Assignment of hyperfine-shifted resonances in low-spin forms of cytochrome c peroxidase by reconstitutions with deuterated hemins

Satterlee, James D.; Erman, James E.; LaMar, Gerd N.; Smith, Kevin M.; Langry, Kevin C.

doi:10.1021/ja00346a001

Cited by 44 publications

(37 citation statements)

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“…Signal B is assigned to 8-CH3 and signals D and I Ha of the 7-propionate moiety, because 3-CH3 and 8-CH3 a] expected and found to be pairwise separated from 1-CH3 ar 5-CH3 (39,44). Analysis of the two-dimensional maps also reveals scaL and dipolar connectivities between signals C and F (crow peak 1).…”

Section: Resultsmentioning

confidence: 92%

Proton NMR investigation into the basis for the relatively high redox potential of lignin peroxidase.

Banci

Bertini

Turano

et al. 1991

Proc. Natl. Acad. Sci. U.S.A.

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Lignin peroxidase shares several sctural features with the well-studied horseradish peroxidase and cytochrome c peroxidase but carries a higher redox potential. Here the heme domain of lignin peroxidase and the lignin peroxidase cyanide adduct was exmined by IH NMR spectroscopy, including nuclear Overhauser effect and two-dimensional measurements, and the mdga were compared with those for horseradish peroxidase and cytochrome c peroxidase. Strctural information was obtained on the orientation of the heme vinyl and propionate groups and the proximal and distal histidines. The shifts of the El proton of the proximal histidine were found to be empirically related to the Fe3+/Fe2' redox potentials.Lignin peroxidase (LiP), secreted by the lignin-degrading fungus Phanerochaete chrysosporium (1, 2), catalyzes the one-electron oxidation of aromatic rings in lignin (3,4 (8,9), (iv) the ability to oxidize phenols and aromatic amines (10), and (v) substrate oxidation at the heme edge rather than at the iron center (11). Many of these characteristics are also shared by cytochrome c peroxidase (CcP; ref. 12). LiP compound I stores a much higher redox potential than HRP compound I, which allows it to oxidize aromatic substrates that are not substrates for HRP and CcP (13,14). In the only direct measurement ofthe redox potential of LiP, Millis et al. (15) proteins (20-23). The purpose of the present study, therefore, was to examine LiP by 1H NMR spectroscopy and to compare the findings with those described for HRP and CcP to relate structure with reactivity. MATERIALS AND METHODSLignin Peroxidase. Phanerochaete chrysosporium Burds (ATCC 24725) was grown in a 100-liter fermentor, essentially as described by Bonnarme and Jeffries (24). Isoenzyme LiP 3 (= H1) was isolated from concentrated culture fluid (25).The cyanide adduct (LiP-CN-) was prepared by titrating with KCN in 10 mM sodium acetate (pH 5).'H NMR Spectroscopy. Spectra of LiP and LiP-CN-[-2 mM in 10 mM sodium acetate (pH 5)] were recorded with Bruker MSL 200 and AMX 600 spectrometers. T, experiments were performed at 200 MHz with the modified driven equilibrium Fourier transform pulse sequence (26). All other spectra at 200 MHz were taken by using a super WEFT (water-eliminated Fourier transform) pulse sequence (27) with a recycle delay of 85 and 220 ms for high-and low-spin species, respectively. The nuclear Overhauser effect (NOE) difference spectra were collected as described (28).Two-dimensional NOE spectroscopy (NOESY) and twodimensional correlated spectroscopy (COSY) spectra were recorded at 600 MHz using presaturation to eliminate the intense water signal. Phase-sensitive NOESY spectra (29) were recorded at mixing times of 15, 50, and 100 ms, using the time proportional phase incrementation method (30). Magnitude COSY (31) experiments provide one of the best sequences for detecting scalar connectivities between paramagnetically shifted signals (32,54). RESULTSNative Protein. Fig. 1, spectrum A, 6956The publication costs of this article were defraye...

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Section: Resultsmentioning

confidence: 92%

Proton NMR investigation into the basis for the relatively high redox potential of lignin peroxidase.

Banci

Bertini

Turano

et al. 1991

Proc. Natl. Acad. Sci. U.S.A.

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“…This protein form, although not physiologically active, has been the most favorable system on which paramagnetic NMR investigations are carried out (35,37,70,71). The short electronic relaxation times and large magnetic anisotropy of the low spin peroxidase cyanide complexes give much sharper and better resolved signals in their proton NMR spectra, providing much more information about the electronic, magnetic, and molecular structural properties of the heme pocket as compared with the native, high spin resting forms (27,38,50,72). In addition, the cyanide adduct of heme peroxidases has been implicated as an important analogue for the active, oxidized low spin enzyme intermediates for which proton NMR spectroscopy is currently inapplicable due to the large resonance line widths (73).…”

Section: Resultsmentioning

confidence: 99%

Two-dimensional NMR Study of the Heme Active Site Structure of Chloroperoxidase

Wang

Tachikawa

et al. 2003

Journal of Biological Chemistry

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“…Making complete heme proton assignments for CcPCN has been an elusive goal since the first attempt in 1983 (40). As detailed under Results and Discussion, the heme-centered paramagnetism, the enzyme's size ( molecular mass ∼34 kDa), and its limited thermal stability have been the primary complicating factors in this effort.…”

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confidence: 99%

Expression, Purification, Characterization, and NMR Studies of Highly Deuterated Recombinant CytochromecPeroxidase

et al. 2001

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Two forms of extensively deuterated S. cerevisiae cytochrome c peroxidase (CcP; EC 1.11.1.5) have been overexpressed in E. coli by growth in highly deuterated medium. One of these ferriheme enzyme forms (recDCcP) was produced using >97% deuterated growth medium and was determined to be approximately 84% deuterated. The second form [recD(His)CcP] was grown in the same highly deuterated medium that had been supplemented with excess histidine (at natural hydrogen isotope abundance) and was also approximately 84% deuterated. This resulted in direct histidine incorporation without isotope scrambling. Both of these enzymes along with the corresponding recombinant native CcP (recNATCcP), which was expressed in a standard medium with normal hydrogen isotope abundance, consisted of 294 amino acid polypeptide chains having the identical sequence to the yeast-isolated enzyme, without any N-terminal modifications. Comparative characterizations of all three enzymes have been carried out for the resting-state, high-spin forms and in the cyanide-ligated, low-spin forms. The primary physical methods employed were electrophoresis, UV-visible spectroscopy, hydrogen peroxide reaction kinetics, mass spectrometry, and (1)H NMR spectroscopy. The results indicate that high-level deuteration does not significantly alter CcP's reactivity or spectroscopy. As an example of potential NMR uses, recDCcPCN and recD(His)CcPCN have been used to achieve complete, unambiguous, stereospecific (1)H resonance assignments for the heme hyperfine-shifted protons, which also allows the heme side chain conformations to be assessed. Assigning these important active-site protons has been an elusive goal since the first NMR spectra on this enzyme were reported 18 years ago, due to a combination of the enzyme's comparatively large size, paramagnetism, and limited thermal stability.

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Assignment of hyperfine-shifted resonances in low-spin forms of cytochrome c peroxidase by reconstitutions with deuterated hemins

Cited by 44 publications

References 1 publication

Proton NMR investigation into the basis for the relatively high redox potential of lignin peroxidase.

Proton NMR investigation into the basis for the relatively high redox potential of lignin peroxidase.

Two-dimensional NMR Study of the Heme Active Site Structure of Chloroperoxidase

Expression, Purification, Characterization, and NMR Studies of Highly Deuterated Recombinant CytochromecPeroxidase

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