Reinhard Kappl - Academia.edu (original) (raw)
Papers by Reinhard Kappl
Biochemistry Usa, Oct 1, 2008
Quercetinase (QueD) of Streptomyces sp. FLA is an enzyme of the monocupin family and catalyzes th... more Quercetinase (QueD) of Streptomyces sp. FLA is an enzyme of the monocupin family and catalyzes the 2,4-dioxygenolytic cleavage of the flavonol quercetin. After expression of the queD gene in Escherichia coli, high specific QueD activity was found in crude cell extracts when the growth medium was supplemented with NiCl 2 or CoCl 2, but not when Mn (2+), Fe (2+), Cu (2+), or Zn (2+) was added. The metal occupancy of Ni- and Co-QueD purified from these cells was </=50%, presumably due to strong overproduction of QueD in E. coli. Circular dichroism spectroscopy indicated the same folded structure with a high content of beta-sheet for the Ni and Co protein. The apparent kinetic constants for quercetin of Ni-QueD ( k cat = 40.1 s (-1), and K m = 5.75 microM) and Co-QueD ( k cat = 7.6 s (-1), and K m = 0.96 muM) indicate similar catalytic efficiencies; however, the approximately 5-fold lower apparent K m value of Ni-QueD for dioxygen suggests that the nickel enzyme performs better under physiological conditions. The pH dependence of k cat,app indicates that an ionizable group with a p K a near 6.8 has to be deprotonated for catalysis. Electron paramagnetic resonance spectra of resting Co-QueD are indicative of a high-spin ( S = (3)/ 2) Co (2+) species in a tetrahedral or trigonal-bipyramidal coordination geometry. Anoxic binding of quercetin to QueD drastically altered the hyperfine pattern at g approximately 6 without changing the valence state of the Co(II) center and elicited a hypsochromic shift of UV-vis absorption band I of quercetin. On the basis of spectroscopic data, and considering the organic chemistry of flavonols, a nonredox role of the metal center in catalysis is discussed.
Circulation, Nov 22, 2011
J Biol Inorg Chem, 1998
CW ENDOR (X-band) spectra for the purple mixed-valence [Cu(1.5+)...Cu(1.5+)], S = 1/2, CuA site i... more CW ENDOR (X-band) spectra for the purple mixed-valence [Cu(1.5+)...Cu(1.5+)], S = 1/2, CuA site in nitrous oxide reductase were obtained after insertion of 65Cu or both 65Cu and 15N-histidine. The 14N/15N isotopic substitution allowed for an unambiguous deconvolution of proton and nitrogen hyperfine couplings in the spectra. A single nitrogen coupling with a value of 12.9 ± 0.4 MHz for 14N was detected. Its anisotropy
Biochemistry Usa, 1998
Based on its metallo-cofactor, the manganese-dependent ribonucleotide reductase (Mn-RRase) respon... more Based on its metallo-cofactor, the manganese-dependent ribonucleotide reductase (Mn-RRase) responsible for delivery of DNA precursors in the Mn-requiring Gram-positive bacterium Corynebacterium (formerly BreVibacterium) ammoniagenes ATCC 6872 is no longer considered as a simple analogue of the aerobic Fe-RRase of Escherichia coli but as the prototype of the class IV enzymes (1). Deliberate dissociation of the Mn-RRase holoenzyme and an improved sample preparation of the dimeric CA2 protein allowed further characterization of the inherent metallo-cofactor by Q-band electron paramagnetic resonance (EPR) spectroscopy. At 40 K, a distinct hyperfine sextet (I ) 5 / 2 , 55 Mn) pattern with a weak zero-field splitting was detected in the CA2 protein prepared from manganese-sufficient cells displaying high RRase activity as expected. This Q-band Mn(II) signal was absent in the apo-CA2 protein obtained from manganese-depleted cells devoid of this enzymatic activity. The presence of a mixed valence manganese cluster in the C. ammoniagenes RRase is excluded since no complex multiline EPR signals were detected in the CA2 protein even at very low (8 K) temperature. The observed Mn(II) spectrum indicates a proteinbound manganese which was modified in the presence of 5.7 mM p-methoxyphenol, but is insensitive toward 10 mM EDTA. Thus, the manganese appeared to be either strictly bound or buried within a hydrophobic pocket of the CA2 protein, inaccessible for EDTA.
