Bradyrhizobium japonicum porphobilinogen synthase uses two Mg(II) and monovalent cations

Bradyrhizobium japonicum porphobilinogen synthase (B. japonicum PBGS) has been purified and characterized from an overexpression system in an Escherichia coli host (Chauhan, S., and O'Brian, M. R. (1995) J. Biol. Chem. 270, 19823-19827). B. japonicum PBGS defines a new class of PBGS protein, type IV (classified by metal ion content), which utilizes a catalytic MgA present at a stoichiometry of 4/octamer, an allosteric MgC present at a stoichiometry of 8/octamer, and a monovalent metal ion, K+. However, the divalent MgB or ZnB present in some other PBGS is not present in B. japonicum PBGS. Under optimal conditions, the Kd for MgA is <0.2 microM, and the Kd for MgC is about 40 microM. The response of B. japonicum PBGS activity to monovalent and divalent cations is mutually dependent and varies dramatically with pH. B. japonicum PBGS is also found to undergo a dynamic equilibrium between active multimeric species and inactive monomers under assay conditions, a kinetic characteristic not reported for other PBGSs. B. japonicum PBGS is the first PBGS that has been rigorously demonstrated to lack a catalytic ZnA. However, consistent with prior predictions, B. japonicum PBGS can bind Zn(II) (presumably as ZnA) at a stoichiometry of 4/octamer with a Kd of 200 microM; but this high concentration is outside a physiologically significant range.     

Chromium(III) modification of the first metal binding site of phosphoenolpyruvate carboxykinase

Chicken liver phosphoenolpyruvate carboxykinase (PEPCK) is activated by Cr2+ as the sole activator under anaerobic conditions. PEPCK was modified with Cr3+, starting with either Cr2+ or Cr3+. Cr3+ has the distinct advantage of being a paramagnetic cation that could serve as a paramagnetic probe. Activators Mn2+, Mg2+, and Co2+ protect against Cr3+ incorporation. EPR, CD, and fluorescence studies indicate that Cr3+ was incorporated into the cation binding site of PEPCK. The water proton relaxation rate (PRR) and fluorescence binding studies showed that Cr3+(n1)-PEPCK forms enzyme-substrate complexes similar to those observed for the Mn2+(n1)-PEPCK complex (n1 represents the metal "enzyme binding site" as opposed to the metal "nucleotide binding site"). Cr3+(n1)-PEPCK requires an additional divalent cation for activity, an indication of two metal sites on PEPCK. Cr3+(n1)-PEPCK retains 15% residual activity as compared to unmodified PEPCK and demonstrates normal Michaelis-Menten kinetics. This is the first report of an active Cr3+-modified enzyme complex.     

The structure of the MnIIADP-nitrate-lyxose complex at the active site of hexokinase

The coordination scheme of Mn2+ in the hexokinase-MnIIADP-nitrate-lyxose complex has been determined by electron paramagnetic resonance (EPR) spectroscopy with 17O-enriched ligands. Nitrate binds to the active site of hexokinase when MnIIADP and a sugar substrate or analogue are present. The binding of nitrate enhances inhibition by glucose when ADP is present and narrows the EPR signals of the enzyme-bound MnIIADP complex in the presence of sugar substrates or analogues. Experiments using regiospecifically 17O-enriched ADP, 17O-enriched nitrate, and 17O-enriched water establish the coordination scheme of Mn2+. The EPR experiments show that ADP is a beta-monodentate ligand and that nitrate binds directly to Mn2+. Four water molecules complete the coordination sphere of the enzyme-bound Mn2+. The dissociation constant (Kd approximately 8 mM) of nitrate for the complex with enzyme, MnIIADP, and lyxose was obtained from titration experiments. These results suggest that nitrate-stabilized, dead-end complexes of hexokinase may be useful in stabilizing the closed conformation of this "hinge-bending" enzyme for crystallographic experiments.     

