The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform

Ann F Walker - One of the best experts on this subject based on the ideXlab platform.

  • determination of the principal g values of type i or highly anisotropic low spin hals Ferriheme centers in frozen solutions
    Journal of Magnetic Resonance, 2013
    Co-Authors: Andrei V Astashkin, Ann F Walker
    Abstract:

    Abstract Continuous wave (CW) electron paramagnetic resonance (EPR) spectroscopy of highly-anisotropic low spin (HALS) ferric heme centers in frozen solutions is not a very informative approach because usually only one feature is reliably observed in the spectra, that at the maximal principal g-value of, typically, 3.3–3.79. The other two EPR turning points are severely broadened by g-strain and are not easily observed in the first-derivative CW EPR spectra. In this work, we have explored the potential of alternative EPR techniques, the electron spin echo (ESE) field sweep and electron spin transient nutation (TN), for obtaining information about the g-tensors of such systems, using as an example a typical HALS ferric heme center, [FeIII(15N-coproporphyrin)(CN)2]. The analysis of the experimental g-tensor of [FeIII(15N-coproporphyrin)(CN)2]− has shown that the widths of the underlying energy distributions for this HALS center are comparable to those found for the rhombic bis-imidazole complex. The greater effect on the g-value distributions for HALS centers is determined by near degeneracy of two of the three lower-energy d-orbitals, dyz and dxz, which contain the unpaired electron.

  • nuclear inelastic scattering and mossbauer spectroscopy as local probes for ligand binding modes and electronic properties in proteins vibrational behavior of a Ferriheme center inside a β barrel protein
    Journal of the American Chemical Society, 2012
    Co-Authors: Beate Moeser, Hongjun Zhang, Ann F Walker, Robert E Berry, Hauke Paulsen, Adam Janoschka, Juliusz A Wolny, Igor Filippov, Aleksandr I Chumakov, Volker Schunemann
    Abstract:

    In this work, we present a study of the influence of the protein matrix on its ability to tune the binding of small ligands such as NO, cyanide (CN(-)), and histamine to the ferric heme iron center in the NO-storage and -transport protein Nitrophorin 2 (NP2) from the salivary glands of the blood-sucking insect Rhodnius prolixus. Conventional Mossbauer spectroscopy shows a diamagnetic ground state of the NP2-NO complex and Type I and II electronic ground states of the NP2-CN(-) and NP2-histamine complex, respectively. The change in the vibrational signature of the protein upon ligand binding has been monitored by Nuclear Inelastic Scattering (NIS), also called Nuclear Resonant Vibrational Spectroscopy (NRVS). The NIS data thus obtained have also been calculated by quantum mechanical (QM) density functional theory (DFT) coupled with molecular mechanics (MM) methods. The calculations presented here show that the heme ruffling in NP2 is a consequence of the interaction with the protein matrix. Structure optimizations of the heme and its ligands with DFT retain the characteristic saddling and ruffling only if the protein matrix is taken into account. Furthermore, simulations of the NIS data by QM/MM calculations suggest that the pH dependence of the binding of NO, but not of CN(-) and histamine, might be a consequence of the protonation state of the heme carboxyls.

  • linear correlation between 1h and 13c chemical shifts of Ferriheme proteins and model Ferrihemes
    Inorganic Chemistry, 2011
    Co-Authors: Fei Yang, Tatiana K Shokhireva, Ann F Walker
    Abstract:

    The (1)H{(13)C} HMQC experiment at natural-abundance (13)C provides a very useful way of determining not only (1)H but also (13)C chemical shifts of most heme substituents, without isotopic labeling of the hemin. This is true both in model low-spin Ferriheme complexes and in low-spin Ferriheme proteins, even when the proton resonances are buried in the protein diamagnetic region, because the carbon shifts are much larger than the proton shifts. In addition, in many cases, the protohemin methyl cross peaks are fairly linearly related to each other, with the slope of the correlation, δ(C)/δ(H), being approximately -2.0 for most low-spin Ferriheme proteins. The reasons why this should be the case, and when it is not, are discussed.

  • 1h and 13c nmr spectroscopic studies of the Ferriheme resonances of three low spin complexes of wild type nitrophorin 2 and nitrophorin 2 v24e as a function of ph
    Journal of Biological Inorganic Chemistry, 2009
    Co-Authors: Fei Yang, Hongjun Zhang, Markus Knipp, Tatiana K Shokhireva, Robert E Berry, Ann F Walker
    Abstract:

    The Ferriheme resonances of the low-spin (S = 1/2) complexes of wild-type (wt) nitrophorin 2 (NP2) and its heme pocket mutant NP2(V24E) with imidazole (ImH), histamine (Hm), and cyanide (CN−) as the sixth ligand have been investigated by NMR spectroscopy as a function of pH (4.0–7.5). For the three wt NP2 complexes, the ratio of the two possible heme orientational isomers, A and B, remains almost unchanged (ratio of A:B approximately 1:6 to 1:5) over this wide pH range. However, strong chemical exchange cross peaks appear in the nuclear Overhauser effect spectroscopy/exchange spectroscopy (NOESY/EXSY) spectra for the heme methyl resonances at low pH (pH* 4.0–5.5), which indicate chemical exchange between two species. We have shown these to be two different exogenous ImH or Hm orientations that are denoted B and B′, with the ImH plane nearly parallel and perpendicular to the ImH plane of the protein-provided His57, respectively. The wt NP2–CN complex also shows EXSY cross peaks due to chemical exchange, which is shown to be a result of interchange between two ruffling distortions of the heme. The same ruffling distortion interchange is also responsible for the ImH and Hm chemical exchange. For the three NP2(V24E) ligand complexes, no EXSY cross peaks are observed, but the A:B ratios change dramatically with pH. The fact that heme favors the A orientation highly for NP2(V24E) at low pH as compared with wt NP2 is believed to be due to the steric effect of the V24E mutation. The existence of the B′ species at lower pH for wt NP2 complexes and the increase in A heme orientation at lower pH for NP2(V24E) are believed to be a result of a change in structure near Glu53 when it is protonated at low pH. 1H{13C} heteronuclear multiple quantum coherence (HMQC) spectra are very helpful for the assignment of heme and nearby protein side chain resonances. Electronic supplementary material The online version of this article (doi:10.1007/s00775-009-0551-3) contains supplementary material, which is available to authorized users.

