Kinetic and crystallographic analysis of the key active site acid/base arginine in a soluble fumarate reductase

There is now overwhelming evidence supporting a common mechanism for fumarate reduction in the respiratory fumarate reductases. The X-ray structures of substrate-bound forms of these enzymes indicate that the substrate is well positioned to accept a hydride from FAD and a proton from an arginine side chain. Recent work on the enzyme from Shewanella frigidimarina [Doherty, M. K., Pealing, S. L., Miles, C. S., Moysey, R., Taylor, P., Walkinshaw, M. D., Reid, G. A., and Chapman, S. K. (2000) Biochemistry 39, 10695-10701] has strengthened the assignment of an arginine (Arg402) as the proton donor in fumarate reduction. Here we describe the crystallographic and kinetic analyses of the R402A, R402K, and R402Y mutant forms of the Shewanella enzyme. The crystal structure of the R402A mutant (2.0 A resolution) shows it to be virtually identical to the wild-type enzyme, apart from the fact that a water molecule occupies the position previously taken by part of the guanidine group of R402. Although structurally similar to the wild-type enzyme, the R402A mutant is inactive under all the conditions that were studied. This implies that a water molecule, in this position in the active site, cannot function as the proton donor for fumarate reduction. In contrast to the R402A mutation, both the R402K and R402Y mutant enzymes are active. Although this activity was at a very low level (at pH 7.2 some 10(4)-fold lower than that for the wild type), it does imply that both lysine and tyrosine can fulfill the role of an active site proton donor, albeit very poorly. The crystal structures of the R402K and R402Y mutant enzymes (2.0 A resolution) show that distances from the lysine and tyrosine side chains to the nearest carbon atom of fumarate are approximately 3.5 A, clearly permitting proton transfer. The combined results from mutagenesis, crystallographic, and kinetic studies provide formidable evidence that R402 acts as both a Lewis acid (stabilizing the build-up of negative charge upon hydride transfer from FAD to fumarate) and a Br?nsted acid (donating the proton to the substrate to complete the formation of succinate).     

Identification of the active site acid/base catalyst in a bacterial fumarate reductase: a kinetic and crystallographic study

The active sites of respiratory fumarate reductases are highly conserved, indicating a common mechanism of action involving hydride and proton transfer. Evidence from the X-ray structures of substrate-bound fumarate reductases, including that for the enzyme from Shewanella frigidimarina [Taylor, P., Pealing, S. L., Reid, G. A., Chapman, S. K., and Walkinshaw, M. D. (1999) Nat. Struct. Biol. 6, 1108-1112], indicates that the substrate is well positioned to accept a hydride from N5 of the FAD. However, the identity of the proton donor has been the subject of recent debate and has been variously proposed to be (using numbering for the S. frigidimarina enzyme) His365, His504, and Arg402. We have used site-directed mutagenesis to examine the roles of these residues in the S. frigidimarina enzyme. The H365A and H504A mutant enzymes exhibited lower k(cat) values than the wild-type enzyme but only by factors of 3-15, depending on pH. This, coupled with the increase in K(m) observed for these enzymes, indicates that His365 and His504 are involved in Michaelis complex formation and are not essential catalytic residues. In fact, examination of the crystal structure of S. frigidimarina fumarate reductase has led to the proposal that Arg402 is the only plausible active site acid. Consistent with this proposal, we report that the R402A mutant enzyme has no detectable fumarate reductase activity. The crystal structure of the H365A mutant enzyme shows that, in addition to the replacement at position 365, there have been some adjustments in the positions of active site residues. In particular, the observed change in the orientation of the Arg402 side chain could account for the decrease in k(cat) seen with the H365A enzyme. These results demonstrate that an active site arginine and not a histidine residue is the proton donor for fumarate reduction.     

The fumarate reductase system as a site of anthelmintic attack inAscaris suum

Various benzimidazole compounds have been shown to be highly eIIective as inhibitors (up to 50% reduction of activity) in vitro of the helminth-specilic enzyme, fumarate reductase, ofAscaris suum. Anthelmintically active and inactive benzimidazoles were similarly effective as inhibitors of enzyme activity. Albendazole-induced inhibition of Iumarate reductase was not observed when the enzyme was preincubated with NADH.     