Calcium induces binding and formation of a spin-coupled dimanganese(II,II) center in the apo-water oxidation complex of photosystem II as precursor to the functional tetra-Mn/Ca cluster

Two new intermediates are described which form in the dark as precursors to the light-induced assembly of the photosynthetic water oxidation complex (WOC) from the inorganic components. Mn2+ binds to the apo-WOC-PSII protein in the absence of calcium at a high-affinity site. By using a hydrophobic chelator to remove Mn2+ and Ca2+ from the WOC and nonspecific Fe3+, a new EPR signal becomes visible upon binding of Mn2+ to this site, characterized by six-line 55Mn hyperfine structure (DeltaHpp = 96 +/- 1 G) and effective g = 8.3. These features indicate a high-spin electronic ground state (S = 5/2) for Mn2+ and a strong ligand field with large anisotropy. This signal is eliminated if excess Ca2+ or Mg2+ is present. A second Mn2+ EPR signal forms in place of this signal upon addition of Ca2+ in the dark. The yield of this Ca-induced Mn signal is optimum at a ratio of 2 Mn/PSII, and saturates with increasing [Ca2+] >/= 8 mM, exhibiting a calcium dissociation constant of KD = 1.4 mM. The EPR signal of the Ca-induced Mn center at 25 K is asymmetric with major g value of approximately 2.04 (DeltaHpp = 380 G) and a shoulder near g approximately 3.1. It also exhibits resolved 55Mn hyperfine splitting with separation DeltaHpp = 42-45 G. These spectral features are diagnostic of a variety of weakly interacting Mn2(II, II) pairs with electronic spins that are magnetic dipolar coupled in the range of intermanganese separations 4.1 +/- 0.4 A, and commonly associated with one or two carboxylate bridges. The calcium requirement for induction of the Mn2(II,II) signal matches the value observed for steady-state O2 evolution (Michaelis constant, KM approximately 1.4 mM), and for light-induced assembly of the WOC by photoactivation. The Ca-induced Mn2(II,II) center is a more efficient electron donor to the photooxidized tyrosine radical, TyrZ+, than is the mononuclear Mn center present in the absence of Ca2+. The Ca-induced Mn2(II,II) signal serves as a precursor for photoactivation of the functional WOC and is abolished by the presence of Mg2+. Formation of the Mn2(II,II) EPR signal by addition of Ca2+ correlates with reduction of flash-induced catalase activity, indicating that calcium modulates the accessibility or reactivity of the Mn2(II,II) core with H2O2. We propose that calcium organizes the binding site for Mn ions in the apo-WOC protein and may even interact directly with the Mn2(II,II) pair via solvent or protein-derived bridging ligands.     

Fosfomycin resistance protein (FosA) is a manganese metalloglutathione transferase related to glyoxalase I and the extradiol dioxygenases

The enzyme conferring resistance to the antibiotic fosfomycin [(1R,2S)-1,2-epoxypropylphosphonic acid] originally reported by Suarez and co-workers [Area, P., Hardisson, C., & Suarez, J. E. (1990) Antimicrob. Agents Chemother. 34, 844-848] is demonstrated in this study to be a metalloglutathione transferase. The apoenzyme is a dimer of 16 kDa subunits. Electron paramagnetic resonance spectroscopy and water proton nuclear magnetic resonance longitudinal relaxation rates suggest that each subunit contains a mononuclear Mn2+ center that interacts strongly with the substrate fosfomycin (Kd = 17 microM) more weakly with the product (Kd = 1.1 mM) and very weakly or not at all with GSH. Inhomogeneous broadening of the EPR signals of enzyme-bound Mn2+ in the presence of H2(17)O indicates that three of the coordination sites on the metal are occupied by water. Sequence alignments, three-dimensional structures, and mechanistic considerations suggest that FosA is related to at least two other metalloenzymes, glyoxalase I and the Mn2+- or Fe2+-containing extradiol dioxygenases. The mechanistic imperative driving the evolution of this previously unidentified superfamily of metalloenzymes is proposed to be bidentate coordination of a substrate or intermediate to the metal center in the enzyme-catalyzed reactions.     