  • assignment of Ferriheme resonances for high and low spin forms of nitrophorin 3 by 1h and 13c nmr spectroscopy and comparison to nitrophorin 2 heme pocket structural similarities and differences
    Inorganica Chimica Acta, 2008
    Co-Authors: Tatiana K Shokhireva, Hongjun Zhang, Robert E Berry, Nikolai V Shokhirev, Ann F Walker
    Abstract:

    Nitrophorin 3 (NP3) is the only one of the four major NO-binding heme proteins found in the saliva of the blood-sucking insect Rhodnius prolixus (also called the Kissing Bug) for which it has not been possible to obtain crystals of diffraction quality for structure determination by X-ray crystallography. Thus we have used NMR spectroscopy, mainly of the hyperfine-shifted Ferriheme substituent resonances, to learn about the similarities and differences in the heme pocket and the iron active site of NP3 as compared to NP2, which has previously been well-characterized by both X-ray crystallography and NMR spectroscopy. Only one residue in the heme pocket differs between the two, F27 of NP2 is Y27 for NP3; in both cases this residue is expected to interact strongly with the 2-vinyl side chain of the B heme rotational isomer or the 4-vinyl of the A heme rotational isomer. Both the high-spin (S = 5/2) aquo complex, NP3-H(2)O, and the low-spin (S = 1/2) N-methylimidazole (NMeIm) complex of NP3 have been studied. It is found that the chemical shifts of the protons of both forms are similar to those of the corresponding NP2 complexes, but with minor differences that indicate a slightly different angle for the proximal histidine (H57) ligand plane. The B heme rotational isomer is preferred by both NP3 and NP2 in both spin states, but to a greater extent when phenylalanine is present at position 27 (A:B = 1:8 for NP2, 1:6 for NP3-Y27F, 1:4 for NP3, and 1:3 for NP2-F27Y). Careful analysis of the 5Me and 8Me shifts of the A and B isomers of the two high-spin nitrophorins leads to the conclusion that the heme environment for the two isomers differs in some way that cannot be explained at the present time. The kinetics of deprotonation of the high-spin complexes of NP2 and NP3 are very different, with NP2 giving well-resolved high-spin aquo and "low-spin" hydroxo proton NMR spectra until close to the end of the titration, while NP3 exhibits broadened (1)H NMR spectra indicative of an intermediate rate of exchange on the NMR timescale between the two forms throughout the titration. The heme methyl shifts of NP2-OH are similar in magnitude and spread to those of NP2-CN, while those of metmyoglobin-hydroxo complexes are much larger in magnitude but not spread. It is concluded that the hydroxo complex of NP2 is likely S = 1/2 with a mixed (d(XY))(2)(d(XZ),d(YZ))(3)/(d(xy))(1)(d(xz),d(yz))(4) electron configuration, while those of met-Mb-OH are likely S = 1/2,3/2 mixed spin systems.

Brian M. Hoffman - One of the best experts on this subject based on the ideXlab platform.

  • Role of the Proximal Cysteine Hydrogen Bonding Interaction in Cytochrome P450 2B4 Studied by Cryoreduction, Electron Paramagnetic Resonance, and Electron-Nuclear Double Resonance Spectroscopy.
    Biochemistry, 2016
    Co-Authors: Roman Davydov, Muralidharan Shanmugam, William A. Gunderson, Naw May Pearl, Brian M. Hoffman
    Abstract:

    Crystallographic studies have shown that the F429H mutation of cytochrome P450 2B4 introduces an H-bond between His429 and the proximal thiolate ligand, Cys436, without altering the protein fold but sharply decreases the enzymatic activity and stabilizes the oxyferrous P450 2B4 complex. To characterize the influence of this hydrogen bond on the states of the catalytic cycle, we have used radiolytic cryoreduction combined with electron paramagnetic resonance (EPR) and (electron–nuclear double resonance (ENDOR) spectroscopy to study and compare their characteristics for wild-type (WT) P450 2B4 and the F429H mutant. (i) The addition of an H-bond to the axial Cys436 thiolate significantly changes the EPR signals of both low-spin and high-spin heme-iron(III) and the hyperfine couplings of the heme-pyrrole 14N but has relatively little effect on the 1H ENDOR spectra of the water ligand in the six-coordinate low-spin Ferriheme state. These changes indicate that the H-bond introduced between His and the proximal ...