For isoquinoline 1-oxidoreductase (IsoOr), the reaction mechanism under turnover conditions was s... more For isoquinoline 1-oxidoreductase (IsoOr), the reaction mechanism under turnover conditions was studied by EPR spectroscopy using rapid-freeze methods. IsoOr displays several EPR-active Mo(V) species including the "very rapid" component found also in xanthine oxidase (XanOx). For IsoOr, unlike XanOx or quinoline 2-oxidoreductase (QuinOr), this species is stable for about 1 h in the absence of an oxidizing substrate [Canne, C., Stephan, I., Finsterbusch, J., Lingens, F., Kappl, R., Fetzner, S., and Hüttermann, J. (1997) Biochemistry 36, 9780-9790]. Under rapid-freeze conditions in the presence of ferricyanide the very rapid species behaves as a kinetically competent intermediate present only during steady-state turnover. To explain the persistence of the very rapid species in IsoOr in the absence of an added oxidant, extremely slow product dissociation is required. This new finding that oxidative conditions facilitate decay of the very rapid signal for IsoOr supports the mechanism of substrate turnover proposed by Lowe, Richards, and Bray [Lowe, D. J., Richards, R. L., and Bray, R. C. (1997) Biochem. Soc. Trans. 25, 774-778]. Additional stopped-flow data reveal that alternative catalytic cycles occur in IsoOr and show that the product dissociates after transfer of a single oxidizing equivalent from ferricyanide. In rapid-freeze measurements magnetic interactions of the very rapid Mo(V) species and the iron-sulfur center FeSI of IsoOr and QuinOr were observed, proving that FeSI is located close to the molybdopterin cofactor in the two proteins. This finding is used to relate the two different iron-sulfur centers of the aldehyde oxidoreductase structure with the EPR-detectable FeS species of the enzymes.
Applied Magnetic Resonance, Oct 10, 2009
Applied Magnetic Resonance, Dec 1, 2001
Electron paramagnetic resonance (EPR) spectroscopy is performed on NO'-ligated hemoand myoglobin ... more Electron paramagnetic resonance (EPR) spectroscopy is performed on NO'-ligated hemoand myoglobin at temperatures between 5 and 300 K. Apart from the standard X-band (9.5 GHz), data are taken at K-(24 GHz), Q-(34 GHz), W-(94 GHz) and Y-band (285 GHz) frequencies, respectively. The spectra are delineated into contributions of two EPR signatures differing in g-tensor symmetry, an axial and a rhombic one. The state contributions are found to differ with temperature and the states show temperature-dependent g-factor variations. In MbNO the axial species (denoted state II) is the only species observed at 300 K with X-band. Analysis of its development with decreasing temperature shows a partial transformation into a rhombic state (I). For this, at an intermediate temperature range (240-100 K), irregular line shapes are apparent, indicating disordered geometries of the Fe-NO-heme group. Regular rhombic lineshapes develop increasingly at temperatures below about 70 K. At 5-10 K, the rhombic state I dominates the EPR spectra but state II is still present. A similar development of spectra is observed in HbNO, for which, however, a rhombic contribution is already present at 300 K. Subunit-associated variations in state contributions and EPR appearance are not resolved at any frequency. Also, lineshape irregularities with decreasing temperature are not manifest in HbNO. These findings are used to present a structure-based picture of states I and II and their transformations and to discuss the origin of the line shape irregularities in MbNO.
Metal Ions in Biological Systems, Feb 1, 2002
Febs Letters, 1991
X-irradiation of the tcrnsry complex of P45O:substrs1c:O, at 77 K products a rcduccd in1crmcdiatc... more X-irradiation of the tcrnsry complex of P45O:substrs1c:O, at 77 K products a rcduccd in1crmcdiatc by clcc1ron addition to 1hc Fc:O, complex which can bc studied by EPR-spcc1roscopy. The EPR spectrum or the new spccics exhibits rhombic symmctrg wi1h g-factors of 2.27. 2.17 and I .95. rcspcclivcly. lncrcasing the tcmpcraturc of 1hc sample IO I90 K rcsulls in loss of inlcnsity or the imcrmcdia1c signals. X-irradia1ion of oxymyoand oxyhemoglobin products similar EPR signals indicaling that ~hc added clcctron is rcsidcm on the Fc:O: complex (Kappl. R., CI al. (1985) Biochim. Biophys. Ac~a 870. 20-30).