A soluble fumarate reductase in Trypanosoma brucei procyclic trypomastigotes

The enzyme NADH-fumarate reductase associated with the membrane fraction of Trypanosoma brucei procyclic trypomastigotes, can be solubilized by more than 50% when increasing the ionic strength to the equivalent of 150 mM KCl. The apparent KMs for NADH (125 microM) and fumarate (50 microM) remain close to those previously reported for the membrane-bound form of this enzyme. Other electron acceptors (i.e. oxygen or cytochrome c) appear to accept electrons in the absence of fumarate (KM for cytochrome c = 50 microM). The drug L-092,201 (Merck, Sharp and Dohme Research Laboratories, Rahway, NJ), an inhibitor of the membrane-bound fumarate reductase, also blocked the solubilized enzyme. Given the relatively high ionic strength of the intracellular environment we propose that, in vivo, the enzyme fumarate reductase is in the mitochondrial matrix or in the soluble fraction of another intracellular compartment.     

Identification of active site residues of Escherichia coli fumarate reductase by site-directed mutagenesis.

Menaquinol-fumarate oxidoreductase of Escherichia coli is a four-subunit membrane-bound complex that catalyzes the final step in anaerobic respiration when fumarate is the terminal electron acceptor. The enzyme is structurally and catalytically similar to succinate dehydrogenase (succinate-ubiquinone oxidoreductase) from both procaryotes and eucaryotes. Both enzymes have been proposed to contain an essential cysteine residue at the active site based on studies with thiol-specific reagents. Chemical modification studies have also suggested roles for essential histidine and arginine residues in catalysis by succinate dehydrogenase. In the present study, a combination of site-directed mutagenesis and chemical modification techniques have been used to investigate the role(s) of the conserved histidine 232, cysteine 247, and arginine 248 residues of the flavorprotein subunit (FrdA) in active site function. A role for His-232 and Arg-248 of FrdA is shown by loss of both fumarate reductase and succino-oxidase activities following site-directed substitution of these particular amino acids. Evidence is also presented that suggests a second arginine residue may form part of the active site. Potential catalytic and substrate-binding roles for arginine are discussed. The effects of removing histidine-232 of FrdA are consistent with its proposed role as a general acid-base catalyst. The fact that succinate oxidation but not fumarate reduction was completely lost, however, might suggest that alternate proton donors substitute for His-232. The data confirm that cysteine 247 of FrdA is responsible for the N-ethylmaleimide sensitivity shown by fumarate reductase but is not required for catalytic activity or the tight-binding of oxalacetate, as previously thought.     

Pseudomonas stutzeri soluble nitrate reductase alphabeta-subunit is a soluble enzyme with a similar electronic structure at the active site as the inner membrane-bound alphabetagamma holoenzyme

A two-subunit (alphabeta) form of dissimilatory nitrate reductase from Pseudomonas stutzeri strain ZoBell was separated from the membrane-residing gamma-subunit by a heat solubilization step. Here we present an optimized purification protocol leading to a soluble alphabeta form with high specific activity (70 U/mg). The soluble form has the stoichiometry alpha(1)beta(1) consisting of the 130 kDa alpha-subunit and the 58 kDa beta-subunit. We did not observe any proteolytic cleavage in the course of the heat solubilization. The enzyme is competively inhibited by azide, but not by chlorate. It exhibits a K(M) value of 3.2 mM for nitrate. We compare the enzymatic and electron paramagnetic resonance (EPR) spectroscopic properties of the alphabeta form with the alphabetagamma holoenzyme which resides in the membrane and can be prepared by detergent extraction. The nearly identical EPR spectra for the Mo(V) signal of both enzyme preparations show that the active site is unaffected by the heat step. The factors influencing the binding of the alpha- and beta-subunit to the gamma-subunit are discussed.     