  • Role of the Proximal Cysteine Hydrogen Bonding Interaction in Cytochrome P450 2B4 Studied by Cryoreduction, Electron Paramagnetic Resonance, and Electron–Nuclear Double Resonance Spectroscopy
    2016
    Co-Authors: Roman Davydov, Brian M. Hoffman, Muralidharan Shanmugam, Naw May Pearl, William A. Gunderson, Lucy Waskell
    Abstract:

    Crystallographic studies have shown that the F429H mutation of cytochrome P450 2B4 introduces an H-bond between His429 and the proximal thiolate ligand, Cys436, without altering the protein fold but sharply decreases the enzymatic activity and stabilizes the oxyferrous P450 2B4 complex. To characterize the influence of this hydrogen bond on the states of the catalytic cycle, we have used radiolytic cryoreduction combined with electron paramagnetic resonance (EPR) and (electron–nuclear double resonance (ENDOR) spectroscopy to study and compare their characteristics for wild-type (WT) P450 2B4 and the F429H mutant. (i) The addition of an H-bond to the axial Cys436 thiolate significantly changes the EPR signals of both low-spin and high-spin heme-iron­(III) and the hyperfine couplings of the heme-pyrrole 14N but has relatively little effect on the 1H ENDOR spectra of the water ligand in the six-coordinate low-spin Ferriheme state. These changes indicate that the H-bond introduced between His and the proximal cysteine decreases the extent of S → Fe electron donation and weakens the Fe­(III)–S bond. (ii) The added H-bond changes the primary product of cryoreduction of the Fe­(II) enzyme, which is trapped in the conformation of the parent Fe­(II) state. In the wild-type enzyme, the added electron localizes on the porphyrin, generating an S = 3/2 state with the anion radical exchange-coupled to the Fe­(II). In the mutant, it localizes on the iron, generating an S = 1/2 Fe­(I) state. (iii) The additional H-bond has little effect on g values and 1H–14N hyperfine couplings of the cryogenerated, ferric hydroperoxo intermediate but noticeably slows its decay during cryoannealing. (iv) In both the WT and the mutant enzyme, this decay shows a significant solvent kinetic isotope effect, indicating that the decay reflects a proton-assisted conversion to Compound I (Cpd I). (v) We confirm that Cpd I formed during the annealing of the cryogenerated hydroperoxy intermediate and that it is the active hydroxylating species in both WT P450 2B4 and the F429H mutant. (vi) Our data also indicate that the added H-bond of the mutation diminishes the reactivity of Cpd I

  • compound i is the reactive intermediate in the first monooxygenation step during conversion of cholesterol to pregnenolone by cytochrome p450scc epr endor cryoreduction annealing studies
    Journal of the American Chemical Society, 2012
    Co-Authors: Roman Davydov, A A Gilep, N V Strushkevich, Sergey A Usanov, Brian M. Hoffman
    Abstract:

    Cytochrome P450scc (CYP11A1) catalyzes conversion of cholesterol (CH) to pregnenolone, the precursor to all steroid hormones. This process proceeds via three sequential monooxygenation reactions: two stereospecific hydroxylations with formation first of 22R-hydroxycholesterol (22-HC) and then 20α,22R-dihydroxycholesterol (20,22-DHC), followed by the C20-C22 bond cleavage. Herein we have employed EPR and ENDOR spectroscopy to characterize the intermediates in the first hydroxylation step by 77K radiolytic one-electron cryoreduction and subsequent annealing of the ternary oxy cytochrome P450scc-cholesterol complex. This approach is fully validated by the demonstration that the cryoreduced ternary complex of oxy-P450scc-CH is catalytically competent and hydroxylates cholesterol to form 22R-HC with no detectable formation of 20-HC, just as occurs under physiological conditions. Cryoreduction of the ternary complex trapped at 77K produces predominantly the hydroperoxy-Ferriheme P450scc intermediate, along with a minor fraction of peroxo-Ferriheme intermediate that converts into a new hydroperoxo-Ferriheme species at 145K. This behavior reveals that the distal pocket of the parent oxy-P450scc-cholesterol complex exhibits an efficient proton delivery network, with an ordered water molecule H-bonded to the distal oxygen of the dioxygen ligand. During annealing of the hydroperoxy-ferric P450scc intermediates at 185K they convert to the primary product complex in which CH has been converted to 22-HC. In this process, the hydroperoxy-ferric intermediate decays with a large sKIE, as expected when proton delivery to the terminal O leads to formation of Compound I (Cpd I). 1H ENDOR measurements of the primary product formed in deuterated solvent show that the heme Fe(III) is coordinated to the 22R-O1H of 22-HC, where the 1H is derived from substrate and exchanges to D after annealing at higher temperatures. These observations establish that Cpd I is agent that hydroxylates CH, rather than the hydroperoxy-ferric heme.

  • Compound I Is the Reactive Intermediate in the First Monooxygenation Step during Conversion of Cholesterol to Pregnenolone by Cytochrome P450scc: EPR/ENDOR/Cryoreduction/Annealing Studies
    2012
    Co-Authors: Roman Davydov, A A Gilep, N V Strushkevich, Sergey A Usanov, Brian M. Hoffman
    Abstract:

