Oxygen activation by a mixed-valent, diiron(II/III) cluster in the glycol cleavage reaction catalyzed by myo-inositol oxygenase

myo-Inositol oxygenase (MIOX) catalyzes the ring-cleaving, four-electron oxidation of its cyclohexan-(1,2,3,4,5,6-hexa)-ol substrate (myo-inositol, MI) to d-glucuronate (DG). The preceding paper [Xing, G., Hoffart, L. M., Diao, Y., Prabhu, K. S., Arner, R. J., Reddy, C. C., Krebs, C., and Bollinger, J. M., Jr. (2006) Biochemistry 45, 5393-5401] demonstrates by M?ssbauer and electron paramagnetic resonance (EPR) spectroscopies that MIOX can contain a non-heme dinuclear iron cluster, which, in its mixed-valent (II/III) and fully oxidized (III/III) states, is perturbed by binding of MI in a manner consistent with direct coordination. In the study presented here, the redox form of the enzyme that activates O(2) has been identified. l-Cysteine, which was previously reported to accelerate turnover, reduces the fully oxidized enzyme to the mixed-valent form, and O(2), the cosubstrate, oxidizes the fully reduced form to the mixed-valent form with a stoichiometry of one per O(2). Both observations implicate the mixed-valent, diiron(II/III) form of the enzyme as the active state. Stopped-flow absorption and freeze-quench EPR data from the reaction of the substrate complex of mixed-valent MIOX [MIOX(II/III).MI] with limiting O(2) in the presence of excess, saturating MI reveal the following cycle: (1) MIOX(II/III).MI reacts rapidly with O(2) to generate an intermediate (H) with a rhombic, g < 2 EPR spectrum; (2) a form of the enzyme with the same absorption features as MIOX(II/III) develops as H decays, suggesting that turnover has occurred; and (3) the starting MIOX(II/III).MI complex is then quantitatively regenerated. This cycle is fast enough to account for the catalytic rate. The DG/O(2) stoichiometry in the reaction, 0.8 +/- 0.1, is similar to the theoretical value of 1, whereas significantly less product is formed in the corresponding reaction of the fully reduced enzyme with limiting O(2). The DG/O(2) yield in the latter reaction decreases as the enzyme concentration is increased, consistent with the hypothesis that initial conversion of the reduced enzyme to the MIOX(II/III).MI complex and subsequent turnover by the mixed-valent form is responsible for the product in this case. The use of the mixed-valent, diiron(II/III) cluster by MIOX represents a significant departure from the mechanisms of other known diiron oxygenases, which all involve activation of O(2) from the II/II manifold.     

Arabidopsis glyoxalase II contains a zinc/iron binuclear metal center that is essential for substrate binding and catalysis

Glyoxalase II participates in the cellular detoxification of cytotoxic and mutagenic 2-oxoaldehydes. Because of its role in chemical detoxification, glyoxalase II has been studied as a potential anti-cancer and/or anti-protozoal target; however, very little is known about the active site and reaction mechanism of this important enzyme. To characterize the active site and kinetic mechanism of the enzyme, a detailed mutational study of Arabidopsis glyoxalase II was conducted. Data presented here demonstrate for the first time that the cytoplasmic form of Arabidopsis glyoxalase II contains an iron-zinc binuclear metal center that is essential for activity. Both metals participate in substrate binding, transition state stabilization, and the hydrolysis reaction. Subtle alterations in the geometry and/or electrostatics of the binuclear center have profound effects on the activity of the enzyme. Additional residues important in substrate binding have also been identified. An overall reaction mechanism for glyoxalase II is proposed based on the mutational and kinetic data from this study and crystallographic data on human glyoxalase II. Information presented here provides new insights into the active site and reaction mechanism of glyoxalase II that can be used for the rational design of glyoxalase II inhibitors.     

L-myo-inosose-1 as a probable intermediate in the reaction catalyzed by myo-inositol oxygenase