Electron Paramagnetic Resonance, 2002
Page 321. 10 Iron Coordination in Metalloproteins: Structural and Electronic Aspects BYJURGEN HUT... more Page 321. 10 Iron Coordination in Metalloproteins: Structural and Electronic Aspects BYJURGEN HUTTERMANN AND REINHARD KAPPL There is still an increasing interest in the application of EPR and its high-resolution ...
Biochimica Et Biophysica Acta, Oct 1, 1988
site did not alter appreciably the pattern of the proton interactions. The t4N couplings of the n... more site did not alter appreciably the pattern of the proton interactions. The t4N couplings of the native specimen indicated equivalent coordination, whereas Zn(||) depletion and CN-addition were found to produce either some or drastic inequivalences, respectively. For N 3 addition to either the native or the Zn(ll)-depleted sample only minor effects on the respective 14N coupling pattern were observed.
J Am Chem Soc, 1999
The ENDOR response of 57Fe nuclei and protons of the high-potential iron-sulfur (HiPIP) protein i... more The ENDOR response of 57Fe nuclei and protons of the high-potential iron-sulfur (HiPIP) protein iso-II from Ectothiorhodospira halophila in frozen solutions, ie, on nonoriented systems, has been exploited to determine electronic and structural details of the oxidized [Fe4S4] 3+ cluster and its protein environment. Two distinct 57Fe hyperfine couplings were resolved and assigned to pairs of highly symmetric ferric and mixed-valence iron ions in agreement with results of Mössbauer and ENDOR studies on related proteins and model ...
Acta Physica Polonica A, 2010
This paper comprises the results obtained by continuous wave (cw)/pulse electron paramagnetic res... more This paper comprises the results obtained by continuous wave (cw)/pulse electron paramagnetic resonance spectroscopy for oxidised coals. The values of EPR linewidths are different for samples of coal treated by different chemical agents. The asymmetric EPR line shape indicates to complex composition of paramagnetic centres in coal. In coal oxidised by HNO 3 big changes of linewidth with temperature were observed. For all samples the spin-lattice relaxation time T 1 was determined by pulse EPR method. The correlation between used chemical agents and the value of T 1 was established.
Proceedings of the National Academy of Sciences of the United States of America, Sep 5, 2013
Fine-tuned regulation of K(+) channel inactivation enables excitable cells to adjust action poten... more Fine-tuned regulation of K(+) channel inactivation enables excitable cells to adjust action potential firing. Fast inactivation present in some K(+) channels is mediated by the distal N-terminal structure (ball) occluding the ion permeation pathway. Here we show that Kv1.4 K(+) channels are potently regulated by intracellular free heme; heme binds to the N-terminal inactivation domain and thereby impairs the inactivation process, thus enhancing the K(+) current with an apparent EC50 value of ∼20 nM. Functional studies on channel mutants and structural investigations on recombinant inactivation ball domain peptides encompassing the first 61 residues of Kv1.4 revealed a heme-responsive binding motif involving Cys13:His16 and a secondary histidine at position 35. Heme binding to the N-terminal inactivation domain induces a conformational constraint that prevents it from reaching its receptor site at the vestibule of the channel pore.
Metal Ions in Biological Systems, Feb 1, 2000
Biochemistry, Dec 1, 2004
1H-3-Hydroxy-4-oxoquinaldine 2,4-dioxygenase (Hod) is a cofactor-less dioxygenase belonging to th... more 1H-3-Hydroxy-4-oxoquinaldine 2,4-dioxygenase (Hod) is a cofactor-less dioxygenase belonging to the alpha/beta hydrolase fold family, catalyzing the cleavage of 1H-3-hydroxy-4-oxoquinaldine (I) and 1H-3-hydroxy-4-oxoquinoline (II) to N-acetyl- and N-formylanthranilate, respectively, and carbon monoxide. Bisubstrate steady-state kinetics and product inhibition patterns of HodC, the C69A protein variant of Hod, suggested a compulsory-order ternary-complex mechanism, in which binding of the organic substrate precedes dioxygen binding, and carbon monoxide is released first. The specificity constants, k(cat)/K(m,A) and k(cat)/K(m,O)()2, were 1.4 x 10(8) and 3.0 x 10(5) M(-1) s(-1) with I and 1.2 x 10(5) and 0.41 x 10(5) M(-1) s(-1) with II, respectively. Whereas HodC catalyzes formation of the dianion of its organic substrate prior to dioxygen binding, HodC-H251A does not, suggesting that H251, which aligns with the histidine of the catalytic triad of the alpha/beta hydrolases, acts as general base in catalysis. Investigation of base-catalyzed dioxygenolysis of I by electron paramagnetic resonance (EPR) spectroscopy revealed formation of a resonance-stabilized radical upon exposure to dioxygen. Since in D(2)O spectral properties are not affected, exchangeable protons are not involved, confirming that the dianion is the reactive intermediate that undergoes single-electron oxidation. We suggest that in the ternary complex of the enzyme, direct single-electron transfer from the substrate dianion to dioxygen may occur, resulting in a radical pair. Based on the estimated spin distribution within the radical anion (observed in the model reaction of I), radical recombination may produce a C4- or C2-hydroperoxy(di)anion. Subsequent intramolecular attack would result in the 2,4-endoperoxy (di)anion that may collapse to the reaction products.