A soluble NADH-dependent fumarate reductase in the reductive tricarboxylic acid cycle of Hydrogenobacter thermophilus TK-6

Fumarate reductase (FRD) is an enzyme that reduces fumarate to succinate. In many organisms, it is bound to the membrane and uses electron donors such as quinol. In this study, an FRD from a thermophilic chemolithoautotrophic bacterium, Hydrogenobacter thermophilus TK-6, was purified and characterized. FRD activity using NADH as an electron donor was not detected in the membrane fraction but was found in the soluble fraction. The purified enzyme was demonstrated to be a novel type of FRD, consisting of five subunits. One subunit showed high sequence identity to the catalytic subunits of known FRDs. Although the genes of typical FRDs are assembled in a cluster, the five genes encoding the H. thermophilus FRD were distant from each other in the genome. Furthermore, phylogenetic analysis showed that the H. thermophilus FRD was located in a distinct position from those of known soluble FRDs. This is the first report of a soluble NADH-dependent FRD in Bacteria and of the purification of a FRD that operates in the reductive tricarboxylic acid cycle.     

Plants water soluble chlorophyll binding proteins act as enzyme-inhibitor pair

The hydrophilic water-soluble chlorophyll binding proteins (WSCP) which form complex with chlorophyll molecules have been numerously isolated from the chloroplasts of plants. Although, their molecular properties have been partly characterized, but their physio-biochemical roles are still unclear in the photosynthesizing organs. In this study, using bioinformatic tools WSCP pair were predicted to act as hydrolase and hydrolase inhibitor towards chlorophyll molecules. To enhance our information regarding the possible functions of WSCP, we cloned WSCP1 and WSCP2 cDNAs from Chenopodium album L. and Brassica oleracea L. leaves and expressed them as soluble maltose-binding fusion proteins in Escherichia coli. The purified fused products were subjected to chlorophyll hydrolyzing activity in vitro. The results showed that WSCP1 and WCSP2 are antagonistically involved in chlorophyll breakdown, while WSCP1 acts as chlorophyll hydrolyzing enzyme (with the hydrolysis rate of about 40% per 12 h), WSCP2 exerts inhibitory activity (with the inhibition rate of about 38% per 12 h) towards chlorophyll hydrolysis. This is the first ever time report speculates the hydrolase/inhibitory roles for WSCP and proposes that the relative activity of WSCP pair might balance and regulate the chlorophyll breakdown process in the photosynthetic apparatus of plants. It may open the new gate to investigate the potent roles of WSCP in plant system.     

Histidine 61: an important heme ligand in the soluble fumarate reductase from Shewanella frigidimarina

An examination of the X-ray structure of the soluble fumarate reductase from Shewanella frigidimarina [Taylor, P., Pealing, S. L., Reid, G. A., Chapman, S. K., and Walkinshaw, M. D. (1999) Nat. Struct. Biol. 6, 1108-1112] shows the presence of four, bis-His-ligated, c-type hemes and one flavin adenine dinucleotide, FAD. The heme groups provide a "molecular wire" for the delivery of electrons to the FAD. Heme IV is closest to the FAD (7.4 A from heme methyl to FAD C7), and His61, a ligand to heme IV, is also close (8.4 A to FAD C7). Electron delivery to the FAD from the heme groups must proceed via heme IV, as hemes I-III are too far from the FAD for feasible electron transfer. To examine the importance of heme IV and its ligation for enzyme function, we have substituted His61 with both methionine and alanine. Here we describe the crystallographic, kinetic, and electrochemical characterization of the H61M and H61A mutant forms of the Shewanella fumarate reductase. The crystal structures of these mutant forms of the enzyme have been determined to 2.1 and 2.2 A resolution, respectively. Substitution of His61 with alanine results in heme IV having only one protein ligand (His86), the sixth coordination position being occupied by an acetate ion derived from the crystal cryoprotectant solution. In the structure of the H61M enzyme, Met61 is found not to ligate the heme iron, a role that is taken by a water molecule. Apart from these features, there are no significant structural alterations as a result of either substitution. Both the H61M-Fcc(3) and H61A-Fcc(3) mutant enzymes are catalytically active but exhibit marked decreases in the value of k(cat) for fumarate reduction with respect to that of the wild type (5- and 10-fold lower, respectively). There is also a significant shift in the pK(a) values for the mutant enzymes, from 7.5 for the wild type to 8.26 for H61M and 9.29 for H61A. The fumarate reductase activity of both mutant enzymes can be recovered to approximately 80% of that seen for the wild type by the addition of exogenous imidazole. In the case of H61A, recovery of activity is also accompanied by a shift of the pK(a) from 9.29 to 7.46 (close, and within experimental error, to that for the wild type). Pre-steady-state kinetic measurements show clearly that rate constants for the fumarate dependent reoxidation of the heme groups are adversely affected by the mutations. The solvent isotope effect for fumarate reduction in the wild-type enzyme has a value of 8.0, indicating that proton delivery is substantially rate limiting. This value falls to 5.6 and 2.2 for the H61M and H61A mutants, respectively, indicating that electron transfer, rather than proton transfer, is becoming more rate-limiting in the mutant enzymes.     