    Cytochrome P450scc (CYP11A1) catalyzes conversion of cholesterol (CH) to pregnenolone, the precursor to all steroid hormones. This process proceeds via three sequential monooxygenation reactions: two stereospecific hydroxylations with formation first of 22R-hydroxycholesterol (22-HC) and then 20α,22R-dihydroxycholesterol (20,22-DHC), followed by C20–C22 bond cleavage. Herein we have employed EPR and ENDOR spectroscopy to characterize the intermediates in the first hydroxylation step by 77 K radiolytic one-electron cryoreduction and subsequent annealing of the ternary oxy-cytochrome P450scc-cholesterol complex. This approach is fully validated by the demonstration that the cryoreduced ternary complex of oxy-P450scc-CH is catalytically competent and hydroxylates cholesterol to form 22-HC with no detectable formation of 20-HC, just as occurs under physiological conditions. Cryoreduction of the ternary complex trapped at 77 K produces predominantly the hydroperoxy-Ferriheme P450scc intermediate, along with a minor fraction of peroxo-Ferriheme intermediate that converts into a new hydroperoxo-Ferriheme species at 145 K. This behavior reveals that the distal pocket of the parent oxy-P450scc-cholesterol complex exhibits an efficient proton delivery network, with an ordered water molecule H-bonded to the distal oxygen of the dioxygen ligand. During annealing of the hydroperoxy-ferric P450scc intermediates at 185 K, they convert to the primary product complex in which CH has been converted to 22-HC. In this process, the hydroperoxy-ferric intermediate decays with a large solvent kinetic isotope effect, as expected when proton delivery to the terminal O leads to formation of Compound I (Cpd I). 1H ENDOR measurements of the primary product formed in deuterated solvent show that the heme Fe­(III) is coordinated to the 22R-O1H of 22-HC, where the 1H is derived from substrate and exchanges to D after annealing at higher temperatures. These observations establish that Cpd I is the agent that hydroxylates CH, rather than the hydroperoxy-ferric heme

  • electrostatic redesign of the myoglobin cytochrome b5 interface to create a well defined docked complex with rapid interprotein electron transfer
    Journal of the American Chemical Society, 2009
    Co-Authors: Peng Xiong, Judith M Nocek, Amanda K K Griffin, Jingyun Wang, Brian M. Hoffman
    Abstract:

    Cyt b5 is the electron-carrier “repair” protein that reduces met-Mb and met-Hb to their O2-carrying ferroheme forms. Studies of electron transfer (ET) between Mb and cyt b5 revealed that they react on a “Dynamic Docking” (DD) energy landscape on which binding and reactivity are uncoupled: binding is weak and involves an ensemble of nearly isoenergetic configurations, only a few of which are reactive; those few contribute negligibly to binding. We set the task of redesigning the surface of Mb so that its reaction with cyt b5 instead would occur on a conventional “simple docking” (SD) energy landscape, on which a complex exhibits a well-defined (set of) reactive binding configuration(s), with binding and reactivity thus no longer being decoupled. We prepared a myoglobin (Mb) triple mutant (D44K/D60K/E85K; Mb(+6)) substituted with Zn-deuteroporphyrin and monitored cytochrome b5 (cyt b5) binding and electron transfer (ET) quenching of the 3ZnMb(+6) triplet state. In contrast, to Mb(WT), the three charge rever...

Markus Knipp - One of the best experts on this subject based on the ideXlab platform.

  • Expression, Purification, and Solid-State NMR Characterization of the Membrane Binding Heme Protein Nitrophorin 7 in Two Electronic Spin States
    2016
    Co-Authors: Sabu Varghese, Fei Yang, Markus Knipp, Hideaki Ogata, Victor Pacheco, Kathrin Wrede, Alexander Medvedev, Henrike Heise
    Abstract:

    The nitrophorins (NPs) comprise a group of NO transporting Ferriheme b proteins found in the saliva of the blood sucking insect Rhodnius prolixus. In contrast to other nitrophorins (NP1–4), the recently identified membrane binding isoform NP7 tends to form oligomers and precipitates at higher concentrations in solution. Hence, solid-state NMR (ssNMR) was employed as an alternative method to gain structural insights on the precipitated protein. We report the expression and purification of 13C,15N isotopically labeled protein together with the first ssNMR characterization of NP7. Because the size of NP7 (21 kDa) still provides a challenge for ssNMR, the samples were reverse labeled with Lys and Val to reduce the number of crosspeaks in two-dimensional spectra. The two electronic spin states with S = 1/2 and S = 0 at the Ferriheme iron were generated by the complexation with imidazole and NO, respectively. ssNMR spectra of both forms are well resolved, which allows for sequential resonance assignments of 22 residues. Importantly, the ssNMR spectra demonstrate that aggregation does not affect the protein fold. Comparison of the spectra of the two electronic spin states allows the determination of paramagnetically shifted cross peaks due to pseudocontact shifts, which assists the assignment of residues close to the heme center

  • complexes of Ferriheme nitrophorin 4 with low molecular weight thiol ate s occurring in blood plasma
    Journal of Inorganic Biochemistry, 2013
    Co-Authors: Koji Nishikawa, Hideaki Ogata, Wolfgang Lubitz, Ozlen F Erdem, Edward J Reijerse, Markus Knipp
    Abstract:

    Abstract Nitrophorins are proteins occurring in the saliva of the blood-sucking insect Rhodnius prolixus to carry NO as a vasodilator and blood-coagulation inhibitor into the victim's tissue. It was suggested that the rate of NO release can be enhanced by the blood-plasma component l -cysteine [J.M.C.Ribeiro, Insect Biochem. Mol. Biol. 26 (1996) 899–905]. However, the mechanism of the reaction is not clear. In the attempt to exploit the reaction in detail, complexes of nitrophorin 4 (NP4) with the thiols 2-mercaptoethanol, l -cysteine, and l -homocysteine and with HS − were formed and characterized under anaerobic conditions using absorption spectroscopy, X-ray crystallography, and EPR spectroscopy. In contrast to met-myoglobin, which is reduced by l -cysteine, all four compounds form low-spin Fe III complexes with NP4. The weak equilibration constants (167–5200 M − 1 ) neither support significant complexation nor the simple displacement of NO in vivo . Both amino acid based thiols form additional H-bonds with side chains of the heme pocket entry. Glutathione and l -methionine did not form a complex, indicating the specificity of the complexes with l -cysteine and l -homocysteine. Continuous wave EPR spectroscopy reveals the simultaneous existence of three low-spin systems in each case that are attributed to various protonation and/or conformational stages in the heme pocket. Electron nuclear double resonance (ENDOR) spectroscopy demonstrates that the thiol sulfurs are, at least in part, protonated. Overall, the results not only demonstrate the good accessibility of the NP4 heme center by biologically relevant thiols, but also represent the first structural characterization of a Ferriheme protein in complex with l -cysteine l -homocysteine.