J Biol Inorg Chem, 2002
Hydrons and electrons are substrates for the enzyme hydrogenase, but cannot be observed in X-ray ... more Hydrons and electrons are substrates for the enzyme hydrogenase, but cannot be observed in X-ray crystal structures. High-resolution 1H electron nuclear double resonance (ENDOR) spectroscopy offers a means to detect the distribution of protons and unpaired electrons. ENDOR spectra were recorded from frozen solutions of the nickel-iron hydrogenases of Desulfovibrio gigas and Desulfomicrobium baculatum, in the "active" state ("Ni-C" EPR signal) and analyzed by orientationally selective simulation methods. The experimental spectra were fitted using a structural model of the nickel-iron centre based on crystallographic results, allowing for differences in electron spin distribution as well as the spatial orientation of the g-matrix ( g-tensor), and anisotropic and isotropic hyperfine couplings of the protons nearest to the nickel ion. ENDOR signals, detected after complete deuterium exchange, were assigned to six protons of the cysteines bound to nickel. The assignment took advantage of the substitution of a selenium for a sulfur ligand, which occurs naturally between the [NiFeSe] and [NiFe] hydrogenases from Dm. baculatum and D. gigas, respectively, and was found to affect just two signals. The four signals with the largest hyperfine couplings, including isotropic contributions from 4.5 to 13.5 MHz, were assigned to the beta-methylene protons of the two terminal cysteine ligands, one of which is substituted by seleno-cysteine in [NiFeSe] hydrogenase. The electron spin is delocalized onto the nickel (50%) and its sulfur ligands, with a higher proportion on the terminal than the bridging ligands. The g-matrix was found to align with the active site in such a way that the g1- g2 plane is nearly coplanar (18.3 degrees) with the plane defined by nickel and three sulfur atoms, and the g2 axis deviates by 22.9 degrees from the vector between nickel and iron. Significantly for the reaction of the enzyme, direct evidence for the binding of hydrons at the active site was obtained by the detection of H/D-exchangeable ENDOR signals.
Appl Magn Reson, 2007
Electron paramagnetic resonance (EPR) and proton ENDOR (electron-nuclear double resonanee) spectr... more Electron paramagnetic resonance (EPR) and proton ENDOR (electron-nuclear double resonanee) spectroscopies were used to analyze the struetural and eleetronic parameters of the oxidized [Fe4S4] cubane clusters in high-potential iron sulfur proteins (HiPIPs) from Ectothiorhodospira halophila (HiPIP I) and Rhodocyclus tenuis. The E. halophila HiPIP I EPR spectra at X-and Q-band revealed a dominant species (simulated with gm~ = 2.1425, g,n, = 2.0315, gmm = 2.0296) anda minor speeies (ea. 5-10% contribution) which was not analyzed further. For R. tenuis HiPIP the EPR spectrum contained a single species only (g~x = 2.1140, g~,, = 2.0392, gm~, = 2.0215), i.e., with gmax significantly smaller than that of the E. halophila protein. Orientation-selected proton ENDOR spectra of HiPIP I of E. halophila were reconstructed by simulation with slight modifications of the crystal structure data. ENDOR from two mutants, F36S and F36G, of E. halophila HiPIP I gave evidente fora common assignment of a HB2 proton of a phenylalanine residue (36 and 44, respectively, in isoenzymes I and II as reported earlier) interacting with the mixed-valence pair iron ions Fe2 and Fe3. For R. tenuis HiPIE the ENDOR spectra were assigned to arise from Fe3 and Fe4 as mixedvalence pair under the assumption of ah unchanged intrinsic g-tensor symmetry. The resulting site specificity of the cubane oxidation was discussed in relation to structural requirements and redox potentials of the two HiPIPs.