Switching an active site helix in dihydrofolate reductase reveals limits to subdomain modularity

To what degree are individual structural elements within proteins modular such that similar structures from unrelated proteins can be interchanged? We study subdomain modularity by creating 20 chimeras of an enzyme, Escherichia coli dihydrofolate reductase (DHFR), in which a catalytically important, 10-residue α-helical sequence is replaced by α-helical sequences from a diverse set of proteins. The chimeras stably fold but have a range of diminished thermal stabilities and catalytic activities. Evolutionary coupling analysis indicates that the residues of this α-helix are under selection pressure to maintain catalytic activity in DHFR. Reversion to phenylalanine at key position 31 was found to partially restore catalytic activity, which could be explained by evolutionary coupling values. We performed molecular dynamics simulations using replica exchange with solute tempering. Chimeras with low catalytic activity exhibit nonhelical conformations that block the binding site and disrupt the positioning of the catalytically essential residue D27. Simulation observables and in vitro measurements of thermal stability and substrate-binding affinity are strongly correlated. Several E. coli strains with chromosomally integrated chimeric DHFRs can grow, with growth rates that follow predictions from a kinetic flux model that depends on the intracellular abundance and catalytic activity of DHFR. Our findings show that although α-helices are not universally substitutable, the molecular and fitness effects of modular segments can be predicted by the biophysical compatibility of the replacement segment.     

Role of His505 in the soluble fumarate reductase from Shewanella frigidimarina

The X-ray structure of the soluble fumarate reductase from Shewanella frigidimarina [Taylor, P., Pealing, S. L., Reid, G. A., Chapman, S. K., and Walkinshaw, M. D. (1999) Nat. Struct. Biol. 6, 1108-1112] clearly shows the presence of an internally bound sodium ion. This sodium ion is coordinated by one solvent water molecule (Wat912) and five backbone carbonyl oxygens from Thr506, Met507, Gly508, Glu534, and Thr536 in what is best described as octahedral geometry (despite the rather long distance from the sodium ion to the backbone oxygen of Met507 (3.1 A)). The water ligand (Wat912) is, in turn, hydrogen bonded to the imidazole ring of His505. This histidine residue is adjacent to His504, a key active-site residue thought to be responsible for the observed pK(a) of the enzyme. Thus, it is possible that His505 may be important in both maintaining the sodium site and in influencing the active site. Here we describe the crystallographic and kinetic characterization of the H505A and H505Y mutant forms of the Shewanella fumarate reductase. The crystal structures of both mutant forms of the enzyme have been solved to 1.8 and 2.0 A resolution, respectively. Both show the presence of the sodium ion in the equivalent position to that found in the wild-type enzyme. The structure of the H505A mutant shows the presence of two water molecules in place of the His505 side-chain which form part of a hydrogen-bonding network with Wat48, a ligand to the sodium ion. The structure of the H505Y mutant shows the hydroxyl group of the tyrosine side-chain hydrogen-bonding to a water molecule which is also a ligand to the sodium ion. Apart from these features, there are no significant structural alterations as a result of either substitution. Both the mutant enzymes are catalytically active but show markedly different pH profiles compared to the wild-type enzyme. At high pH (above 8.5), the wild type and mutant enzymes have very similar activities. However, at low pH (6.0), the H505A mutant enzyme is some 20-fold less active than wild-type. The combined crystallographic and kinetic results suggest that His505 is not essential for sodium binding but does affect catalytic activity perhaps by influencing the pK(a) of the adjacent His504.     

