Sun Un

Leiden University, Leyden, South Holland, Netherlands

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Publications (49)218.63 Total impact

  • Sun Un
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    ABSTRACT: A common feature of a large majority of the manganese metalloenzymes, as well as many synthetic biomimetic complexes, is the bonding between the manganese ion and imidazoles. This interaction was studied by examining the nature and structure of manganese(II) imidazole complexes in frozen aqueous solutions using 285 GHz high magnet-field continuous-wave electron paramagnetic resonance (cw-HFEPR) and 95 GHz pulsed electron-nuclear double resonance (ENDOR) and pulsed electron-double resonance detected nuclear magnetic resonance (PELDOR-NMR). The (55)Mn hyperfine coupling and isotropic g values of Mn(II) in frozen imidazole solutions continuously decreased with increasing imidazole concentration. ENDOR and PELDOR-NMR measurements demonstrated that the structural basis for this behavior arose from the imidazole concentration-dependent distribution of three six-coordinate and two four-coordinate species: [Mn(H2O)6](2+), [Mn(imidazole)(H2O)5](2+), [Mn(imidazole)2(H2O)4](2+), [Mn(imidazole)3(H2O)](2+), and [Mn(imidazole)4](2+). The hyperfine and g values of manganese proteins were also fully consistent with this imidazole effect. Density functional theory methods were used to calculate the structures, spin and charge densities, and hyperfine couplings of a number of different manganese imidazole complexes. The use of density functional theory with large exact-exchange admixture calculations gave isotropic (55)Mn hyperfine couplings that were semiquantitative and of predictive value. The results show that the covalency of the Mn-N bonds play an important role in determining not only magnetic spin parameters but also the structure of the metal binding site. The relationship between the isotropic (55)Mn hyperfine value and the number of imidazole ligands provides a quick and easy test for determining whether a protein binds an Mn(II) ion using histidine residues and, if so, how many are involved. Application of this method shows that as much as 40% of the Mn(II) ions in Deinococcus radiodurans are ligated to two histidines (Tabares, L. C.; Un, S. J. Biol. Chem2013, in press).
    Inorganic Chemistry 03/2013; · 4.59 Impact Factor
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    ABSTRACT: High magnetic-field high-frequency electron paramagnetic resonance techniques were used to measure in situ Mn(II) speciation in Deinococcus radiodurans, a radiation resistant bacteria capable of accumulating high concentrations of Mn(II). It was possible to identify and quantify the evolution of Mn(II) species in intact cells at various stages of growth. Aside from water, 95 GHz high-field electron-nuclear double resonance showed that the Mn(II) ions are bound to histidines and phosphate groups, mostly from fructose-1,6-bisphosphate but also nucleotides. During stationary growth phase, 285 GHz continuous-wave EPR measurements showed that histidine is the most common ligand to Mn(II) and that significant amounts of cellular Mn(II) in D. radiodurans are bound to peptides and proteins. As much as 40% of the total Mn(II) was in manganese superoxide dismutase and it is this protein and not smaller manganese complexes, as has been suggested recently, that is probably the primary defense against superoxide.
    Journal of Biological Chemistry 01/2013; · 4.65 Impact Factor
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    ABSTRACT: The main cofactors that determine the photosystem II (PSII) oxygen evolution activity are borne by the D1 and D2 subunits. In the cyanobacterium Thermosynechococcus elongatus, there are three psbA genes coding for D1. Among the 344 residues constituting D1, there are 21 substitutions between PsbA1 and PsbA3, 31 between PsbA1 and PsbA2, and 27 between PsbA2 and PsbA3. Here, we present the first study of PsbA2-PSII. Using EPR and UV-visible time-resolved absorption spectroscopy, we show that: (i) the time-resolved EPR spectrum of Tyr(Z)(•) in the (S(3)Tyr(Z)(•))' is slightly modified; (ii) the split EPR signal arising from Tyr(Z)(•) in the (S(2)Tyr(Z)(•))' state induced by near-infrared illumination at 4.2 K of the S(3)Tyr(Z) state is significantly modified; and (iii) the slow phases of P(680)(+) reduction by Tyr(Z) are slowed down from the hundreds of μs time range to the ms time range, whereas both the S(1)Tyr(Z)(•) → S(2)Tyr(Z) and the S(3)Tyr(Z)(•) → S(0)Tyr(Z) + O(2) transition kinetics remained similar to those in PsbA(1/3)-PSII. These results show that the geometry of the Tyr(Z) phenol and its environment, likely the Tyr-O···H···Nε-His bonding, are modified in PsbA2-PSII when compared with PsbA(1/3)-PSII. They also point to the dynamics of the proton-coupled electron transfer processes associated with the oxidation of Tyr(Z) being affected. From sequence comparison, we propose that the C144P and P173M substitutions in PsbA2-PSII versus PsbA(1/3)-PSII, respectively located upstream of the α-helix bearing Tyr(Z) and between the two α-helices bearing Tyr(Z) and its hydrogen-bonded partner, His-190, are responsible for these changes.