Biochemistry Usa, Oct 1, 2008
Quercetinase (QueD) of Streptomyces sp. FLA is an enzyme of the monocupin family and catalyzes th... more Quercetinase (QueD) of Streptomyces sp. FLA is an enzyme of the monocupin family and catalyzes the 2,4-dioxygenolytic cleavage of the flavonol quercetin. After expression of the queD gene in Escherichia coli, high specific QueD activity was found in crude cell extracts when the growth medium was supplemented with NiCl 2 or CoCl 2, but not when Mn (2+), Fe (2+), Cu (2+), or Zn (2+) was added. The metal occupancy of Ni- and Co-QueD purified from these cells was </=50%, presumably due to strong overproduction of QueD in E. coli. Circular dichroism spectroscopy indicated the same folded structure with a high content of beta-sheet for the Ni and Co protein. The apparent kinetic constants for quercetin of Ni-QueD ( k cat = 40.1 s (-1), and K m = 5.75 microM) and Co-QueD ( k cat = 7.6 s (-1), and K m = 0.96 muM) indicate similar catalytic efficiencies; however, the approximately 5-fold lower apparent K m value of Ni-QueD for dioxygen suggests that the nickel enzyme performs better under physiological conditions. The pH dependence of k cat,app indicates that an ionizable group with a p K a near 6.8 has to be deprotonated for catalysis. Electron paramagnetic resonance spectra of resting Co-QueD are indicative of a high-spin ( S = (3)/ 2) Co (2+) species in a tetrahedral or trigonal-bipyramidal coordination geometry. Anoxic binding of quercetin to QueD drastically altered the hyperfine pattern at g approximately 6 without changing the valence state of the Co(II) center and elicited a hypsochromic shift of UV-vis absorption band I of quercetin. On the basis of spectroscopic data, and considering the organic chemistry of flavonols, a nonredox role of the metal center in catalysis is discussed.
Circulation, Nov 22, 2011
J Biol Inorg Chem, 1998
CW ENDOR (X-band) spectra for the purple mixed-valence [Cu(1.5+)...Cu(1.5+)], S = 1/2, CuA site i... more CW ENDOR (X-band) spectra for the purple mixed-valence [Cu(1.5+)...Cu(1.5+)], S = 1/2, CuA site in nitrous oxide reductase were obtained after insertion of 65Cu or both 65Cu and 15N-histidine. The 14N/15N isotopic substitution allowed for an unambiguous deconvolution of proton and nitrogen hyperfine couplings in the spectra. A single nitrogen coupling with a value of 12.9 ± 0.4 MHz for 14N was detected. Its anisotropy
Biochemistry Usa, 1998
Based on its metallo-cofactor, the manganese-dependent ribonucleotide reductase (Mn-RRase) respon... more Based on its metallo-cofactor, the manganese-dependent ribonucleotide reductase (Mn-RRase) responsible for delivery of DNA precursors in the Mn-requiring Gram-positive bacterium Corynebacterium (formerly BreVibacterium) ammoniagenes ATCC 6872 is no longer considered as a simple analogue of the aerobic Fe-RRase of Escherichia coli but as the prototype of the class IV enzymes (1). Deliberate dissociation of the Mn-RRase holoenzyme and an improved sample preparation of the dimeric CA2 protein allowed further characterization of the inherent metallo-cofactor by Q-band electron paramagnetic resonance (EPR) spectroscopy. At 40 K, a distinct hyperfine sextet (I ) 5 / 2 , 55 Mn) pattern with a weak zero-field splitting was detected in the CA2 protein prepared from manganese-sufficient cells displaying high RRase activity as expected. This Q-band Mn(II) signal was absent in the apo-CA2 protein obtained from manganese-depleted cells devoid of this enzymatic activity. The presence of a mixed valence manganese cluster in the C. ammoniagenes RRase is excluded since no complex multiline EPR signals were detected in the CA2 protein even at very low (8 K) temperature. The observed Mn(II) spectrum indicates a proteinbound manganese which was modified in the presence of 5.7 mM p-methoxyphenol, but is insensitive toward 10 mM EDTA. Thus, the manganese appeared to be either strictly bound or buried within a hydrophobic pocket of the CA2 protein, inaccessible for EDTA.