Aspartate-407 in Rhodobacter sphaeroides cytochrome c oxidase is not required for proton pumping or manganese binding

Several pathways for proton transport in cytochrome c oxidase have been proposed on the basis of mutational analysis and X-ray structure: at least one for moving "pumped" protons from the interior to exterior of the membrane and a separate route for transporting "substrate" protons from the interior to the binuclear metal center to combine with oxygen to make H2O. According to the crystal structures of cytochrome c oxidase, Asp407 (Rhodobacter sphaeroides numbering) is at the interface of subunit I and subunit II of the oxidase, in a negative patch proposed to be the proton exit site in a pumping pathway, as well as a possible ligand to Mg [Iwata et al. (1995) Nature 376, 660-669]. Three mutants at the Asp407 position of R. sphaeroides cytochrome oxidase, Asp407Ala, Asp407Asn, and Asp407Cys, have been purified and characterized. All showed electron transfer activity, and pH dependence of activity, similar to that of the wild type enzyme and no major structural changes, as evidenced by visible, EPR, and resonance Raman spectroscopy. When reconstituted into artificial vesicles, the purified mutants pumped protons with normal efficiency and responded to the membrane pH and electrical gradients in a manner similar to that of wild type. Furthermore, the EPR spectra and Mn quantitation analysis of mutants grown in high Mn indicated no significant alteration in the Mn/Mg site. These results suggest that Asp407 does not play a critical role in proton translocation or in Mn/Mg binding.     

Binding of different divalent cations to the active site of avian sarcoma virus integrase and their effects on enzymatic activity

Retroviral integrases (INs) contain two known metal binding domains. The N-terminal domain includes a zinc finger motif and has been shown to bind Zn2+, whereas the central catalytic core domain includes a triad of acidic amino acids that bind Mn2+ or Mg2+, the metal cofactors required for enzymatic activity. The integration reaction occurs in two distinct steps; the first is a specific endonucleolytic cleavage step called "processing," and the second is a polynucleotide transfer or "joining" step. Our previous results showed that the metal preference for in vitro activity of avian sarcoma virus IN is Mn2+ > Mg2+ and that a single cation of either metal is coordinated by two of the three critical active site residues (Asp-64 and Asp-121) in crystals of the isolated catalytic domain. Here, we report that Ca2+, Zn2+, and Cd2+ can also bind in the active site of the catalytic domain. Furthermore, two zinc and cadmium cations are bound at the active site, with all three residues of the active site triad (Asp-64, Asp-121, and Glu-157) contributing to their coordination. These results are consistent with a two-metal mechanism for catalysis by retroviral integrases. We also show that Zn2+ can serve as a cofactor for the endonucleolytic reactions catalyzed by either the full-length protein, a derivative lacking the N-terminal domain, or the isolated catalytic domain of avian sarcoma virus IN. However, polynucleotidyl transferase activities are severely impaired or undetectable in the presence of Zn2+. Thus, although the processing and joining steps of integrase employ a similar mechanism and the same active site triad, they can be clearly distinguished by their metal preferences.     

Spectroscopic comparisons of the pH dependencies of Fe-substituted (Mn)superoxide dismutase and Fe-superoxide dismutase