    Journal of Biological Chemistry 02/2012; 287(16):13336-47. · 4.65 Impact Factor
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    ABSTRACT: One of the most puzzling questions of manganese and iron superoxide dismutases (SODs) is what is the basis for their metal-specificity. This review summarizes our findings on the Mn(II) electronic structure of SODs and related synthetic models using high-field high-frequency electron paramagnetic resonance (HFEPR), a technique that is able to achieve a very detailed and quantitative information about the electronic structure of the Mn(II) ions. We have used HFEPR to compare eight different SODs, including iron, manganese and cambialistic proteins. This comparative approach has shown that in spite of their high structural homology each of these groups have specific spectroscopic and biochemical characteristics. This has allowed us to develop a model about how protein and metal interactions influence protein pK, inhibitor binding and the electronic structure of the manganese center. To better appreciate the thermodynamic prerequisites required for metal discriminatory SOD activity and their relationship to HFEPR spectroscopy, we review the work on synthetic model systems that functionally mimic Mn-and FeSOD. Using a single ligand framework, it was possible to obtain metal-discriminatory "activity" as well as variations in the HFEPR spectra that parallel those found in the proteins. Our results give new insights into protein-metal interactions from the perspective of the Mn(II) and new steps towards solving the puzzle of metal-specificity in SODs.
    Biochimica et Biophysica Acta 10/2009; 1804(2):308-17. · 4.66 Impact Factor
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    ABSTRACT: A high-field electron paramagnetic resonance (HFEPR) study of oxalate decarboxylase (OxdC) is reported. OxdC breaks down oxalate to carbon dioxide and formate and possesses two distinct manganese(II) binding sites, referred to as site-1 and -2. The Mn(II) zero-field interaction was used to probe the electronic state of the metal ion and to examine chemical/mechanistic roles of each of the Mn(II) centers. High magnetic-fields were exploited not only to resolve the two sites, but also to measure accurately the Mn(II) zero-field parameters of each of the sites. The spectra exhibited surprisingly complex behavior as a function of pH. Six different species were identified based on their zero-field interactions, two corresponding to site-1 and four states to site-2. The assignments were verified using a mutant that only affected site-1. The speciation data determined from the HFEPR spectra for site -2 was consistent with a simple triprotic equilibrium model, while the pH dependence of site-1 could be described by a single pK(a). This pH dependence was independent of the presence of the His-tag and of whether the preparations contained 1.2 or 1.6 Mn per subunit. Possible structures of the six species are proposed based on spectroscopic data from model complexes and existing protein crystallographic structures obtained at pH 8 are discussed. Although site-1 has been identified as the active site and no role has been assigned to site-2, the pronounced changes in the electronic structure of the latter and its pH behavior, which also matches the pH-dependent activity of this enzyme, suggests that even if the conversion of oxalate to formate is carried out at site-1, site-2 likely plays a catalytically relevant role.