In previous investigations, it was necessary to have Fe(II) and cysteine present in order to assay the catalytic activity of purified hog kidney myo-inositol oxygenase. In the present study it was found that, if this purified nonheme iron enzyme is slowly frozen in solution with glutathione and stored at -20 degrees C, it is fully active in the absence of activators if catalase is present to remove adventitious H2O2. With this simpler assay system it was possible to clarify the effects of several variables on the enzymic reaction. Thus, the maximum velocity is pH-dependent with a maximum around pH 9.5, but the apparent Km for myo-inositol (air atmosphere) remains constant at 5.0 mM throughout a broad pH range. The enzyme is quite specific for its substrate myo-inositol, is very sensitive to oxidants and reductants, but is not affected by a variety of complexing agents, nucleotides, sulfhydryl reagents, etc. In other experiments it was found that L-myo-inosose-1, a potential intermediate in the enzymic reaction, is a potent competitive inhibitor (Ki = 62 microM), while other inososes and a solution thought to contain D-glucodialdehyde, another potential intermediate, are weak inhibitors. Also, both a kinetic deuterium isotope effect (kH/kD = 2.1) and a tritium isotope effect (kH/kT = 7.5) are observed for the enzymic reaction when [1-2H]- and [1-3H]-myo-inositol are used as reactants. These latter results are considered strong evidence that the oxygenase reaction proceeds by a pathway involving L-myo-inosose-1 as an intermediate rather than by an alternative pathway that would have D-glucodialdehyde as the intermediate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Propagation of Saccharomyces cerevisiae [PSI+] prion is impaired by factors that regulate Hsp70 substrate binding

The Saccharomyces cerevisiae [PSI(+)] prion is believed to be a self-propagating cytoplasmic amyloid. Earlier characterization of HSP70 (SSA1) mutations suggested that [PSI(+)] propagation is impaired by alterations that enhance Ssa1p's substrate binding. This impairment is overcome by second-site mutations in Ssa1p's conserved C-terminal motif (GPTVEEVD), which mediates interactions with tetratricopeptide repeat (TPR) cochaperones. Sti1p, a TPR cochaperone homolog of mammalian Hop1 (Hsp70/90 organizing protein), activates Ssa1p ATPase, which promotes substrate binding by Ssa1p. Here we find that in SSA1-21 cells depletion of Sti1p improved [PSI(+)] propagation, while excess Sti1p weakened it. In contrast, depletion of Fes1p, a nucleotide exchange factor for Ssa1p that facilitates substrate release, weakened [PSI(+)] propagation, while overproducing Fes1p improved it. Therefore, alterations of Hsp70 cochaperones that promote or prolong Hsp70 substrate binding impair [PSI(+)] propagation. We also find that the GPTVEEVD motif is important for physical interaction with Hsp40 (Ydj1p), another Hsp70 cochaperone that promotes substrate binding but is dispensable for viability. We further find that depleting Cpr7p, an Hsp90 TPR cochaperone and CyP-40 cyclophilin homolog, improved [PSI(+)] propagation in SSA1 mutants. Although Cpr7p and Sti1p are Hsp90 cochaperones, we provide evidence that Hsp90 is not involved in [PSI(+)] propagation, suggesting that Sti1p and Cpr7p functionally interact with Hsp70 independently of Hsp90.     

A transferrin-like protein that does not bind iron is induced by iron deficiency in the alga Dunaliella salina

Iron deficiency induces two major transferrin-like proteins in the plasma membrane (Pm) of the halotolerant alga Dunaliella salina. TTf, a 150-kDa protein, previously identified as a salt-induced triplicated transferrin, having iron-binding characteristics resembling animal transferrins, and a 100-kDa protein designated idi-100 (for iron-deficiency-induced 100 kDa protein). According to the predicted amino acid sequence of idi-100, it is only 30% identical to TTf and differs from it in having two, rather than three, homologous internal repeats and in a lower conservation of canonical iron/bicarbonate binding residues. Both are localized in the outer surface of the membrane; however, TTf can be dissociated from the membrane by treatment with EDTA, whereas release of idi-100 requires detergents. The accumulation of idi-100 under iron deficiency lags behind that of TTf and in contrast to TTf, it is not induced by high salinity, suggesting that induction of idi-100 requires lower Fe threshold levels than that of TTf. In contrast to TTf, idi-100 does not bind Fe; however, there are indications for interactions with bicarbonate ions. These results suggest that despite their common resemblance to transferrins, their similar subcellular localization and their induction by iron deficiency, idi-100 and TTf fulfill different functions.     

Serine-376 contributes to the binding of substrate by ribulose-bisphosphate carboxylase/oxygenase from Anacystis nidulans.

Site-directed mutagenesis was used to change Ser376 in the active site of ribulose-1,5-bisphosphate carboxylase/oxygenase from the cyanobacterium Anacystis nidulans to Cys, Thr, or Ala. When expressed in Escherichia coli and purified, the mutant enzymes exhibited carboxylase activities that were reduced by 99% or more with respect to the activity of the wild-type enzyme. The Km values for ribulose bisphosphate at pH 8.0, 30 degrees C, were elevated from 46 microM for wild-type enzyme to 287, 978, and 81 microM for mutants in which Cys, Thr, or Ala, respectively, replaced Ser376. The Cys and Thr variants were almost devoid of oxygenase activity whereas the Ala variant had 16% as much oxygenase as wild-type enzyme, suggesting that this mutation had greatly elevated the oxygenase:carboxylase ratio.     