A proton delivery pathway in the soluble fumarate reductase from Shewanella frigidimarina

The mechanism for fumarate reduction by the soluble fumarate reductase from Shewanella frigidimarina involves hydride transfer from FAD and proton transfer from the active-site acid, Arg-402. It has been proposed that Arg-402 forms part of a proton transfer pathway that also involves Glu-378 and Arg-381 but, unusually, does not involve any bound water molecules. To gain further insight into the importance of this proton pathway we have perturbed it by substituting Arg-381 by lysine and methionine and Glu-378 by aspartate. Although all the mutant enzymes retain measurable activities, there are orders-of-magnitude decreases in their k(cat) values compared with the wild-type enzyme. Solvent kinetic isotope effects show that proton transfer is rate-limiting in the wild-type and mutant enzymes. Proton inventories indicate that the proton pathway involves multiple exchangeable groups. Fast scan protein-film voltammetric studies on wild-type and R381K enzymes show that the proton transfer pathway delivers one proton per catalytic cycle and is not required for transporting the other proton, which transfers as a hydride from the reduced, protonated FAD. The crystal structures of E378D and R381M mutant enzymes have been determined to 1.7 and 2.1 A resolution, respectively. They allow an examination of the structural changes that disturb proton transport. Taken together, the results indicate that Arg-381, Glu-378, and Arg-402 form a proton pathway that is completely conserved throughout the fumarate reductase/succinate dehydrogenase family of enzymes.     

Chemical modification of an essential lysine at the active site of enoyl-CoA reductase in fatty acid synthetase

Fatty acid synthetase from goose uropygial gland was inactivated by treatment with pyridoxal 5′-phosphate. Malonyl-CoA and acetyl-CoA did not protect the enzyme whereas NADPH provided about 70% protection against this inactivation. 2′-Monophospho-ADP-ribose was nearly as effective as NADPH while 2′-AMP, 5′-AMP, ADP-ribose, and NADH were ineffective suggesting that pyridoxal 5′-phosphate modified a group that interacts with the 5′-pyrophosphoryl group of NADPH and that the 2′-phosphate is necessary for the binding of the coenzyme to the enzyme. Of the seven component activities catalyzed by fatty acid synthetase only the enoyl-CoA reductase activity was inhibited. Inactivation of both the overall activity and enoyl-CoA reductase of fatty acid synthetase by this compound was reversed by dialysis or dilution but not after reduction with NaBH₄. The modified protein showed a characteristic Schiff base absorption (maximum at 425 nm) that disappeared on reduction with NaBH₄ resulting in a new absorption spectrum with a maximum at 325 nm. After reduction the protein showed a fluorescence spectrum with a maximum at 394 nm. Reduction of pyridoxal phosphate-treated protein with NaB³H₄ resulted in incorporation of ³H into the protein and paper chromatography of the acid hydrolysate of the modified protein showed only one fluorescent spot which was labeled and ninhydrin positive and had an R_f identical to that of authentic N⁶-pyridoxyllysine. When [4-³H]pyridoxal phosphate was used all of the ³H, incorporated into the protein, was found in pyridoxyllysine. All of these results strongly suggest that pyridoxal phosphate inhibited fatty acid synthetase by forming a Schiff base with the ?-amino group of lysine in the enoyl-CoA reductase domain of the enzyme. The number of lysine residues modified was estimated with [4-³H]pyridoxal-5′-phosphate/NaBH₄ and by pyridoxal-5′-phosphate/NaB³H₄. Scatchard analysis showed that modification of two lysine residues per subunit resulted in complete inactivation of the overall activity and enoyl-CoA reductase of fatty acid synthetase. NADPH prevented the inactivation of the enzyme by protecting one of these two lysine residues from modification. The present results are consistent with the hypothesis that each subunit of the enzyme contains an enoyl-CoA reductase domain in which a lysine residue, at or near the active site, interacts with NADPH.     