For isoquinoline 1-oxidoreductase (IsoOr), the reaction mechanism under turnover conditions was s... more For isoquinoline 1-oxidoreductase (IsoOr), the reaction mechanism under turnover conditions was studied by EPR spectroscopy using rapid-freeze methods. IsoOr displays several EPR-active Mo(V) species including the "very rapid" component found also in xanthine oxidase (XanOx). For IsoOr, unlike XanOx or quinoline 2-oxidoreductase (QuinOr), this species is stable for about 1 h in the absence of an oxidizing substrate [Canne, C., Stephan, I., Finsterbusch, J., Lingens, F., Kappl, R., Fetzner, S., and Hüttermann, J. (1997) Biochemistry 36, 9780-9790]. Under rapid-freeze conditions in the presence of ferricyanide the very rapid species behaves as a kinetically competent intermediate present only during steady-state turnover. To explain the persistence of the very rapid species in IsoOr in the absence of an added oxidant, extremely slow product dissociation is required. This new finding that oxidative conditions facilitate decay of the very rapid signal for IsoOr supports the mechanism of substrate turnover proposed by Lowe, Richards, and Bray [Lowe, D. J., Richards, R. L., and Bray, R. C. (1997) Biochem. Soc. Trans. 25, 774-778]. Additional stopped-flow data reveal that alternative catalytic cycles occur in IsoOr and show that the product dissociates after transfer of a single oxidizing equivalent from ferricyanide. In rapid-freeze measurements magnetic interactions of the very rapid Mo(V) species and the iron-sulfur center FeSI of IsoOr and QuinOr were observed, proving that FeSI is located close to the molybdopterin cofactor in the two proteins. This finding is used to relate the two different iron-sulfur centers of the aldehyde oxidoreductase structure with the EPR-detectable FeS species of the enzymes.
Applied Magnetic Resonance, Oct 10, 2009
Applied Magnetic Resonance, Dec 1, 2001
Electron paramagnetic resonance (EPR) spectroscopy is performed on NO'-ligated hemoand myoglobin ... more Electron paramagnetic resonance (EPR) spectroscopy is performed on NO'-ligated hemoand myoglobin at temperatures between 5 and 300 K. Apart from the standard X-band (9.5 GHz), data are taken at K-(24 GHz), Q-(34 GHz), W-(94 GHz) and Y-band (285 GHz) frequencies, respectively. The spectra are delineated into contributions of two EPR signatures differing in g-tensor symmetry, an axial and a rhombic one. The state contributions are found to differ with temperature and the states show temperature-dependent g-factor variations. In MbNO the axial species (denoted state II) is the only species observed at 300 K with X-band. Analysis of its development with decreasing temperature shows a partial transformation into a rhombic state (I). For this, at an intermediate temperature range (240-100 K), irregular line shapes are apparent, indicating disordered geometries of the Fe-NO-heme group. Regular rhombic lineshapes develop increasingly at temperatures below about 70 K. At 5-10 K, the rhombic state I dominates the EPR spectra but state II is still present. A similar development of spectra is observed in HbNO, for which, however, a rhombic contribution is already present at 300 K. Subunit-associated variations in state contributions and EPR appearance are not resolved at any frequency. Also, lineshape irregularities with decreasing temperature are not manifest in HbNO. These findings are used to present a structure-based picture of states I and II and their transformations and to discuss the origin of the line shape irregularities in MbNO.
Metal Ions in Biological Systems, Feb 1, 2002
Febs Letters, 1991
X-irradiation of the tcrnsry complex of P45O:substrs1c:O, at 77 K products a rcduccd in1crmcdiatc... more X-irradiation of the tcrnsry complex of P45O:substrs1c:O, at 77 K products a rcduccd in1crmcdiatc by clcc1ron addition to 1hc Fc:O, complex which can bc studied by EPR-spcc1roscopy. The EPR spectrum or the new spccics exhibits rhombic symmctrg wi1h g-factors of 2.27. 2.17 and I .95. rcspcclivcly. lncrcasing the tcmpcraturc of 1hc sample IO I90 K rcsulls in loss of inlcnsity or the imcrmcdia1c signals. X-irradia1ion of oxymyoand oxyhemoglobin products similar EPR signals indicaling that ~hc added clcctron is rcsidcm on the Fc:O: complex (Kappl. R., CI al. (1985) Biochim. Biophys. Ac~a 870. 20-30).