We have compared the active sites of Escherichia coli Fe-substituted (Mn)superoxide dismutase [Fe-sub-(Mn)SOD] and Fe-SOD to elucidate the basis for the inactivity of Fe-sub-(Mn)SOD, despite its apparent similarity to Fe-SOD. The active site of (reduced) Fe2+-sub-(Mn)SOD is qualitatively similar to that of native Fe2+-SOD, indicating similar active site structures and coordination environments for Fe2+. Its nativelike pK is indicative of nativelike local electrostatics, and consistent with Fe2+-sub-(Mn)SOD's retention of ability to reduce O2*- [Vance and Miller (1998) J. Am. Chem. Soc. 120(3), 461-467]. The active site of (oxidized) Fe3+-sub-(Mn)SOD differs from that of Fe3+-SOD with respect to the EPR signals produced at both neutral and high pH, indicating different coordination environments for Fe3+. Although Fe3+-sub-(Mn)SOD binds the small anions N3- and F-, the KD for N3- is tighter than that of Fe3+-SOD, suggesting that the (Mn)SOD protein favors anion binding more than does the (Fe)SOD protein. The EPR spectral consequences of binding F- are reminiscent of those observed upon binding the first F- to Fe3+-SOD, but the EPR spectrum obtained upon binding N3- is different, consistent with crystallographic observation of a different binding mode for N3- in Thermus thermophilus Mn-SOD than Fe-SOD [Lah, M., et al. (1995) Biochemistry 34, 1646-1660]. We find a pK of 8.5 to be associated with dramatic changes in the EPR spectrum. In addition, we confirm the pK between 6 and 7 that has previously been reported based on changes in the optical signal and N3- binding [Yamakura, F., et al. (1995) Eur. J. Biochem. 227, 700-706]. However, this latter pK appears to be associated with much subtler changes in the EPR spectrum. The non-native pKs observed in Fe3+-sub-(Mn)SOD and the differences in the Fe3+ coordination indicated by the EPR spectra are consistent with Fe3+-sub-(Mn)SOD's inability to oxidize O2*- and suggest that its low E degrees is due to perturbation of the oxidized state.     

Light-induced high-spin signals from the oxygen evolving center in Ca2+-depleted photosystem II studied by dual mode electron paramagnetic resonance spectroscopy

Illuminating Ca2+-depleted photosystem (PS) II membranes generated two new EPR signals at g = 11 and 15 by perpendicular and parallel polarization modes, respectively. Two turnovers of the oxygen evolving center (OEC) beyond the modified S2' state are required for the appearance of these signals. The formation of the signals correlated with that of an asymmetric (singlet-like) EPR signal observed at g approximately 2. Spectral simulation indicated that both signals arose from a transition between |2(+/-)> levels with intradoublet splitting of Delta = 0.276 cm-1 in an S = 2 spin system. Furthermore, the two signals in parallel and perpendicular modes were formed at the same time, indicating that the same metal center was responsible. The molecular z-axis of the S = 2 spin system for the signals was almost parallel to the plane of thylakoid membranes. These results indicate that the Mn cluster in the photosynthetic oxygen evolving center is the source of the new EPR species which may be a Mn(IV)-Mn(IV) or Mn(III)-Mn(III) dimer or a Mn(III) monomer. Redox events of the Mn cluster in the Ca2+-depleted PS II are discussed based on these observations.     

A Mn(II)-Mn(III) EPR signal arises from the interaction of NO with the S1 state of the water-oxidizing complex of photosystem II

It was shown recently [Goussias, C., Ioannidis, N., and Petrouleas, V. (1997) Biochemistry 36, 9261-9266] that incubation of photosystem II preparations with NO at -30 degrees C in the dark results in the formation of a new intermediate of the water-oxidizing complex. This is characterized by an EPR signal centered at g = 2 with prominent manganese hyperfine structure. We have examined the detailed structure of the signal using difference EPR spectroscopy. This is facilitated by the observations that NO can be completely removed without decrease or modification of the signal, and illumination at 0 degree C eliminates the signal. The signal spans 1600 G and is characterized by sharp hyperfine structure. 14NO and 15NO cw EPR combined with pulsed ENDOR and ESEEM studies show no detectable contributions of the nitrogen nucleus to the spectrum. The spectrum bears similarities to the experimental spectrum of the Mn(II)-Mn(III) catalase [Zheng, M., Khangulov, S. V., Dismukes, G. C., and Barynin, V. V. (1994) Inorg. Chem. 33, 382-387]. Simulations allowing small variations in the catalase-tensor values result in an almost accurate reproduction of the NO-induced signal. This presents strong evidence for the assignment of the latter to a magnetically isolated Mn(II)-Mn(III) dimer. Since the starting oxidation states of Mn are higher than II, we deduce that NO acts effectively as a reductant, e.g., Mn(III)-Mn(III) + NO--> Mn(II)-Mn(III) + NO+. The temperature dependence of the nonsaturated EPR-signal intensity in the range 2-20 K indicates that the signal results from a ground state. The cw microwave power saturation data in the range 4-8 K can be interpreted assuming an Orbach relaxation mechanism with an excited state at delta = 42 K. Assuming antiferromagnetic coupling, -2JS1.S2, between the two manganese ions, J is estimated to be 10 cm-1. The finding that an EPR signal from the Mn cluster of PSII can be clearly assigned to a magnetically isolated Mn(II)-Mn(III) dimer bears important consequences in interpreting the structure of the Mn cluster. Although the signal is not currently assigned to a particular S state, it arises from a state lower than S1, possibly lower than S0, too.     