A fluorimetric study of substrate and effector binding of D-ribulose-1,5-biphosphate carboxylase/oxygenase from spinach

2-p-toluidino-naphthalene-6-sulfonate (TNS) is a sensitive fluorescent reporter group for the detection of the events at the reaction centres of the ribulose biphosphate carboxylase/oxygenase from spinach. The formation of binary complexes of the carboxylase with substrates and effectors is associated with significant changes (ΔF) of the fluorescence emission of the enzyme-TNS-complex. This indicates substrate and effector induced conformational changes of the enzyme. From the concentration dependence of ΔF the following dissociation constants for ribulose biphosphate (RuBP) and Mg²⁺ were determined: K(RuBP) = 0,5 μM and K(Mg²⁺) = 1 mM. Sugar phosphates, e.g. 6-phosphogluconate, which show regulatory effects in the carboxylation and oxygenation of RuBP, function antagonistically to RuBP, presumably by competition with RuBP for its allosteric binding site.     

Precursor maltose-binding protein is active in binding substrate.

Precursor maltose-binding protein synthesized in vitro was shown to be active in binding maltose by affinity chromatography.     

Steady-state kinetics of substrate binding and iron release in tomato ACC oxidase

1-Aminocyclopropane-1-carboxylate oxidase (ACC oxidase) catalyzes the last step in the biosynthetic pathway of the plant hormone, ethylene. This unusual reaction results in the oxidative ring cleavage of 1-aminocyclopropane carboxylate (ACC) into ethylene, cyanide, and CO2 and requires ferrous ion, ascorbate, and molecular oxygen for catalysis. A new purification procedure and assay method have been developed for tomato ACC oxidase that result in greatly increased enzymatic activity. This method allowed us to determine the rate of iron release from the enzyme and the effect of the activator, CO2, on this rate. Initial velocity studies support an ordered kinetic mechanism where ACC binds first followed by O2; ascorbate can bind after O2 or possibly before ACC. This kinetic mechanism differs from one recently proposed for the ACC oxidase from avocado.     

Thioredoxin reductase system mediates iron binding in IscA and iron delivery for the iron-sulfur cluster assembly in IscU

IscA is a key member of the iron-sulfur cluster assembly machinery found in bacteria and eukaryotes. Previously, IscA was characterized as an alternative iron-sulfur cluster assembly scaffold, as purified IscA can host transient iron-sulfur clusters. However, recent studies indicated that IscA is an iron-binding protein that can provide iron for the iron-sulfur cluster assembly in a proposed scaffold IscU (Ding H., Clark, R. J., and Ding, B. (2004) J. Biol. Chem. 279, 37499-37504). To further elucidate the roles of IscA in the biogenesis of iron-sulfur clusters, we reevaluate the iron binding activity of IscA under physiologically relevant conditions. The results indicate that in the presence of the thioredoxin reductase system, Escherichia coli IscA binds iron with an iron association constant of 2.0 x 10(19) M(-1) in vitro. Whereas all three components (thioredoxin 1, thioredoxin reductase and NADPH) in the thioredoxin reductase system are essential for mediating the iron binding in IscA, only catalytic amounts of thioredoxin 1 and thioredoxin reductase are required. In contrast, IscU fails to bind iron in the presence of the thioredoxin reductase system, suggesting that the iron binding in IscA is specific. Nevertheless, the thioredoxin reductase system can promote the iron-sulfur cluster assembly in IscU in the presence of the iron-loaded IscA, cysteine desulfurase (IscS), and L-cysteine, demonstrating a physiologically relevant system for the biogenesis of iron-sulfur clusters. The results provide additional evidence for the hypothesis that IscA is capable of recruiting intracellular "free" iron and delivering the iron for the iron-sulfur cluster assembly in IscU.     

