial taxa. Conventionalized germ‐free mice subjected to selenium diets gave similar results and showed an increased diversity of the bacterial population in animals fed with higher levels of selenium. Germ‐free mice fed selenium diets modified their selenoproteome expression similar to control mice but showed higher levels and activity of glutathione peroxidase 1 and methionine‐R‐sulfoxide reductase 1 in the liver, suggesting partial sequestration of selenium by the gut microorganisms, limiting its availability for the host. These changes in the selenium status were independent of the levels of other trace elements. The data show that dietary selenium affects both composition of the intestinal microflora and colonization of the gastrointestinal tract, which, in turn, influence the host selenium status and selenoproteome expression.—Kasaikina, M. V., Kravtsova, M. A., Lee, B. C., Seravalli, J., Peterson, D. A., Walter, J., Legge, R., Benson, A. K., Hatfield, D. L., Gladyshev, V. N. Dietary selenium affects host selenoproteome expression by influencing the gut microbiota. FASEB J. 25, 2492–2499 (2011). www.fasebj.org

Crystallographic Snapshots of Cyanide- and Water-Bound C-Clusters from Bifunctional Carbon Monoxide Dehydrogenase/Acetyl-CoA Synthase

Nickel-containing carbon monoxide dehydrogenases (CODHs) reversibly catalyze the oxidation of carbon monoxide to carbon dioxide and are of vital importance in the global carbon cycle. The unusual catalytic CODH C-cluster has been crystallographically characterized as either a NiFe4S4 or a NiFe4S5 metal center, the latter containing a fifth, additional sulfide that bridges Ni and a unique Fe site. To determine whether this bridging sulfide is catalytically relevant and to further explore the mechanism of the C-cluster, we obtained crystal structures of the 310 kDa bifunctional CODH/acetyl-CoA synthase complex from Moorella thermoacetica bound both with a substrate H2O/OH− molecule and with a cyanide inhibitor. X-ray diffraction data were collected from native crystals and from identical crystals soaked in a solution containing potassium cyanide. In both structures, the substrate H2O/OH− molecule exhibits binding to the unique Fe site of the C-cluster. We also observe cyanide binding in a bent conformation to Ni of the C-cluster, adjacent the substrate H2O/OH− molecule. Importantly, the bridging sulfide is not present in either structure. As these forms of the C-cluster represent the coordination environment immediately before the reaction takes place, our findings do not support a fifth, bridging sulfide playing a catalytic role in the enzyme mechanism. The crystal structures presented here, along with recent structures of CODHs from other organisms, have led us toward a unified mechanism for CO oxidation by the C-cluster, the catalytic center of an environmentally important enzyme.

Visualizing molecular juggling within a B12-dependent methyltransferase complex

Derivatives of vitamin B12 are used in methyl group transfer in biological processes as diverse as methionine synthesis in humans and CO2 fixation in acetogenic bacteria. This seemingly straightforward reaction requires large, multimodular enzyme complexes that adopt multiple conformations to alternately activate, protect and perform catalysis on the reactive B12 cofactor. Crystal structures determined thus far have provided structural information for only fragments of these complexes, inspiring speculation about the overall protein assembly and conformational movements inherent to activity. Here we present X-ray crystal structures of a complete 220 kDa complex that contains all enzymes responsible for B12-dependent methyl transfer, namely the corrinoid iron–sulphur protein and its methyltransferase from the model acetogen Moorella thermoacetica. These structures provide the first three-dimensional depiction of all protein modules required for the activation, protection and catalytic steps of B12-dependent methyl transfer. In addition, the structures capture B12 at multiple locations between its ‘resting’ and catalytic positions, allowing visualization of the dramatic protein rearrangements that enable methyl transfer and identification of the trajectory for B12 movement within the large enzyme scaffold. The structures are also presented alongside in crystallo spectroscopic data, which confirm enzymatic activity within crystals and demonstrate the largest known conformational movements of proteins in a crystalline state. Taken together, this work provides a model for the molecular juggling that accompanies turnover and helps explain why such an elaborate protein framework is required for such a simple, yet biologically essential reaction.

High-Resolution Imaging of Selenium in Kidneys: A Localized Selenium Pool Associated with Glutathione Peroxidase 3