Structures of normal single-stranded DNA and deoxyribo-3'-S-phosphorothiolates bound to the 3'-5' exonucleolytic active site of DNA polymerase I from Escherichia coli

The interaction of a divalent metal ion with a leaving 3' oxygen is a central component of several proposed mechanisms of phosphoryl transfer. In support of this are recent kinetic studies showing that thiophilic metal ions (e.g., Mn2+) stimulate the hydrolysis of compounds in which sulfur takes the place of the leaving oxygen. To examine the structural basis of this phenomenon, we have solved four crystal structures of single-stranded DNA's containing either oxygen or sulfur at a 3'-bridging position bound in conjunction with various metal ions at the 3'-5' exonucleolytic active site of the Klenow fragment (KF) of DNA polymerase I from Escherichia coli. Two structures of normal ssDNA bound to KF in the presence of Zn2+ and Mn2+ or Zn2+ alone were refined at 2.6- and 2.25-A resolution, respectively. They serve as standards for comparison with other Mn2+- and Zn2+-containing structures. In these cases, Mn2+ and Zn2+ bind at metal ion site B in a nearly identical position to Mg2+ (Brautigam and Steitz (1998) J. Mol. Biol. 277, 363-377). Two structures of KF bound to a deoxyoligonucleotide that contained a 3'-bridging sulfur at the scissile phosphate were refined at 2.03-A resolution. Although the bridging sulfur compounds bind in a manner very similar to that of the normal oligonucleotides, the presence of the sulfur changes the metal ion binding properties of the active site such that Mn2+ and Zn2+ are observed at metal ion site B, but Mg2+ is not. It therefore appears that the ability of the bridging sulfur compounds to exclude nonthiophilic metal ions from metal ion site B explains the low activity of KF exonuclease on these substrates in the presence of Mg2+ (Curley et al. (1997) J. Am. Chem. Soc. 119, 12691-12692) and that the 3'-bridging atom of the substrate is influencing the binding of metal ion B prior to catalysis.     

Electron paramagnetic resonance spectroscopic measurement of Mn2+ binding affinities to the hammerhead ribozyme and correlation with cleavage activity

Efficient phosphodiester bond cleavage activity by the hammerhead ribozyme requires divalent cations. Toward understanding this metal ion requirement, the Mn2+-binding properties of hammerhead model ribozymes have been investigated under dilute solution conditions, using electron paramagnetic resonance spectroscopy (EPR) to detect free Mn2+ in the presence of added ribozyme. Numbers and affinities of bound Mn2+ were obtained at pH 7.8 (5 mM triethanolamine) in the presence of 0, 0.1, and 1.0 M NaCl for an RNA-DNA model consisting of a 13-nucleotide DNA "substrate" hybridized to a 34-nucleotide RNA "enzyme" [Pley, H. W., Flaherty, K. M., and McKay, D. B. (1994) Nature 372, 68-74]. In 0.1 M NaCl, two classes of Mn2+ sites are found with n1 = 3.7 +/- 0.4, Kd(1) = 4 +/- 1 microM (type 1) and n2 = 5.2 +/- 0.4, Kd(2) = 460 +/- 130 microM (type 2). The high-affinity type 1 sites are confirmed for an active RNA-RNA hybrid (34-nucleotide RNA enzyme:13-nucleotide RNA substrate) by EPR measurements at low Mn2+ concentrations. Decreasing NaCl concentration results in an increased number of bound Mn2+ per hammerhead. By contrast, a binding titration in 1 M NaCl indicates that a single Mn2+ site with apparent Kd approximately 10 microM is populated in low concentrations of Mn2+, and apparent cooperative effects at higher Mn2+ concentrations result in population of a similar total number of Mn2+ sites (n1 = 8-10) as found in 0.1 M NaCl. Mn2+-dependent activity profiles are similar for the active RNA-RNA hybrid in 0.1 and 1 M NaCl. Correlation with binding affinities determined by EPR indicates that hammerhead activity in 0.1 M NaCl is only observed after all four of the high-affinity Mn2+ sites are occupied, rises with population of the type 2 sites, and is independent of Mn2+ concentrations corresponding to > 8-9 Mn2+ bound per hammerhead. Equivalent measurements in 1 M NaCl demonstrate a rise in activity with the cooperative transition observed in the Mn2+ binding curve. These measurements indicate that, over this NaCl concentration range, hammerhead ribozyme activity is influenced by population of a specific set of divalent cation sites.     