Failure of apoptosis-inducing factor to act as neuroglobin reductase

Neuroglobin (Ngb) is a hexacoordinate globin expressed in the nervous system of vertebrates, where it protects neurons against hypoxia. Ferrous Ngb has been proposed to favor cell survival by scavenging NO and/or reducing cytochrome c released into the cytosol during hypoxic stress. Both catalytic functions require an as yet unidentified Ngb-reductase activity. Such an activity was detected both in tissue homogenates of human brain and liver and in Escherichia coli extracts. Since NADH:flavorubredoxin oxidoreductase from E. coli, that was shown to reduce ferric Ngb, shares sequence similarity with the human apoptosis-inducing factor (AIF), AIF has been proposed by us as a candidate Ngb reductase. In this study, we tested this hypothesis and show that the Ngb-reductase activity of recombinant human AIF is negligible and hence incompatible with such a physiological function.     

Engineering of the iron site in ribonucleotide reductase to a self-hydroxylating monooxygenase.

Protein R2 of ribonucleotide reductase contains a dinuclear ferric iron center adjacent to a tyrosyl radical in the interior of the protein matrix. A patch of hydrophobic residues surrounds the iron-radical cofactor. Its importance during the oxidative generation of the iron-radical cofactor was investigated by site-directed mutagenesis of Phe-208 to tyrosine. The mutant protein R2 F208Y has prominent absorption bands at 460 and 720 nm reminiscent of those in ferric-catecholate complexes. Resonance Raman spectroscopy shows that the iron center of R2 F208Y contains a bidentate catechol ligand. The mechanism for generation of this protein-derived dihydroxyphenylalanine may be similar to the catalytic cycle of methane monooxygenase.     

Mitochondrial fumarate reductase as a target of chemotherapy: from parasites to cancer cells

Recent research on respiratory chain of the parasitic helminth, Ascaris suum has shown that the mitochondrial NADH-fumarate reductase system (fumarate respiration), which is composed of complex I (NADH-rhodoquinone reductase), rhodoquinone and complex II (rhodoquinol-fumarate reductase) plays an important role in the anaerobic energy metabolism of adult parasites inhabiting hosts. The enzymes in these parasite-specific pathways are potential target for chemotherapy. We isolated a novel compound, nafuredin, from Aspergillus niger, which inhibits NADH-fumarate reductase in helminth mitochondria at nM order. It competes for the quinone-binding site in complex I and shows high selective toxicity to the helminth enzyme. Moreover, nafuredin exerts anthelmintic activity against Haemonchus contortus in in vivo trials with sheep indicating that mitochondrial complex I is a promising target for chemotherapy. In addition to complex I, complex II is a good target because its catalytic direction is reverse of succinate-ubiquionone reductase in the host complex II. Furthermore, we found atpenin and flutolanil strongly and specifically inhibit mitochondrial complex II. Interestingly, fumarate respiration was found not only in the parasites but also in some types of human cancer cells. Analysis of the mitochondria from the cancer cells identified an anthelminthic as a specific inhibitor of the fumarate respiration. Role of isoforms of human complex II in the hypoxic condition of cancer cells and fetal tissues is a challenge. This article is part of a Special Issue entitled Biochemistry of Mitochondria, Life and Intervention 2010.     

Physiological role of soluble fumarate reductase in redox balancing during anaerobiosis in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, there are two isoenzymes of fumarate reductase (FRDS1 and FRDS2), encoded by the FRDS and OSM1 genes, respectively. Simultaneous disruption of these two genes results in a growth defect of the yeast under anaerobic conditions, while disruption of the OSM1 gene causes slow growth. However, the metabolic role of these isoenzymes has been unclear until now. In the present study, we found that the anaerobic growth of the strain disrupted for both the FRDS and OSM1 genes was fully restored by adding the oxidized form of methylene blue or phenazine methosulfate, which non-enzymatically oxidize cellular NADH to NAD(+). When methylene blue was added at growth-limiting concentrations, growth was completely arrested after exhaustion of oxidized methylene blue. In the double-disrupted strain, the accumulation of succinate in the supernatant was markedly decreased during anaerobic growth in the presence of methylene blue. These results suggest that fumarate reductase isoenzymes are required for the reoxidation of intracellular NADH under anaerobic conditions, but not aerobic conditions.     