AIM Recent advances in quantitative methods and sensitive imaging techniques of trace elements provide opportunities to uncover and explain their biological roles. In particular, the distribution of selenium in tissues and cells under both physiological and pathological conditions remains unknown. In this work, we applied high-resolution synchrotron X-ray fluorescence microscopy (XFM) to map selenium distribution in mouse liver and kidney. RESULTS Liver showed a uniform selenium distribution that was dependent on selenocysteine tRNA([Ser]Sec) and dietary selenium. In contrast, kidney selenium had both uniformly distributed and highly localized components, the latter visualized as thin circular structures surrounding proximal tubules. Other parts of the kidney, such as glomeruli and distal tubules, only manifested the uniformly distributed selenium pattern that co-localized with sulfur. We found that proximal tubule selenium localized to the basement membrane. It was preserved in Selenoprotein P knockout mice, but was completely eliminated in glutathione peroxidase 3 (GPx3) knockout mice, indicating that this selenium represented GPx3. We further imaged kidneys of another model organism, the naked mole rat, which showed a diminished uniformly distributed selenium pool, but preserved the circular proximal tubule signal. INNOVATION We applied XFM to image selenium in mammalian tissues and identified a highly localized pool of this trace element at the basement membrane of kidneys that was associated with GPx3. CONCLUSION XFM allowed us to define and explain the tissue topography of selenium in mammalian kidneys at submicron resolution.

Water-gas shift reaction catalyzed by redox enzymes on conducting graphite platelets

The water-gas shift (WGS) reaction (CO + H(2)O <==> CO(2) + H(2)) is of major industrial significance in the production of H(2) from hydrocarbon sources. High temperatures are required, typically in excess of 200 degrees C, using d-metal catalysts on oxide supports. In our study the WGS process is separated into two half-cell electrochemical reactions (H(+) reduction and CO oxidation), catalyzed by enzymes attached to a conducting particle. The H(+) reduction reaction is catalyzed by a hydrogenase, Hyd-2, from Escherichia coli, and CO oxidation is catalyzed by a carbon monoxide dehydrogenase (CODH I) from Carboxydothermus hydrogenoformans. This results in a highly efficient heterogeneous catalyst with a turnover frequency, at 30 degrees C, of at least 2.5 s(-1) per minimum functional unit (a CODH/Hyd-2 pair) which is comparable to conventional high-temperature catalysts.

Pulse-Chase Studies of the Synthesis of Acetyl-CoA by Carbon Monoxide Dehydrogenase/Acetyl-CoA Synthase: EVIDENCE FOR A RANDOM MECHANISM OF METHYL AND CARBONYL ADDITION*

Carbon monoxide dehydrogenase/acetyl-CoA synthase catalyzes acetyl-CoA synthesis from CO, CoA, and a methylated corrinoid iron-sulfur protein, which acts as a methyl donor. This reaction is the last step in the Wood-Ljungdahl pathway of anaerobic carbon fixation. The binding sequence for the three substrates has been debated for over a decade. Different binding orders imply different mechanisms (i.e. paramagnetic versus diamagnetic mechanisms). Ambiguity arises because CO and CoA can each undergo isotopic exchange with acetyl-CoA, suggesting that either of these two substrates could be the last to bind to the acetyl-CoA synthase active site. Furthermore, carbonylation, CoA binding, and methyl transfer can all occur in the absence of the other two substrates. Here, we report pulse-chase studies, which unambiguously establish the order in which the three substrates bind. Although a CoA pulse is substantially diluted by excess CoA in the chase, isotope recovery of a pulse of labeled CO or methyl group is unaffected by the presence of excess unlabeled CO or methyl group in the chase. These results demonstrate that CoA is the last substrate to bind and that CO and the methyl group bind randomly as the first substrate in acetyl-CoA synthesis. Up to 100% of the methyl groups and CoA and up to 60–70% of the CO employed in the pulse phase can be trapped in the product acetyl-CoA.

Reduced Utilization of Selenium by Naked Mole Rats Due to a Specific Defect in GPx1 Expression

Naked mole rat (MR) Heterocephalus glaber is a rodent model of delayed aging because of its unusually long life span (>28 years). It is also not known to develop cancer. In the current work, tissue imaging by x-ray fluorescence microscopy and direct analyses of trace elements revealed low levels of selenium in the MR liver and kidney, whereas MR and mouse brains had similar selenium levels. This effect was not explained by uniform selenium deficiency because methionine sulfoxide reductase activities were similar in mice and MR. However, glutathione peroxidase activity was an order of magnitude lower in MR liver and kidney than in mouse tissues. In addition, metabolic labeling of MR cells with 75Se revealed a loss of the abundant glutathione peroxidase 1 (GPx1) band, whereas other selenoproteins were preserved. To characterize the MR selenoproteome, we sequenced its liver transcriptome. Gene reconstruction revealed standard selenoprotein sequences except for GPx1, which had an early stop codon, and SelP, which had low selenocysteine content. When expressed in HEK 293 cells, MR GPx1 was present in low levels, and its expression could be rescued neither by removing the early stop codon nor by replacing its SECIS element. In addition, GPx1 mRNA was present in lower levels in MR liver than in mouse liver. To determine if GPx1 deficiency could account for the reduced selenium content, we analyzed GPx1 knock-out mice and found reduced selenium levels in their livers and kidneys. Thus, MR is characterized by the reduced utilization of selenium due to a specific defect in GPx1 expression.