    The Journal of Physical Chemistry B 08/2009; 113(26):9016-25. · 3.61 Impact Factor
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    ABSTRACT: The catalytic cycle of numerous enzymes involves the coupling between proton transfer and electron transfer. Yet, the understanding of this coordinated transfer in biological systems remains limited, likely because its characterization relies on the controlled but experimentally challenging modifications of the free energy changes associated with either the electron or proton transfer. We have performed such a study here in Photosystem II. The driving force for electron transfer from Tyr(Z) to P(680)(*+) has been decreased by approximately 80 meV by mutating the axial ligand of P(680), and that for proton transfer upon oxidation of Tyr(Z) by substituting a 3-fluorotyrosine (3F-Tyr(Z)) for Tyr(Z). In Mn-depleted Photosystem II, the dependence upon pH of the oxidation rates of Tyr(Z) and 3F-Tyr(Z) were found to be similar. However, in the pH range where the phenolic hydroxyl of Tyr(Z) is involved in a H-bond with a proton acceptor, the activation energy of the oxidation of 3F-Tyr(Z) is decreased by 110 meV, a value which correlates with the in vitro finding of a 90 meV stabilization energy to the phenolate form of 3F-Tyr when compared to Tyr (Seyedsayamdost et al. J. Am. Chem. Soc. 2006, 128,1569-1579). Thus, when the phenol of Y(Z) acts as a H-bond donor, its oxidation by P(680)(*+) is controlled by its prior deprotonation. This contrasts with the situation prevailing at lower pH, where the proton acceptor is protonated and therefore unavailable, in which the oxidation-induced proton transfer from the phenolic hydroxyl of Tyr(Z) has been proposed to occur concertedly with the electron transfer to P(680)(*+). This suggests a switch between a concerted proton/electron transfer at pHs < 7.5 to a sequential one at pHs > 7.5 and illustrates the roles of the H-bond and of the likely salt-bridge existing between the phenolate and the nearby proton acceptor in determining the coupling between proton and electron transfer.
    Journal of the American Chemical Society 04/2009; 131(12):4425-33. · 10.68 Impact Factor
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    ABSTRACT: Superoxide dismutases (SODs) catalyze the disproportionation of superoxide to dioxygen and hydrogen peroxide. The active metal sites of iron and manganese superoxide dismutases are structurally indistinguishable from each other. Despite the structural homology, these enzymes exhibit a high degree of metal selective activity suggesting subtle redox tuning of the active site. The redox tuning model, however, up to now has been challenged by the existence of so-called cambialistic SODs that function with either metal ion. We have prepared and investigated two sets of manganese complexes in which groups of varying electron-withdrawing character, as measured by their Hammett constants sigma Para, have been introduced into the ligands. We observed that the Mn(III)/Mn(II) reduction potential for the series based on 4'-X-terpyridine ligands together with the corresponding values for the iron-substituted 4'-X-terpyridine complexes changed linearly with sigma Para. The redox potential of the iron and manganese complexes could be varied by as much as 600 mV by the 4'-substitution with the manganese complexes being slightly more sensitive to the substitution than iron. The difference was such that in the case where the 4'-substituent was a pyrrolidine group both the manganese and the iron complex were thermodynamically competent to catalytically disproportionate superoxide, making this particular ligand "cambialistic". Taking our data and those available from the literature together, it was found that in addition to the electron-withdrawing capacity of the 4'-substituents the overall charge of the Mn(II) complexes plays a major role in tuning the redox potential, about 600 mV per charge unit. The ion selectivity in Mn and FeSODs and the occurrence of cambialistic SODs are discussed in view of these results. We conclude that the more distant electrostatic contributions may be the source of metal specific enzymatic activity.
    Inorganic Chemistry 05/2008; 47(7):2897-908. · 4.59 Impact Factor
  • Journal of the American Chemical Society 12/2007; 129(45):13825-7. · 10.68 Impact Factor
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    ABSTRACT: Superoxide dismutases (SODs) are proteins specialized in the depletion of superoxide from the cell through disproportionation of this anion into oxygen and hydrogen peroxide. We have used high-field electron paramagnetic resonance (HFEPR) to test a two-site binding model for the interaction of manganese-SODs with small anions. Because tyrosine-34 was thought to act as a gate between these two sites in this model, a tyrosine to phenylalanine mutant of the superoxide dismutase from R. capsulatus was constructed. Although the replacement slightly reduced activity, HFEPR measurements demonstrated that the electronic structure of the Mn(II) center was unaffected by the mutation. In contrast, the mutation had a profound effect on the interactions of fluoride and azide with the Mn(II) center. It was concluded that the absence of tyrosine-34 prevented the close approach of these anions to the metal ion. This mutation also enhanced the formation of a hexacoordinated water-Mn(II)SOD complex at low temperatures. Together, these results showed that the role of Y34 is unlikely to involve redox tuning but rather is important in regulating the equilibria between the anionic substrate in solution and the two binding sites near the metal. These observations further supported the originally proposed mutually exclusive two-binding-site model.