  • insertion of an h bonding residue into the distal pocket of the Ferriheme protein nitrophorin 4 effect on nitrite iron coordination and nitrite disproportionation
    Chemistry & Biodiversity, 2012
    Co-Authors: Hideaki Ogata, Markus Knipp
    Abstract:

    Heme proteins are important entities for the metabolism of nitrite. Inspection of the structural features of the reported hemoprotein-nitrite crystal structures reveals that, except for nitrophorin 4 (NP4), H-bonding to the nitrite ligand is accomplished via histidine or arginine residues. These H-bonds probably play an important role for the nitrite coordination and/or reactivities. In nitrophorins, which catalyze the nitrite disproportionation reaction, such a residue is missing. Here, we report on the L130R mutant of the NP isoprotein NP4 that provides the Arg130 residue as part of the flexible G-H loop as a potential H-bonding residue in the distal heme pocket. Similar to the wild-type protein, nitrite remains N-bonded in the crystal structure of NP4(L130R). However, spectroscopic investigations show that, in solution, a second ligand-rotational orientation exists, which is in fast-exchange equilibrium with the normal, parallel ligand orientation. Moreover, the nitrite disproportionation is inhibited in NP4(L130R). Comparison with another, also less active mutant NP4(D30N) suggests that the displacement of H(2)O molecules from the heme cavity prevents the proton donation pathway through Asp30.

  • Heterogeneous kinetics of the carbon monoxide association and dissociation reaction to nitrophorin 4 and 7 coincide with structural heterogeneity of the gate-loop.
    Journal of the American Chemical Society, 2012
    Co-Authors: Stefania Abbruzzetti, Hideaki Ogata, Stefano Bruno, Cristiano Viappiani, Markus Knipp
    Abstract:

    NO is an important signaling molecule in human tissue. However, the mechanisms by which this molecule is controlled and directed are currently little understood. Nitrophorins (NPs) comprise a group of Ferriheme proteins originating from blood-sucking insects that are tailored to protect and deliver NO via coordination to and release from the heme iron. Therefore, the kinetics of the association and dissociation reactions were studied in this work using the ferroheme–CO complexes of NP4, NP4(D30N), and NP7 as isoelectronic models for the Ferriheme–NO complexes. The kinetic measurements performed by nanosecond laser-flash-photolysis and stopped-flow are accompanied by resonance Raman and FT-IR spectroscopy to characterize the carbonyl species. Careful analysis of the CO rebinding kinetics reveals that in NP4 and, to a larger extent, NP7 internal gas binding cavities are located, which temporarily trap photodissociated ligands. Moreover, changes in the free energy barriers throughout the rebinding and releas...

  • identification of the native n terminus of the membrane attaching Ferriheme protein nitrophorin 7 from rhodnius prolixus
    Analytical Biochemistry, 2012
    Co-Authors: Markus Knipp, Rodrigo P Soares, Marco H Pereira
    Abstract:

    All species of the genus Rhodnius have a characteristic red coloration in their salivary glands due to the presence of heme proteins. Some of these secreted proteins, known as nitrophorins (NPs), are responsible for many of the antihemostatic activities of Rhodnius saliva such as anticoagulant and antihistamine. Several NPs have been described (NP1-4 and NP7), where NP7 is the only one with affinity to phospholipid membranes. Computational prediction suggested that NP7 also has an extended N-terminal tail on signal peptide cleavage; however, the complementary DNA does not allow the determination of the exact site of signal peptidase cleavage. On the other hand, according to previous studies, the exact length of the N-terminus has important consequences for the nitric oxide binding properties of NP7. Here, a method was developed to select phospholipid membrane-attaching proteins from homogenized tissue for analysis by mass spectrometry. The method was used to determine the exact N-terminus of the Ferriheme protein NP7 from homogenates of the salivary glands of 5th instar nymphal stages of Rhodnius prolixus.

Roman Davydov - One of the best experts on this subject based on the ideXlab platform.

  • Role of the Proximal Cysteine Hydrogen Bonding Interaction in Cytochrome P450 2B4 Studied by Cryoreduction, Electron Paramagnetic Resonance, and Electron-Nuclear Double Resonance Spectroscopy.
    Biochemistry, 2016
    Co-Authors: Roman Davydov, Muralidharan Shanmugam, William A. Gunderson, Naw May Pearl, Brian M. Hoffman
    Abstract:

    Crystallographic studies have shown that the F429H mutation of cytochrome P450 2B4 introduces an H-bond between His429 and the proximal thiolate ligand, Cys436, without altering the protein fold but sharply decreases the enzymatic activity and stabilizes the oxyferrous P450 2B4 complex. To characterize the influence of this hydrogen bond on the states of the catalytic cycle, we have used radiolytic cryoreduction combined with electron paramagnetic resonance (EPR) and (electron–nuclear double resonance (ENDOR) spectroscopy to study and compare their characteristics for wild-type (WT) P450 2B4 and the F429H mutant. (i) The addition of an H-bond to the axial Cys436 thiolate significantly changes the EPR signals of both low-spin and high-spin heme-iron(III) and the hyperfine couplings of the heme-pyrrole 14N but has relatively little effect on the 1H ENDOR spectra of the water ligand in the six-coordinate low-spin Ferriheme state. These changes indicate that the H-bond introduced between His and the proximal ...