Polar distribution dynamics of Con A binding sites in embryo sacs is temporally coupled by the fertilization process

The binding site distribution of concanavalin agglutinin (Con A) and wheat germ agglutinin (WGA) on embryo sacs at various developmental stages of Torenia fournieri L was studied by using a cooled Charge Coupled Device (CCD) and fluorescent Con A and WGA probes. The distribution patterns of Con A and WGA binding sites on embryo sacs changed during the fertilization process. The fluorescent signal indicating Con A binding sites was distributed evenly on the surface of the embryo sac wall before anthesis, was much denser on the micropylar end of the embryo sac wall and looked like a corona on the day of anthesis. After pollination, stronger fluorescence was present on the micropylar end of the embryo sac wall and the filiform apparatus (FA), showing an obvious polar distribution. When the pollen tube entered the embryo sac and reached a synergid, the fluorescence was still concentrated on the micropylar end and FA, and started to appear on the synergid. After fertilization, the polar distribution of the fluorescence gradually disappeared and an even distribution pattern was observed again on the embryo sac wall. These results revealed that the dynamic distribution of Con A binding sites was temporally coupled with the process of fertilization. WGA binding site distribution on the embryo sac was also investigated and showed a simple pattern but also regularly changed during the process of fertilization. The variation of these lectin binding sites during the fertilization process suggests that lectin binding site interactions may play a role in the process.     

Heme oxygenase activity causes transient hypersensitivity to oxidative ultraviolet A radiation that depends on release of iron from heme

Heme oxygenase (HO) breaks down heme to iron, biliverdin, and carbon monoxide, and activity of this enzyme increases in many tissues and cell types after exposure to oxidative stress. There is evidence that increased HO activity is involved in long-term protective mechanisms against oxidative stress. We studied the effect of artificially overexpressed HO activity on the cytotoxicity of oxidative ultraviolet A (UVA) radiation after loading human cells with the HO substrate ferric heme (hemin). In contrast to the reported long-term protection attributed to HO activity, cells overexpressing HO activity were hypersensitive to UVA radiation shortly after heme treatment when compared with control cells. Cells overexpressing HO activity showed an increased rate of heme consumption and a higher level of accumulated free chelatable iron when compared with control cells. The hypersensitivity of cells overexpressing HO to UVA radiation after heme treatment was apparently caused by the increased accumulation of chelatable iron, because the iron chelator desferrioxamine strongly reduced the hypersensitivity. One day after the heme treatment, cells overexpressing HO activity were no longer hypersensitive to UVA radiation. We conclude that increased HO activity can temporarily increase the sensitivity of cells to oxidative stress by releasing iron from heme.     

A Novel indole compound that inhibits Pseudomonas aeruginosa growth by targeting MreB is a substrate for MexAB-OprM

Drug efflux systems contribute to the intrinsic resistance of Pseudomonas aeruginosa to many antibiotics and biocides and hamper research focused on the discovery and development of new antimicrobial agents targeted against this important opportunistic pathogen. Using a P. aeruginosa PAO1 derivative bearing deletions of opmH, encoding an outer membrane channel for efflux substrates, and four efflux pumps belonging to the resistance nodulation/cell division class including mexAB-oprM, we identified a small-molecule indole-class compound (CBR-4830) that is inhibitory to growth of this efflux-compromised strain. Genetic studies established MexAB-OprM as the principal pump for CBR-4830 and revealed MreB, a prokaryotic actin homolog, as the proximal cellular target of CBR-4830. Additional studies establish MreB as an essential protein in P. aeruginosa, and efflux-compromised strains treated with CBR-4830 transition to coccoid shape, consistent with MreB inhibition or depletion. Resistance genetics further suggest that CBR-4830 interacts with the putative ATP-binding pocket in MreB and demonstrate significant cross-resistance with A22, a structurally unrelated compound that has been shown to promote rapid dispersion of MreB filaments in vivo. Interestingly, however, ATP-dependent polymerization of purified recombinant P. aeruginosa MreB is blocked in vitro in a dose-dependent manner by CBR-4830 but not by A22. Neither compound exhibits significant inhibitory activity against mutant forms of MreB protein that bear mutations identified in CBR-4830-resistant strains. Finally, employing the strains and reagents prepared and characterized during the course of these studies, we have begun to investigate the ability of analogues of CBR-4830 to inhibit the growth of both efflux-proficient and efflux-compromised P. aeruginosa through specific inhibition of MreB function.     

To be, or not to be two sites: that is the question about LeuT substrate binding

Transport proteins of the neurotransmitter sodium symporter (NSS) family regulate the extracellular concentration of several neurotransmitters in the central nervous system. The only member of this family for which atomic-resolution structural data are available is the prokaryotic homologue LeuT. This protein has been used as a model system to study the molecular mechanism of transport of the NSS family. In this Journal Club, we discuss two strikingly different LeuT transport mechanisms: one involving a single high-affinity substrate binding site and one recently proposed alternative involving two high-affinity substrate binding sites that are allosterically coupled.     