MMP-9 from TNF alpha-stimulated keratinocytes binds to cell membranes and type I collagen: a cause for extended matrix degradation in inflammation?

The in vitro selected lead-dependent ribozyme is among the smallest and simplest of the known catalytic RNA motifs and has a unique metal ion specificity for divalent lead. The conformation and dynamics of this ribozyme are analyzed here by NMR and chemical probing experiments. Complete assignments of the 1H, 13C, and 15N resonances have been made, and the NMR chemical shift changes in the presence of Pb2+, Mg2+ or high concentrations of Na+ show that there is no significant structural change upon addition of either activating (Pb2+) or inhibitory (Mg2+) divalent ions. The 13C NMR relaxation data indicate substantial dynamic fluctuations on various time-scales for active-site residues in this ribozyme. The combination of chemical probing and NMR experiments reveals a picture of the active site for the lead-dependent ribozyme that has both ordered and dynamic features.     

Heme binding by a bacterial repressor protein, the gene product of the ferric uptake regulation (fur) gene of Escherichia coli

The fur gene product, Fur, of Escherichia coli is a repressor when it binds Fe(II). Since heme and iron metabolism are closely linked and Fur is rich in histidine, a ligand for heme, the binding of heme to Fur was investigated. The oxidized Fur-heme complex is stable and low spin with a Soret maximum at 404 nm and no 620-nm band. CO coordinates with the reduced heme-Fur complex, causing a shift from 412 nm to 410 nm, and stabilizes it, increasing the half-life from 5 to 15 min. Circular dichroism (CD) spectra in the Soret region show heme bound in an asymmetric environment in Fur, both in the oxidized and reduced-CO forms. Quenching of tyrosine fluorescence by heme revealed rapid, tight binding (Kd < 1 microM) with an unusual stoichiometry of 1 heme:1 Fur dimer. Fur binds Mn(II), a model ligand for the endogenous Fe(II), much more weakly (Kd > 80 microM). Far-ultraviolet CD spectroscopy showed that the alpha-helix content of apo-Fur decreases slightly with heme binding, but increases with Mn(II) binding. Competition experiments indicated that heme interacts with Fur dimers at the same site as Mn(II) and can displace the metal. In contrast to Mn(II), Zn(II) did not quench the tyrosine fluoroescence of Fur, affected the CD spectrum less than Mn(II), but did bind in a manner which prevented heme from binding. In sum, Fur not only binds heme and Zn(II) with sufficient affinity to be biologically relevant, but the interactions that occur between these ligands and their effects on Mn(II) binding need to be taken into account when addressing the biological function of Fur.     

Determination of vitamin C in effervescent tablets containing other vitamins together with trace elements

A simple, rapid method is reported for the determination of vitamin C in effervescent tablets containing other vitamins and several trace elements such as Mg(II), Zn(II), Fe(II), Mn(II), Cu(II) and Mo(VI). The procedure was developed on the basis of the bromate ion-iodide ion-ascorbic acid clock reaction (Landolt reaction). Interference effects of air oxygen and metal ions and the role of pH are discussed in detail.     