    Biochemistry 09/2007; 46(32):9320-7. · 3.38 Impact Factor
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    ABSTRACT: The recombinant form of the extrinsic 23 kDa protein (psbP) of Photosystem II (PSII) was studied with respect to its capability to bind Mn. The stoichiometry was determined to be one manganese bound per protein. A very high binding constant, K(A)=10(-17) M(-1), was determined by dialysis of the Mn containing protein against increasing EDTA concentration. High Field EPR spectroscopy was used to distinguish between specific symmetrically ligated Mn(II) from those non-specifically Mn(II) attached to the protein surface. Upon Mn binding PsbP exhibited fluorescence emission with maxima at 415 and 435 nm when tryptophan residues were excited. The yield of this blue fluorescence was variable from sample to sample. It was likely that different conformational states of the protein were responsible for this variability. The importance of Mn binding to PsbP in the context of photoactivation of PSII is discussed.
    Biochimica et Biophysica Acta 07/2007; 1767(6):583-8. · 4.66 Impact Factor
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    ABSTRACT: The Mn4Ca cluster of photosystem II (PSII) goes through five sequential oxidation states (S0-S4) in the water oxidation process that also involves a tyrosine radical intermediate (TyrZ*). An S2TyrZ* state in which the Mn4Ca cluster and TyrZ* are magnetically coupled to each other and which is characterized by a distinct "split-signal" EPR spectrum can be generated in acetate-treated PSII. This state was examined by high-field EPR (HFEPR) in PSII from Thermosynechococcus elongatus isolated from a D2-Tyr160Phe mutant to avoid spectral contributions from TyrD*. In contrast to the same state in plants, both antiferromagnetic and ferromagnetic spin-spin couplings were observed. The intrinsic g values of TyrZ* in the coupled state were directly measured from the microwave frequency dependence of the HFEPR spectrum. The TyrZ* gx value in the antiferromagnetic centers was 2.0083, indicating that the coupled radical was in a less electropositive environment than in Mn-depleted PSII. Two gx values were found in the ferromagnetically coupled centers, 2.0069 and 2.0079. To put these values in perspective, the second redox-active tyrosine, TyrD*, was examined in various electrostatic environments. The TyrD* gx value changed from 2.0076 in the wild type to 2.0095 when the hydrogen bond from histidine 189 to TyrD* was removed using the D2-His189Leu mutant, indicating a change to a significantly less electropositive environment. BLY3P/6-31+G** density functional calculations on the hydrogen-bonded p-ethylphenoxy radical-imidazole supermolecular model complex showed that the entire range of Tyr* gx values, from 2.0065 to 2.0095, could be explained by the combined effects of hydrogen bonding and the dielectric constant of the local protein environment.