  • Role of the Proximal Cysteine Hydrogen Bonding Interaction in Cytochrome P450 2B4 Studied by Cryoreduction, Electron Paramagnetic Resonance, and Electron–Nuclear Double Resonance Spectroscopy
    2016
    Co-Authors: Roman Davydov, Brian M. Hoffman, Muralidharan Shanmugam, Naw May Pearl, William A. Gunderson, Lucy Waskell
    Abstract:

    Crystallographic studies have shown that the F429H mutation of cytochrome P450 2B4 introduces an H-bond between His429 and the proximal thiolate ligand, Cys436, without altering the protein fold but sharply decreases the enzymatic activity and stabilizes the oxyferrous P450 2B4 complex. To characterize the influence of this hydrogen bond on the states of the catalytic cycle, we have used radiolytic cryoreduction combined with electron paramagnetic resonance (EPR) and (electron–nuclear double resonance (ENDOR) spectroscopy to study and compare their characteristics for wild-type (WT) P450 2B4 and the F429H mutant. (i) The addition of an H-bond to the axial Cys436 thiolate significantly changes the EPR signals of both low-spin and high-spin heme-iron­(III) and the hyperfine couplings of the heme-pyrrole 14N but has relatively little effect on the 1H ENDOR spectra of the water ligand in the six-coordinate low-spin Ferriheme state. These changes indicate that the H-bond introduced between His and the proximal cysteine decreases the extent of S → Fe electron donation and weakens the Fe­(III)–S bond. (ii) The added H-bond changes the primary product of cryoreduction of the Fe­(II) enzyme, which is trapped in the conformation of the parent Fe­(II) state. In the wild-type enzyme, the added electron localizes on the porphyrin, generating an S = 3/2 state with the anion radical exchange-coupled to the Fe­(II). In the mutant, it localizes on the iron, generating an S = 1/2 Fe­(I) state. (iii) The additional H-bond has little effect on g values and 1H–14N hyperfine couplings of the cryogenerated, ferric hydroperoxo intermediate but noticeably slows its decay during cryoannealing. (iv) In both the WT and the mutant enzyme, this decay shows a significant solvent kinetic isotope effect, indicating that the decay reflects a proton-assisted conversion to Compound I (Cpd I). (v) We confirm that Cpd I formed during the annealing of the cryogenerated hydroperoxy intermediate and that it is the active hydroxylating species in both WT P450 2B4 and the F429H mutant. (vi) Our data also indicate that the added H-bond of the mutation diminishes the reactivity of Cpd I

  • compound i is the reactive intermediate in the first monooxygenation step during conversion of cholesterol to pregnenolone by cytochrome p450scc epr endor cryoreduction annealing studies
    Journal of the American Chemical Society, 2012
    Co-Authors: Roman Davydov, A A Gilep, N V Strushkevich, Sergey A Usanov, Brian M. Hoffman
    Abstract:

    Cytochrome P450scc (CYP11A1) catalyzes conversion of cholesterol (CH) to pregnenolone, the precursor to all steroid hormones. This process proceeds via three sequential monooxygenation reactions: two stereospecific hydroxylations with formation first of 22R-hydroxycholesterol (22-HC) and then 20α,22R-dihydroxycholesterol (20,22-DHC), followed by the C20-C22 bond cleavage. Herein we have employed EPR and ENDOR spectroscopy to characterize the intermediates in the first hydroxylation step by 77K radiolytic one-electron cryoreduction and subsequent annealing of the ternary oxy cytochrome P450scc-cholesterol complex. This approach is fully validated by the demonstration that the cryoreduced ternary complex of oxy-P450scc-CH is catalytically competent and hydroxylates cholesterol to form 22R-HC with no detectable formation of 20-HC, just as occurs under physiological conditions. Cryoreduction of the ternary complex trapped at 77K produces predominantly the hydroperoxy-Ferriheme P450scc intermediate, along with a minor fraction of peroxo-Ferriheme intermediate that converts into a new hydroperoxo-Ferriheme species at 145K. This behavior reveals that the distal pocket of the parent oxy-P450scc-cholesterol complex exhibits an efficient proton delivery network, with an ordered water molecule H-bonded to the distal oxygen of the dioxygen ligand. During annealing of the hydroperoxy-ferric P450scc intermediates at 185K they convert to the primary product complex in which CH has been converted to 22-HC. In this process, the hydroperoxy-ferric intermediate decays with a large sKIE, as expected when proton delivery to the terminal O leads to formation of Compound I (Cpd I). 1H ENDOR measurements of the primary product formed in deuterated solvent show that the heme Fe(III) is coordinated to the 22R-O1H of 22-HC, where the 1H is derived from substrate and exchanges to D after annealing at higher temperatures. These observations establish that Cpd I is agent that hydroxylates CH, rather than the hydroperoxy-ferric heme.