VO2+(IV) complexes with pyruvate carboxylase: activation of oxaloacetate decarboxylation and EPR properties of enzyme-VO2+ complexes

Chicken liver pyruvate carboxylase catalyzes a nonclassical ping-pong mechanism in which the carboxylation of biotin at subsite 1 of the active site is coupled to the biotin-dependent carboxylation of pyruvate at subsite 2. The functions of two divalent cation cofactors and at least one monovalent cation cofactor in catalysis are not well understood. The oxyvanadyl cation, VO2+ does not support phosphoryl transfer at the first subsite, and uncouples the decarboxylation of oxaloacetate at subsite 2 from the formation of ATP at subsite 1. Stimulation of this oxaloacetate decarboxylase activity in the presence of substrates and cofactors of the first subsite, including VO2+, VOADP-, Pi, and acetyl CoA, suggests that these cofactors and substrates induce the movement of carboxybiotin from the second subsite to the first subsite, where it is decarboxylated. VO2+ EPR has provided evidence for enzymic and nucleotide divalent cation binding sites within the first subsite. The EPR properties of enzyme bound VO2+ were altered by bicarbonate, suggesting that this substrate ligands directly to VO2+ at the enzymic metal site. Fluorescence quenching experiments suggest that a monovalent cation may interact with bicarbonate at the first subsite as well. The results of this study provide evidence that (i) the extrinsic metal ion cofactors interact with the substrates at the first subsite, and that (ii) divalent cations play a role in coupling catalysis at the two nonoverlapping subsites by inducing the decarboxylation of carboxybiotin at the first subsite.     

Characterization of ligands of a high-affinity metal-binding site in the latent chloroplast F1-ATPase by EPR spectroscopy of bound VO2+

Vanadyl, (V = O)2+, is able to substitute for Mg2+ as a cofactor for ATPase activity catalyzed by the chloroplast F1-ATPase (CF1). Mg2+-dependent ATPase activity was also observed with CF1 that contained VO(2+)-ATP bound specifically to the noncatalytic N2 site. Modulation of the Mg(2+)-ATPase activity induced by VO2+ bound at this site indicates that the metal bound to the noncatalytic site affects catalytic activity. When CF1 is depleted of nucleotides from all but the N1 site, a single Mg2+ remains bound at a site designated M1. Addition of VO2+ to the depleted protein gives rise to an EPR spectrum characteristic of a CF1-bound VO2+ species. The binding curve of the VO2+ complex to latent, nucleotide-depleted CF1 was determined by the integrated intensities of the -5/2 parallel peak in the EPR spectrum as calibrated using atomic absorption spectroscopy. Under these conditions, VO2+ binds cooperatively to approximately two sites designated M2 and M3. Three-pulse ESEEM spectra of the CF1-VO2+ complex contain two intense modulations with frequencies and field-dependent behavior that show that they are from a directly coordinated 14N nucleus. Analysis of the bound VO2+ by ENDOR spectroscopy revealed the presence of a single group of protons associated with an equatorial amino or water ligand that is exchangeable with solvent. Using the additivity relation for hyperfine coupling, the most probable set of equatorial ligands to the VO2+ bound to CF1 under these conditions consists of one lysine nitrogen, two carboxyl oxygens from aspartate or glutamate, and one water.     

The C2 catalytic domain of adenylyl cyclase contains the second metal ion (Mn2+) binding site