    Biochemistry 04/2007; 46(11):3138-50. · 3.38 Impact Factor
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    ABSTRACT: The reactive intermediates formed in the catalase-peroxidase from Synechocystis PCC6803 upon reaction with peroxyacetic acid, and in the absence of peroxidase substrates, are the oxoferryl-porphyrin radical and two subsequent protein-based radicals that we have previously assigned to a tyrosyl (Tyr()) and tryptophanyl (Trp()) radicals by using multifrequency Electron Paramagnetic Resonance (EPR) spectroscopy combined with deuterium labeling and site-directed mutagenesis. In this work, we have further investigated the Trp() in order to identify the site for the tryptophanyl radical formation, among the 26 Trp residues of the enzyme and to possibly understand the protein constraints that determine the selective formation of this radical. Based on our previous findings about the absence of the Trp() intermediate in four of the Synechocystis catalase-peroxidase variants on the heme distal side (W122F, W106A, H123Q, and R119A) we constructed new variants on Trp122 and Trp106 positions. Trp122 is very close to the iron on the heme distal side while Trp106 belongs to a short stretch (11 amino acid residues on the enzyme surface) that is highly conserved in catalase-peroxidases. We have used EPR spectroscopy to characterize the changes on the heme microenvironment induced by these mutations as well as the chemical nature of the radicals formed in each variant. Our findings identify Trp106 as the tryptophanyl radical site in Synechocystis catalase-peroxidase. The W122H and W106Y variants were specially designed to mimic the hydrogen-bond interactions of the naturally occurring Trp residues. These variants clearly demonstrated the important role of the extensive hydrogen-bonding network of the heme distal side, in the formation of the tryptophanyl radical. Moreover, the fact that W106Y is the only Synechocystis catalase-peroxidase variant of the distal heme side that recovers a catalase activity comparable to the WT enzyme, strongly indicates that the integrity of the extensive hydrogen-bonding network is also essential for the catalatic activity of the enzyme.
    Journal of Inorganic Biochemistry 06/2006; 100(5-6):1091-9. · 3.20 Impact Factor
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    ABSTRACT: The effect of the substrate analogues azide and fluoride on the manganese(II) zero-field interactions of different manganese-containing superoxide dismutases (SOD) was measured using high-field electron paramagnetic resonance spectroscopy. Two cambialistic types, proteins that are active with manganese or iron, were studied along with two that were only active with iron and another that was only active with manganese. It was found that azide was able to coordinate directly to the pentacoordinated Mn(II) site of only the MnSOD from Escherichia coli and the cambialistic SOD from Rhodobacter capsulatus. The formation of a hexacoordinate azide-bound center was characterized by a large reduction in the Mn(II) zero-field interaction. In contrast, all five SODs were affected by fluoride, but no evidence for hexacoordinate Mn(II) formation was detected. For both azide and fluoride, the extent of binding was no more than 50%, implying either that a second binding site was present or that binding was self-limiting. Only the Mn(II) zero-field interactions of the two SODs that had little or no activity with manganese were found to be significantly affected by pH, the manganese-substituted iron superoxide dismutase from E. coli and the Gly155Thr mutant of the cambialistic SOD from Porphyromonas gingivalis. A model for anion binding and the observed pK involving tyrosine-34 is presented.
    Biochemistry 03/2006; 45(6):1919-29. · 3.38 Impact Factor
  • Sun Un
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    ABSTRACT: Electron paramagnetic resonance (EPR) spectroscopy has been extensively used to identify and characterize protein-based redox active amino acid radicals based on their g-values and hyperfine couplings. To better understand how these parameters depend on the electronic structure and environment of the radical, the theoretical g-values and proton hyperfine tensors of three models corresponding to the tyrosyl, tryptophanyl and glycyl radicals were calculated using Gaussian 03. The g-values were determined using the B3LYP/6-31+G(D,P) combination of density functional and basis set, while the hyperfine tensors were determined using the B3LYP/EPR-III and PBE0/EPR-III combinations. Comparisons are made to measured values. It was found that by appropriately accounting for hydrogen bonds and the dielectric constant of the environment, good agreement could be achieved between the calculated and measured g-values. In all three cases, the g-anisotropy arose from significant spin density on a nitrogen or oxygen atom. The calculated hyperfine tensors for the three radicals did not differ significantly from previous calculations. In the case of the tyrosyl radical, it is shown for the first time that the para-position substituent that is opposite of the C-O group can break the symmetry of the phenyl ring, leading to different hyperfine tensors for the two large ortho proton couplings. For the tyrosyl and tryptophanyl models, the calculated hyperfine couplings to hydrogen-bonding protons were in very good agreement with measured values for both the tyrosyl and tryptophanyl models.