  • Compound I Is the Reactive Intermediate in the First Monooxygenation Step during Conversion of Cholesterol to Pregnenolone by Cytochrome P450scc: EPR/ENDOR/Cryoreduction/Annealing Studies
    2012
    Co-Authors: Roman Davydov, A A Gilep, N V Strushkevich, Sergey A Usanov, Brian M. Hoffman
    Abstract:

    Cytochrome P450scc (CYP11A1) catalyzes conversion of cholesterol (CH) to pregnenolone, the precursor to all steroid hormones. This process proceeds via three sequential monooxygenation reactions: two stereospecific hydroxylations with formation first of 22R-hydroxycholesterol (22-HC) and then 20α,22R-dihydroxycholesterol (20,22-DHC), followed by C20–C22 bond cleavage. Herein we have employed EPR and ENDOR spectroscopy to characterize the intermediates in the first hydroxylation step by 77 K radiolytic one-electron cryoreduction and subsequent annealing of the ternary oxy-cytochrome P450scc-cholesterol complex. This approach is fully validated by the demonstration that the cryoreduced ternary complex of oxy-P450scc-CH is catalytically competent and hydroxylates cholesterol to form 22-HC with no detectable formation of 20-HC, just as occurs under physiological conditions. Cryoreduction of the ternary complex trapped at 77 K produces predominantly the hydroperoxy-Ferriheme P450scc intermediate, along with a minor fraction of peroxo-Ferriheme intermediate that converts into a new hydroperoxo-Ferriheme species at 145 K. This behavior reveals that the distal pocket of the parent oxy-P450scc-cholesterol complex exhibits an efficient proton delivery network, with an ordered water molecule H-bonded to the distal oxygen of the dioxygen ligand. During annealing of the hydroperoxy-ferric P450scc intermediates at 185 K, they convert to the primary product complex in which CH has been converted to 22-HC. In this process, the hydroperoxy-ferric intermediate decays with a large solvent kinetic isotope effect, as expected when proton delivery to the terminal O leads to formation of Compound I (Cpd I). 1H ENDOR measurements of the primary product formed in deuterated solvent show that the heme Fe­(III) is coordinated to the 22R-O1H of 22-HC, where the 1H is derived from substrate and exchanges to D after annealing at higher temperatures. These observations establish that Cpd I is the agent that hydroxylates CH, rather than the hydroperoxy-ferric heme

  • a superoxo ferrous state in a reduced oxy ferrous hemoprotein and model compounds
    Journal of the American Chemical Society, 2003
    Co-Authors: Roman Davydov, James D Satterlee, Hiroshi Fujii, Alexandra Sauermasarwa, Daryle H Busch, Brian M. Hoffman
    Abstract:

    Cryoreduction of the [FeO2]6 (n = 6 is the number of electrons in 3d orbitals on Fe and π* orbitals on O2) dioxygen-bound ferroheme through γ irradiation at 77 K generates an [FeO2]7 reduced oxy-heme. Numerous investigations have examined [FeO2]7 centers that have been characterized as peroxo-ferric centers, denoted [FeO2]per7, in which a Ferriheme binds a dianionic peroxo-ligand. The generation of such an intermediate can be understood heuristically if the [FeO2]6 parent is viewed as a superoxo-ferric center and the injected electron localizes on the O−O moiety. We here report EPR/ENDOR experiments which show quite different properties for the [FeO2]7 centers produced by cryoreduction of monomeric oxy-hemoglobin (oxy-GMH3) from Glycera dibranchiata, which is unlike mammalian “globins” in having a leucine in place of the distal histidine; of frozen aprotic solutions of oxy-ferrous octaethyl porphyrin; and of the oxy-ferrous complex of the heme model, cyclidene. These [FeO2]7 centers are characterized as “...

Volker Schunemann - One of the best experts on this subject based on the ideXlab platform.

  • nuclear inelastic scattering and mossbauer spectroscopy as local probes for ligand binding modes and electronic properties in proteins vibrational behavior of a Ferriheme center inside a β barrel protein
    Journal of the American Chemical Society, 2012
    Co-Authors: Beate Moeser, Hongjun Zhang, Ann F Walker, Robert E Berry, Hauke Paulsen, Adam Janoschka, Juliusz A Wolny, Igor Filippov, Aleksandr I Chumakov, Volker Schunemann
    Abstract:

    In this work, we present a study of the influence of the protein matrix on its ability to tune the binding of small ligands such as NO, cyanide (CN(-)), and histamine to the ferric heme iron center in the NO-storage and -transport protein Nitrophorin 2 (NP2) from the salivary glands of the blood-sucking insect Rhodnius prolixus. Conventional Mossbauer spectroscopy shows a diamagnetic ground state of the NP2-NO complex and Type I and II electronic ground states of the NP2-CN(-) and NP2-histamine complex, respectively. The change in the vibrational signature of the protein upon ligand binding has been monitored by Nuclear Inelastic Scattering (NIS), also called Nuclear Resonant Vibrational Spectroscopy (NRVS). The NIS data thus obtained have also been calculated by quantum mechanical (QM) density functional theory (DFT) coupled with molecular mechanics (MM) methods. The calculations presented here show that the heme ruffling in NP2 is a consequence of the interaction with the protein matrix. Structure optimizations of the heme and its ligands with DFT retain the characteristic saddling and ruffling only if the protein matrix is taken into account. Furthermore, simulations of the NIS data by QM/MM calculations suggest that the pH dependence of the binding of NO, but not of CN(-) and histamine, might be a consequence of the protonation state of the heme carboxyls.

  • models of the membrane bound cytochromes mossbauer spectra of crystalline low spin Ferriheme complexes having axial ligand plane dihedral angles ranging from 0 to 90
    Journal of the American Chemical Society, 2006
    Co-Authors: Thomas Teschner, Ann F Walker, Volker Schunemann, Liliya A Yatsunyk, H Winkler, Hauke Paulsen, Robert W Scheidt, A X Trautwein
    Abstract:

    Crystalline samples of four low-spin Fe(III) octaalkyltetraphenylporphyrinate and two low-spin Fe(III) tetramesitylporphyrinate complexes, all of which are models of the bis-histidine-coordinated c...