Membrane-bound mammalian adenylyl cyclase isoforms contain two internally homologous cytoplasmic domains (C1 and C2). When expressed separately, C1 and C2 are catalytically inactive, but conversion of ATP to cAMP is observed if C1 and C2 are combined. By analogy with DNA polymerases, adenylyl cyclases are thought to require two divalent metal ions for nucleotide binding and phosphodiester formation; however, only one Mg2+ ion (liganded to C1) has been visualized in the recently solved crystal structure of a C1-C2 complex [Tesmer, J. J. G., Sunahara, R. K., Gilman, A. G., and Sprang, S. R. (1997) Science 278, 1907-1916]. Here, we have studied the binding of ATP to IIC2 (from type II adenylyl cyclase) using ATP analogues [2',3'-dialdehyde ATP (oATP), a quasi-irreversible inhibitor that is covalently incorporated via reduction of a Schiff base, the photoaffinity ligand 8-azido-ATP (8N3-ATP), and trinitrophenyl-ATP (TNP-ATP), a fluorescent analogue] and fluorescein isothiocyanate (FITC). [alpha-32P]oATP and 8N-[alpha-32P]ATP are specifically incorporated into IIC2. Labeling of IIC2 by [alpha-32P]oATP and by FITC is greatly enhanced by Mn2+ and to a much lesser extent by Mg2+. Similarly, TNP-ATP binds to IIC2 as determined by fluorescence enhancement, and this binding is promoted by Mn2+. Thus, a second metal ion binding site (preferring Mn2+) is contained within the C2 domain, and this finding highlights the analogy in the reaction catalyzed by DNA polymerases and adenylyl cyclases.     

The reaction pathway of the isomerization of D-xylose catalyzed by the enzyme D-xylose isomerase: a theoretical study

Different pathways of the metal-induced isomerization of D-xylose to D-xylulose are investigated and compared in detail using energy minimization and molecular dynamics simulation. Two theoretical models are constructed for the reaction: in vacuum and in the enzyme D-xylose isomerase. The vacuum model is constructed based on the X-ray structure of the active site of D-xylose isomerase. It contains the atoms directly involved in the reaction and is studied using a semi-empirical molecular orbital method (PM3). The model in the enzyme includes the effects of the enzyme environment on the reaction using a combined quantum mechanical and molecular mechanical potential. For both models, the structures of the reactants, products, and intermediate complexes along the isomerization pathway are optimized. The effects of the position of the "catalytic Mg2+ ion" on the energies of the reactions are studied. The results indicate: 1) in vacuum, the isomerization reaction is favored when the catalytic metal cation is at site A, which is remote from the substrate; 2) in the enzyme, the catalytic metal cation, starting from site A, moves and stays at site B, which is close to the substrate; analysis of the charge redistribution of the active site during the catalytic process shows that the metal ion acts as a Lewis acid to polarize the substrate and catalyze the hydride shift; these results are consistent with previous experimental observations; and 3) Lys183 plays an important role in the isomerization reaction. The epsilon-NH3+ group of its side chain can provide a proton to the carboxide ion of the substrate to form a hydroxyl group after the hydride shift step. This role of Lys183 has not been suggested before. Based on our calculations, we believe that this is a reasonable mechanism and consistent with site-directed mutation experiments.     

Dioxygen inactivation of pyruvate formate-lyase: EPR evidence for the formation of protein-based sulfinyl and peroxyl radicals

We here report EPR studies that provide evidence for radical intermediates generated from the glycyl radical of activated pyruvate formate-lyase (PFL) during the process of oxygen-dependent enzyme inactivation, radical quenching, and protein fragmentation. Upon exposure of active PFL to air, a long-lived radical intermediate was generated, which exhibits an EPR spectrum assigned to a sulfinyl radical (RSO*). The EPR spectrum of a sulfinyl radical was also generated from the activated C418A mutant of PFL, indicating that Cys 418 is not the site of sulfinyl radical formation. Exposure of the activated C419A mutant or C418AC419A double mutant to air on the other hand, resulted in a new EPR spectrum that we assign to the alpha-carbon peroxyl radical (ROO*) of the active-site glycine, G734. These findings suggest that C419 is the site of sulfinyl radical formation and that replacement of this cysteine with alanine results in the accumulation of the carbon peroxyl radical. The results also support the proposal that the peroxyl radical and the sulfinyl radical are intermediates in the oxygen-dependent inactivation and cleavage of the protein. Moreover, these observations are consistent with the hypothesis that C419 and G734 are in close proximity in the activated enzyme and may participate in a glycyl/thiyl radical equilibrium. A mechanism that accounts for the formation of the radical intermediates is proposed.