    Magnetic Resonance in Chemistry 12/2005; 43 Spec no.:S229-36. · 1.53 Impact Factor
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    ABSTRACT: The spin-other-orbit (SOO) contribution to the g-tensor (DeltagSOO) of electron paramagnetic resonance arises due to the interaction of electron-spin magnetic moment with the magnetic field produced by the orbital motion of other electrons. A similar mechanism is responsible for the leading term in nuclear magnetic-shielding tensors sigma. We demonstrate that analogous to sigma, paramagnetic DeltagSOO contribution exhibits a pronounced dependence on the choice of the magnetic-field gauge. The gauge corrections to DeltagSOO are similar in magnitude, and opposite in sign, to the paramagnetic SOO term. We calculate gauge-invariant DeltagSOO values using gauge-including atomic orbitals and density-functional theory. For organic radicals, complete gauge-invariant DeltagSOO values typically amount to less than 500 parts per million (ppm), and are small compared to other g-tensor contributions. For the first-row transition-metal compounds, DeltagSOO may contribute several thousand ppm to the g-tensor, but are negligible compared to the remaining deviations from experiment. With popular choices for the magnetic-field gauge, the individual gauge-variant contributions may be an order of magnitude higher, and do not provide a reliable estimation of DeltagSOO.
    The Journal of Chemical Physics 07/2005; 122(21):214101. · 3.12 Impact Factor
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    ABSTRACT: To gain new insights into the function of photosystem II (PSII) herbicides DCMU (a urea herbicide) and bromoxynil (a phenolic herbicide), we have studied their effects in a better understood system, the bacterial photosynthetic reaction center of the terbutryn-resistant mutant T4 of Blastochloris (Bl.) viridis. This mutant is uniquely sensitive to these herbicides. We have used redox potentiometry and time-resolved absorption spectroscopy in the nanosecond and microsecond time scale. At room temperature the P(+)(*)Q(A)(-)(*) charge recombination in the presence of bromoxynil was faster than in the presence of DCMU. Two phases of P(+)(*)Q(A)(-)(*) recombination were observed. In accordance with the literature, the two phases were attributed to two different populations of reaction centers. Although the herbicides did induce small differences in the activation barriers of the charge recombination reactions, these did not explain the large herbicide-induced differences in the kinetics at ambient temperature. Instead, these were attributed to a change in the relative amplitude of the phases, with the fast:slow ratio being approximately 3:1 with bromoxynil and approximately 1:2 with DCMU at 300 K. Redox titrations of Q(A) were performed with and without herbicides at pH 6.5. The E(m) was shifted by approximately -75 mV by bromoxynil and by approximately +55 mV by DCMU. As the titrations were done over a time range that is assumed to be much longer than that for the transition between the two different populations, the potentials measured are considered to be a weighted average of two potentials for Q(A). The influence of the herbicides can thus be considered to be on the equilibrium of the two reaction center forms. This may also be the case in photosystem II.
    Biochemistry 05/2005; 44(15):5931-9. · 3.38 Impact Factor
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    ABSTRACT: Two different temperature dependences of the manganese(II) high-field electron paramagnetic resonance spectrum of manganese superoxide dismutase from E. coli were observed. In the 25-200 K range, the zero-field interaction steadily decreased with increasing temperature. This was likely due to the thermal expansion of the protein. From these results, it was possible to deduce an approximately r(-)(2.5) dependence of Mn(II) zero-field interaction on ligand-metal distance. At temperatures above 240 K, a distinct six-line component was detected, the amplitude of which decreased with increasing temperature. On the basis of similarities to the six-line spectrum observed for the azide-complexed E. coli manganese superoxide dismutase, the newly detected six-line spectrum was assigned to a hexacoordinate Mn(II) center resulting from the coordination of a nearby water molecule to the normally five-coordinate center. The changes in enthalpy and entropy characterizing the hexacoordinate-pentacoordinate equilibrium in the 240-268 K range were -5 kcal/mol and -24 cal/mol.K, respectively. The structural implications of the zero-field parameters of the newly found hexacoordinate form in comparison to those of the Mn(II) centers in concanavalin-A and manganese-containing R. spheroides photosynthetic reaction centers and the values predicted by the superposition model are discussed.