Structure of a glutathione conjugate bound to the active site of aldose reductase

Aldose reductase (AR) is a monomeric NADPH-dependent oxidoreductase that catalyzes the reduction of aldehydes, ketones, and aldo-sugars. AR has been linked to the development of hyperglycemic injury and is a clinical target for the treatment of secondary diabetic complications. In addition to reducing glucose, AR is key regulator of cell signaling through it's reduction of aldehydes derived from lipoproteins and membrane phospholipids. AR catalyzes the reduction of glutathione conjugates of unsaturated aldehydes with higher catalytic efficiency than free aldehydes. The X-ray structure of human AR holoenzyme in complex with the glutathione analogue S-(1,2-dicarboxyethyl) glutathione (DCEG) was determined at a resolution of 1.94 A. The distal carboxylate group of DCEG's dicarboxyethyl moiety interacted with the conserved AR anion binding site residues Tyr48, His110, and Trp111. The bound DCEG's glutathione backbone adopted the low-energy Y-shape form. The C-terminal carboxylate of DCEG glutathione's glycine formed hydrogen bonds to Leu301 and Ser302, while the remaining interactions between DCEG and AR were hydrophobic, permitting significant flexibility of the AR and glutathione (GS) analogue interaction. The observed conformation and interactions of DCEG with AR were consistent with our previously published molecular dynamics model of glutathionyl-propanal binding to AR. The current structure identifies major interactions of glutathione conjugates with the AR active-site residues.     

Intermolecular electron transfer in two-iron superoxide reductase: a putative role for the desulforedoxin center as an electron donor to the iron active site

Superoxide reductase (SOR) is a superoxide detoxification system present in some microorganisms. Its active site consists of an unusual mononuclear iron center with an FeN4S1 coordination which catalyzes the one-electron reduction of superoxide to form hydrogen peroxide. Different classes of SORs have been described depending on the presence of an additional rubredoxin-like, desulforedoxin iron center, whose function has remained unknown until now. In this work, we investigated the mechanism of the reduction of the SOR iron active site using the NADPH:flavodoxin oxidoreductase from Escherichia coli, which was previously shown to efficiently transfer electrons to the Desulfoarculus baarsii SOR. When present, the additional rubredoxin-like iron center could function as an electronic relay between cellular reductases and the iron active site for superoxide reduction. This electron transfer was mainly intermolecular, between the rubredoxin-like iron center of one SOR and the iron active site of another SOR. These data provide the first experimental evidence for a possible role of the rubredoxin-like iron center in the superoxide detoxifying activity of SOR.     

Structural assembly of the active site in an aldo-keto reductase by NADPH cofactor

A 1.9 A resolution X-ray structure of the apo-form of Corynebacterium 2,5-diketo-d-gluconic acid reductase A (2,5-DKGR A), a member of the aldo-keto reductase superfamily, has been determined by molecular replacement using the NADPH-bound form of the same enzyme as the search model. 2,5-DKGR A catalyzes the NADPH-dependent stereo-specific reduction of 2,5-diketo-d-gluconate (2,5-DKG) to 2-keto-l-gulonate, a precursor in the industrial production of vitamin C. An atomic-resolution structure for the apo-form of the enzyme, in conjunction with our previously reported high-resolution X-ray structure for the holo-enzyme and holo/substrate model, allows a comparative analysis of structural changes that accompany cofactor binding. The results show that regions of the active site undergo coordinated conformational changes of up to 8 A. These conformational changes result in the organization and structural rearrangement of residues associated with substrate binding and catalysis. Thus, NADPH functions not only to provide a hydride ion for catalytic reduction, but is also a critical structural component for formation of a catalytically competent form of DKGR A.