  • low spin Ferriheme models of the cytochromes correlation of molecular structure with epr and mossbauer spectral parameters
    Hyperfine Interactions, 2004
    Co-Authors: Thomas Teschner, A X Trautwein, Volker Schunemann, Liliya A Yatsunyk, F. A. Walker
    Abstract:

    The magnetic Mossbauer spectra of a series of low-spin Ferriheme complexes have been investigated and compared with their EPR spectral parameters and molecular structures. To date there has been little systematic analysis of either estimated or fitted values of the hyperfine coupling constants for low-spin Ferriheme centers and no meaningful correlation has been established between the Mossbauer parameters and the axial ligands of such species. With the results of the present study, we have been able to find correlations of molecular structures with iron-orbital splittings, g-tensor values derived from EPR signals and magnetic hyperfine interaction components A zz obtained from magnetic Mossbauer spectra. These correlations should be useful to future workers in the field of heme-containing enzymes.

  • models of the bis histidine coordinated ferricytochromes mossbauer and epr spectroscopic studies of low spin iron iii tetrapyrroles of various electronic ground states and axial ligand orientations
    Journal of Biological Inorganic Chemistry, 2003
    Co-Authors: Rudiger Benda, A X Trautwein, Volker Schunemann, Sheng Cai, Jayapal Reddy Polam, Todd C Watson, Tatjana Kh Shokhireva, Ann F Walker
    Abstract:

    The EPR and magnetic Mossbauer spectra of a series of axial ligand complexes of tetrakis(2,6- dimethoxyphenyl)porphyrinatoiron(III), ((2,6-(OMe)2)4 TPPFeL2) + , where L=N-methylimidazole, 2-methyli- midazole, or 4-(dimethylamino)pyridine, of one axial ligand complex of tetraphenylporphyrin, the bis(4-cy- anopyridine) complex (TPPFe(4-CNPy)2) + , and of one axial ligand complex of tetraphenylchlorin, (TPCFe(ImH)2) + , where ImH=imidazole, have been investigated and compared to those of low-spin Fe(III) porphyrinates and Ferriheme proteins reported in the literature. On the basis of this and previous comple- mentary spectroscopic investigations, three types of complexes have been identified: those having (dxy) 2 (dxz,dyz) 3 electronic ground states with axial ligands aligned in perpendicular planes (Type I), those having (dxy) 2 (dxz,dyz) 3 electronic ground states with axial ligands aligned in parallel planes (Type II), and those having the novel (dxz,dyz) 4 (dxy) 1 electronic ground state (Type III). A subset of the latter type, with planar axial ligands aligned parallel to each other or strong macrocycle asymmetry that yield rhombic EPR spectra, cannot be created using the porphyrinate ligand. Type I centers are characterized by ''largegmax'' EPR spectra with g>3.2 and well-resolved, widely spread magnetic Mossbauer spectra having Azz/gNlN>680 kG, with Axx negative in sign but much smaller in magnitude than Azz, while Type II centers have well-resolved rhombic EPR spectra with gzz=2.4-3.1 and also less-resolved magnetic Mossbauer spectra, and usually have Azz/gNlNin the range of 440-660 kG (but in certain cases as small as 180 kG) and Axx again negative in sign but only somewhat smaller (but occasionally larger in magnitude) than Azz, and Type III centers have axial EPR spectra with g?� 2.6 or smaller and gk<1.0-1.95, but often not resolved, and less-resolved magnetic Mossbauer spectra having Azz/gNlNin the range of 270-400 kG, and Axx again negative in sign but much smaller in magnitude than Azz. An exception to this rule is (TPPFe(4- CNPy)2) + , which has Axx/gNlN=)565 kG, Ayy/gNlN= 629 kG, and Azz/gNlN=4 kG. A subset of Type II complexes (Type II¢) have rhombicities (V/D) much greater than 0.67 and Azz/gNlNranging from 320 to 170 kG, with Axx also negative but with the magnitude of Axx significantly larger than that of Azz. These clas- sifications are also observed for a variety of Ferriheme proteins, and they lead to linear correlations between Azz and either Axx,gzz ,o rV/D for Types I and II (but not for Azz versus V/D for Type II¢). Not enough data are yet available on Type III complexes to determine what, if any, correlations may be observed.

  • nuclear resonant forward scattering using synchrotron radiation applied to study the low spin Ferriheme complex tppfe nh2pzh 2 cl
    2002
    Co-Authors: Rudiger Benda, A X Trautwein, Volker Schunemann, Christian Herta, H Winkler, Y Shvydko, F. A. Walker
    Abstract:

    We have characterised the low-spin Ferriheme complex bis(3-aminopyrazole)tetraphenyl-porphyrinatoiron(III) chloride ([TPPFe(NH2PzH)2]Cl) by nuclear resonant forward scattering (NFS) using synchrotron radiation at 4.2K in fields up to 6T. The analysis of the field dependant NFS spectra obtained with the g-tensor determined by previous pulsed EPR measurements [1] (g=(1.87, 2.28, 2.39)) yields the hyperfine parameters η = 0.0; β = 90°; y = 90°; ΔEQ = −2.56 mm/s and A/μngn = (−46.9, 9.5, 17.6) T. This investigation shows that NFS provides higher accuracy in determining hyperfine parameters than conventional Mossbauer spectroscopy, if the delay time is long enough and the effective thickness is relatively high (in the present case t ≈ 150 ns and teff ≈ 10).