    Journal of the American Chemical Society 05/2005; 127(16):6039-47. · 10.68 Impact Factor
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    Angewandte Chemie International Edition 03/2005; 44(10):1536-40. · 11.34 Impact Factor
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    ABSTRACT: The Mn(II) high-magnetic-field electron paramagnetic resonance (HFEPR) spectra of five different superoxide dismutases (SODs) were measured at 190 and 285 GHz. The native E. coli manganese SOD was found to be distinct from the other SODs by virtue of its large zero-field E-value. The two wild-type cambialistic proteins from Porphyromonas gingivalis and Rhodobacter capsulatus were also distinct. However, the Gly155Thr mutant of the P. gingivalis SOD changed the Mn(II) spectrum so that it closely resembled the spectrum of manganese reconstituted E. coli iron SOD. This observation paralleled enzyme activity measurements that show that this mutation causes the loss of activity with manganese and enhanced activity with iron indicating a conversion from a cambialistic to an iron-specific protein. The Mn(II) magnetic parameters were determined by simultaneously fitting the multifrequency data. Simulations were carried out by numerically diagonalizing the spin Hamiltonian and explicitly calculating all possible transition probabilities. The relationship between the Mn(II) zero-field interaction and structure of the metal binding site is also discussed.
    Journal of the American Chemical Society 04/2004; 126(9):2720-6. · 10.68 Impact Factor
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    ABSTRACT: Catalase-peroxidases are bifunctional heme enzymes with a high structural homology to peroxidases from prokaryotic origin and a catalatic activity comparable to monofunctional catalases. These unique features of catalase-peroxidases make them good systems to study and understand the role of alternative electron pathways both in catalases and peroxidases. In particular, it is of interest to study the poorly understood role of tyrosyl and tryptophanyl radicals as alternative cofactors in the catalytic cycle of catalases and peroxidases. In this work, we have used a powerful combination of multifrequency EPR spectroscopy, isotopic labeling of tryptophan and tyrosine residues, and site-directed mutagenesis to unequivocally identify the reactive intermediates formed by the wild-type Synechocystis PCC6803 catalase-peroxidase. Selected variants of the heme distal and proximal sides of the Synechocystis enzyme were investigated. Variants on the aromatic residues of the short stretch located relatively close to the heme and spanning the distal and proximal sides were also investigated. In the wild-type enzyme, the EPR signal of the catalases and peroxidases (typical) Compound I intermediate [Fe(IV)=O por.+] was observed. Two protein-based radical intermediates were also detected and identified as a Tyr. and a Trp. . The site of Trp. is proposed to be Trp 106, a residue belonging to the conserved short stretch in catalase-peroxidases and located at a 7-8 A distance to the heme propionate groups. An extensive hydrogen-bonding network on the heme distal side, involving Trp122, His123, Arg119, seven structural waters, the heme 6-propionate group, and Trp106, is proposed to have a key role on the formation of the tryptophanyl radical. We used high-field EPR spectroscopy (95-285 GHz) to resolve the g-anisotropy of the protein-based radicals in Synechocystis catalase-peroxidase. The broad gx component of the HF EPR spectrum of the Tyr. in Synechocystis catalase-peroxidase was consistent with a distributed electropositive protein environment to the tyrosyl radical.
    Journal of the American Chemical Society 12/2003; 125(46):14093-102. · 10.68 Impact Factor

Publication Stats

677 Citations
218.63 Total Impact Points

Institutions

  • 2009
    • Leiden University
      Leyden, South Holland, Netherlands
  • 2007
    • University of Michigan
      • Department of Chemistry
      Ann Arbor, MI, United States
  • 1998–2007
    • Atomic Energy and Alternative Energies Commission
      • Bioenergetics, Structural Biology, and Mechanisms (SB2SM/UMR 8221CNRS)
      Gif-sur-Yvette, Ile-de-France, France
  • 1994–2005
    • French National Centre for Scientific Research
      Lutetia Parisorum, Île-de-France, France
  • 1997–2001
    • Institut de Génétique et de Biologie Moléculaire et Cellulaire
      Strasburg, Alsace, France
    • Freie Universität Berlin
      Berlín, Berlin, Germany
  • 1992–1993
    • Massachusetts Institute of Technology
      • Department of Chemistry
      Cambridge, MA, United States