Electron Paramagnetic Resonance, 2002
Page 321. 10 Iron Coordination in Metalloproteins: Structural and Electronic Aspects BYJURGEN HUT... more Page 321. 10 Iron Coordination in Metalloproteins: Structural and Electronic Aspects BYJURGEN HUTTERMANN AND REINHARD KAPPL There is still an increasing interest in the application of EPR and its high-resolution ...
Biochimica Et Biophysica Acta, Oct 1, 1988
site did not alter appreciably the pattern of the proton interactions. The t4N couplings of the n... more site did not alter appreciably the pattern of the proton interactions. The t4N couplings of the native specimen indicated equivalent coordination, whereas Zn(||) depletion and CN-addition were found to produce either some or drastic inequivalences, respectively. For N 3 addition to either the native or the Zn(ll)-depleted sample only minor effects on the respective 14N coupling pattern were observed.
J Am Chem Soc, 1999
The ENDOR response of 57Fe nuclei and protons of the high-potential iron-sulfur (HiPIP) protein i... more The ENDOR response of 57Fe nuclei and protons of the high-potential iron-sulfur (HiPIP) protein iso-II from Ectothiorhodospira halophila in frozen solutions, ie, on nonoriented systems, has been exploited to determine electronic and structural details of the oxidized [Fe4S4] 3+ cluster and its protein environment. Two distinct 57Fe hyperfine couplings were resolved and assigned to pairs of highly symmetric ferric and mixed-valence iron ions in agreement with results of Mössbauer and ENDOR studies on related proteins and model ...
Acta Physica Polonica A, 2010
This paper comprises the results obtained by continuous wave (cw)/pulse electron paramagnetic res... more This paper comprises the results obtained by continuous wave (cw)/pulse electron paramagnetic resonance spectroscopy for oxidised coals. The values of EPR linewidths are different for samples of coal treated by different chemical agents. The asymmetric EPR line shape indicates to complex composition of paramagnetic centres in coal. In coal oxidised by HNO 3 big changes of linewidth with temperature were observed. For all samples the spin-lattice relaxation time T 1 was determined by pulse EPR method. The correlation between used chemical agents and the value of T 1 was established.
Proceedings of the National Academy of Sciences of the United States of America, Sep 5, 2013
Fine-tuned regulation of K(+) channel inactivation enables excitable cells to adjust action poten... more Fine-tuned regulation of K(+) channel inactivation enables excitable cells to adjust action potential firing. Fast inactivation present in some K(+) channels is mediated by the distal N-terminal structure (ball) occluding the ion permeation pathway. Here we show that Kv1.4 K(+) channels are potently regulated by intracellular free heme; heme binds to the N-terminal inactivation domain and thereby impairs the inactivation process, thus enhancing the K(+) current with an apparent EC50 value of ∼20 nM. Functional studies on channel mutants and structural investigations on recombinant inactivation ball domain peptides encompassing the first 61 residues of Kv1.4 revealed a heme-responsive binding motif involving Cys13:His16 and a secondary histidine at position 35. Heme binding to the N-terminal inactivation domain induces a conformational constraint that prevents it from reaching its receptor site at the vestibule of the channel pore.
Metal Ions in Biological Systems, Feb 1, 2000
Biochemistry, Dec 1, 2004
1H-3-Hydroxy-4-oxoquinaldine 2,4-dioxygenase (Hod) is a cofactor-less dioxygenase belonging to th... more 1H-3-Hydroxy-4-oxoquinaldine 2,4-dioxygenase (Hod) is a cofactor-less dioxygenase belonging to the alpha/beta hydrolase fold family, catalyzing the cleavage of 1H-3-hydroxy-4-oxoquinaldine (I) and 1H-3-hydroxy-4-oxoquinoline (II) to N-acetyl- and N-formylanthranilate, respectively, and carbon monoxide. Bisubstrate steady-state kinetics and product inhibition patterns of HodC, the C69A protein variant of Hod, suggested a compulsory-order ternary-complex mechanism, in which binding of the organic substrate precedes dioxygen binding, and carbon monoxide is released first. The specificity constants, k(cat)/K(m,A) and k(cat)/K(m,O)()2, were 1.4 x 10(8) and 3.0 x 10(5) M(-1) s(-1) with I and 1.2 x 10(5) and 0.41 x 10(5) M(-1) s(-1) with II, respectively. Whereas HodC catalyzes formation of the dianion of its organic substrate prior to dioxygen binding, HodC-H251A does not, suggesting that H251, which aligns with the histidine of the catalytic triad of the alpha/beta hydrolases, acts as general base in catalysis. Investigation of base-catalyzed dioxygenolysis of I by electron paramagnetic resonance (EPR) spectroscopy revealed formation of a resonance-stabilized radical upon exposure to dioxygen. Since in D(2)O spectral properties are not affected, exchangeable protons are not involved, confirming that the dianion is the reactive intermediate that undergoes single-electron oxidation. We suggest that in the ternary complex of the enzyme, direct single-electron transfer from the substrate dianion to dioxygen may occur, resulting in a radical pair. Based on the estimated spin distribution within the radical anion (observed in the model reaction of I), radical recombination may produce a C4- or C2-hydroperoxy(di)anion. Subsequent intramolecular attack would result in the 2,4-endoperoxy (di)anion that may collapse to the reaction products.