Rpe65 is a retinyl ester binding protein that presents insoluble substrate to the isomerase in retinal pigment epithelial cells

Photon capture by a rhodopsin pigment molecule induces 11-cis to all-trans isomerization of its retinaldehyde chromophore. To restore light sensitivity, the all-trans-retinaldehyde must be chemically re-isomerized by an enzyme pathway called the visual cycle. Rpe65, an abundant protein in retinal pigment epithelial (RPE) cells and a homolog of beta-carotene dioxygenase, appears to play a role in this pathway. Rpe65-/- knockout mice massively accumulate all-trans-retinyl esters but lack 11-cis-retinoids and rhodopsin visual pigment in their retinas. Mutations in the human RPE65 gene cause a severe recessive blinding disease called Leber's congenital amaurosis. The function of Rpe65, however, is unknown. Here we show that Rpe65 specifically binds all-trans-retinyl palmitate but not 11-cis-retinyl palmitate by a spectral-shift assay, by co-elution during gel filtration, and by co-immunoprecipitation. Using a novel fluorescent resonance energy transfer (FRET) binding assay in liposomes, we demonstrate that Rpe65 extracts all-trans-retinyl esters from phospholipid membranes. Assays of isomerase activity reveal that Rpe65 strongly stimulates the enzymatic conversion of all-trans-retinyl palmitate to 11-cis-retinol in microsomes from bovine RPE cells. Moreover, we show that addition of Rpe65 to membranes from rpe65-/- mice, which possess no detectable isomerase activity, restores isomerase activity to wild-type levels. Rpe65 by itself, however, has no intrinsic isomerase activity. These observations suggest that Rpe65 presents retinyl esters as substrate to the isomerase for synthesis of visual chromophore. This proposed function explains the phenotype in mice and humans lacking Rpe65.     

Evidence that bicarbonate is not the substrate in photosynthetic oxygen evolution

It is widely accepted that the oxygen produced by photosystem II of cyanobacteria, algae, and plants is derived from water. Earlier proposals that bicarbonate may serve as substrate or catalytic intermediate are almost forgotten, though not rigorously disproved. These latter proposals imply that CO2 is an intermediate product of oxygen production in addition to O2. In this work, we investigated this possible role of exchangeable HCO3- in oxygen evolution in two independent ways. (1) We studied a possible product inhibition of the electron transfer into the catalytic Mn4Ca complex during the oxygen-evolving reaction by greatly increasing the pressure of CO2. This was monitored by absorption transients in the near UV. We found that a 3,000-fold increase of the CO2 pressure over ambient conditions did not affect the UV transient, whereas the S(3) --> S(4) --> S(0) transition was half-inhibited by raising the O2 pressure only 10-fold over ambient, as previously established. (2) The flash-induced O2 and CO2 production by photosystem II was followed simultaneously with membrane inlet mass spectrometry under approximately 15% H2(18)O enrichment. Light flashes that revealed the known oscillatory O2 release failed to produce any oscillatory CO2 signal. Both types of results exclude that exchangeable bicarbonate is the substrate for (and CO2 an intermediate product of) oxygen evolution by photosynthesis. The possibility that a tightly bound carbonate or bicarbonate is a cofactor of photosynthetic water oxidation has remained.     

COMMD1 expression is controlled by critical residues that determine XIAP binding

COMMD {COMM [copper metabolism Murr1 (mouse U2af1-rs1 region 1)] domain-containing} proteins participate in several cellular processes, ranging from NF-kappaB (nuclear factor kappaB) regulation, copper homoeostasis, sodium transport and adaptation to hypoxia. The best-studied member of this family is COMMD1, but relatively little is known about its regulation, except that XIAP [X-linked IAP (inhibitor of apoptosis)] functions as its ubiquitin ligase. In the present study, we identified that the COMM domain of COMMD1 is required for its interaction with XIAP, and other COMMD proteins can similarly interact with IAPs. Two conserved leucine repeats within the COMM domain were found to be critically required for XIAP binding. A COMMD1 mutant which was unable to bind to XIAP demonstrated a complete loss of basal ubiquitination and great stabilization of the protein. Underscoring the importance of IAP-mediated ubiquitination, we found that long-term expression of wild-type COMMD1 results in nearly physiological protein levels as a result of increased ubiquitination, but this regulatory event is circumvented when a mutant form that cannot bind XIAP is expressed. In summary, our findings indicate that COMMD1 expression is controlled primarily by protein ubiquitination, and its interaction with IAP proteins plays an essential role.