J Biol Inorg Chem, 2002
Hydrons and electrons are substrates for the enzyme hydrogenase, but cannot be observed in X-ray ... more Hydrons and electrons are substrates for the enzyme hydrogenase, but cannot be observed in X-ray crystal structures. High-resolution 1H electron nuclear double resonance (ENDOR) spectroscopy offers a means to detect the distribution of protons and unpaired electrons. ENDOR spectra were recorded from frozen solutions of the nickel-iron hydrogenases of Desulfovibrio gigas and Desulfomicrobium baculatum, in the "active" state ("Ni-C" EPR signal) and analyzed by orientationally selective simulation methods. The experimental spectra were fitted using a structural model of the nickel-iron centre based on crystallographic results, allowing for differences in electron spin distribution as well as the spatial orientation of the g-matrix ( g-tensor), and anisotropic and isotropic hyperfine couplings of the protons nearest to the nickel ion. ENDOR signals, detected after complete deuterium exchange, were assigned to six protons of the cysteines bound to nickel. The assignment took advantage of the substitution of a selenium for a sulfur ligand, which occurs naturally between the [NiFeSe] and [NiFe] hydrogenases from Dm. baculatum and D. gigas, respectively, and was found to affect just two signals. The four signals with the largest hyperfine couplings, including isotropic contributions from 4.5 to 13.5 MHz, were assigned to the beta-methylene protons of the two terminal cysteine ligands, one of which is substituted by seleno-cysteine in [NiFeSe] hydrogenase. The electron spin is delocalized onto the nickel (50%) and its sulfur ligands, with a higher proportion on the terminal than the bridging ligands. The g-matrix was found to align with the active site in such a way that the g1- g2 plane is nearly coplanar (18.3 degrees) with the plane defined by nickel and three sulfur atoms, and the g2 axis deviates by 22.9 degrees from the vector between nickel and iron. Significantly for the reaction of the enzyme, direct evidence for the binding of hydrons at the active site was obtained by the detection of H/D-exchangeable ENDOR signals.
Appl Magn Reson, 2007
Electron paramagnetic resonance (EPR) and proton ENDOR (electron-nuclear double resonanee) spectr... more Electron paramagnetic resonance (EPR) and proton ENDOR (electron-nuclear double resonanee) spectroscopies were used to analyze the struetural and eleetronic parameters of the oxidized [Fe4S4] cubane clusters in high-potential iron sulfur proteins (HiPIPs) from Ectothiorhodospira halophila (HiPIP I) and Rhodocyclus tenuis. The E. halophila HiPIP I EPR spectra at X-and Q-band revealed a dominant species (simulated with gm~ = 2.1425, g,n, = 2.0315, gmm = 2.0296) anda minor speeies (ea. 5-10% contribution) which was not analyzed further. For R. tenuis HiPIP the EPR spectrum contained a single species only (g~x = 2.1140, g~,, = 2.0392, gm~, = 2.0215), i.e., with gmax significantly smaller than that of the E. halophila protein. Orientation-selected proton ENDOR spectra of HiPIP I of E. halophila were reconstructed by simulation with slight modifications of the crystal structure data. ENDOR from two mutants, F36S and F36G, of E. halophila HiPIP I gave evidente fora common assignment of a HB2 proton of a phenylalanine residue (36 and 44, respectively, in isoenzymes I and II as reported earlier) interacting with the mixed-valence pair iron ions Fe2 and Fe3. For R. tenuis HiPIE the ENDOR spectra were assigned to arise from Fe3 and Fe4 as mixedvalence pair under the assumption of ah unchanged intrinsic g-tensor symmetry. The resulting site specificity of the cubane oxidation was discussed in relation to structural requirements and redox potentials of the two HiPIPs.