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Title: Proton NMR assignments of heme contracts and catalytically implicated amino acids in cyanide-ligated cytochrome c peroxidase determined from one- and two-dimensional nuclear Overhauser effects

Abstract

Proton NMR assignments of the heme pocket and catalytically relevant amino acid protons have been accomplished for cyanide-ligated yeast cytochrome c peroxidase. This form of the protein, while not enzymatically active itself, is the best model available (that displays a resolvable proton NMR spectrum) for the six-coordinate low-spin active intermediates, compounds I and II. The assignments were made with a combination of one- and two-dimensional nuclear Overhauser effect methods and demonstrate the utility of NOESY experiments for paramagnetic proteins of relatively large size (M{sub r} 34,000). Assignments of both isotope exchangeable and nonexchangeable proton resonances were obtained by using enzyme preparations in both 90% H{sub 2}O/10% D{sub 2}O and, separately, in 99.9% D{sub 2}O solvent systems. Complete resonance assignments have been achieved for the proximal histidine, His-175, and His-52, which is a member of the catalytic triad on the distal side of the heme. In addition, partial assignments are reported for Trp-51 and Arg-48, catalytically important residues, both on the distal side. Aside from His-175, partial assignments for amino acids on the proximal side of the heme are proposed for the alanines at primary sequence positions 174 and 176 and for Thr-180 and Leu-232.

Authors:
 [1];  [2]
  1. (Washington State Univ., Pullman (USA))
  2. (Northern Illinois Univ., DeKalb (USA))
Publication Date:
OSTI Identifier:
5594898
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemistry; (United States); Journal Volume: 30:18
Country of Publication:
United States
Language:
English
Subject:
62 RADIOLOGY AND NUCLEAR MEDICINE; AMINO ACIDS; NUCLEAR MAGNETIC RESONANCE; HEME; PEROXIDASES; MOLECULAR STRUCTURE; ARGININE; CYTOCHROME OXIDASE; CYTOCHROMES; HEAVY WATER; HISTIDINE; MOLECULAR WEIGHT; OVERHAUSER EFFECT; PROTONS; TRYPTOPHAN; YEASTS; AROMATICS; AZAARENES; AZOLES; BARYONS; CARBOXYLIC ACIDS; ELEMENTARY PARTICLES; ENZYMES; EUMYCOTA; FERMIONS; FUNGI; HADRONS; HAEM DEHYDROGENASES; HETEROCYCLIC ACIDS; HETEROCYCLIC COMPOUNDS; HYDROGEN COMPOUNDS; IMIDAZOLES; INDOLES; MAGNETIC RESONANCE; MICROORGANISMS; NUCLEONS; ORGANIC ACIDS; ORGANIC COMPOUNDS; ORGANIC NITROGEN COMPOUNDS; OXIDOREDUCTASES; OXYGEN COMPOUNDS; PIGMENTS; PLANTS; PORPHYRINS; PROTEINS; PYRROLES; RESONANCE; WATER; 550601* - Medicine- Unsealed Radionuclides in Diagnostics

Citation Formats

Satterlee, J.D., and Erman, J.E.. Proton NMR assignments of heme contracts and catalytically implicated amino acids in cyanide-ligated cytochrome c peroxidase determined from one- and two-dimensional nuclear Overhauser effects. United States: N. p., 1991. Web. doi:10.1021/bi00232a005.
Satterlee, J.D., & Erman, J.E.. Proton NMR assignments of heme contracts and catalytically implicated amino acids in cyanide-ligated cytochrome c peroxidase determined from one- and two-dimensional nuclear Overhauser effects. United States. doi:10.1021/bi00232a005.
Satterlee, J.D., and Erman, J.E.. 1991. "Proton NMR assignments of heme contracts and catalytically implicated amino acids in cyanide-ligated cytochrome c peroxidase determined from one- and two-dimensional nuclear Overhauser effects". United States. doi:10.1021/bi00232a005.
@article{osti_5594898,
title = {Proton NMR assignments of heme contracts and catalytically implicated amino acids in cyanide-ligated cytochrome c peroxidase determined from one- and two-dimensional nuclear Overhauser effects},
author = {Satterlee, J.D. and Erman, J.E.},
abstractNote = {Proton NMR assignments of the heme pocket and catalytically relevant amino acid protons have been accomplished for cyanide-ligated yeast cytochrome c peroxidase. This form of the protein, while not enzymatically active itself, is the best model available (that displays a resolvable proton NMR spectrum) for the six-coordinate low-spin active intermediates, compounds I and II. The assignments were made with a combination of one- and two-dimensional nuclear Overhauser effect methods and demonstrate the utility of NOESY experiments for paramagnetic proteins of relatively large size (M{sub r} 34,000). Assignments of both isotope exchangeable and nonexchangeable proton resonances were obtained by using enzyme preparations in both 90% H{sub 2}O/10% D{sub 2}O and, separately, in 99.9% D{sub 2}O solvent systems. Complete resonance assignments have been achieved for the proximal histidine, His-175, and His-52, which is a member of the catalytic triad on the distal side of the heme. In addition, partial assignments are reported for Trp-51 and Arg-48, catalytically important residues, both on the distal side. Aside from His-175, partial assignments for amino acids on the proximal side of the heme are proposed for the alanines at primary sequence positions 174 and 176 and for Thr-180 and Leu-232.},
doi = {10.1021/bi00232a005},
journal = {Biochemistry; (United States)},
number = ,
volume = 30:18,
place = {United States},
year = 1991,
month = 5
}
  • The structural changes in the heme macrocycle and substituents caused by binding of Ca{sup 2+} to the diheme cytochrome c peroxidase from Paracoccuspantotrophus were clarified by resonance Raman spectroscopy of the inactive filly oxidized form of the enzyme. The changes in the macrocycle vibrational modes are consistent with a Ca{sup 2+}-dependent increase in the out-of-plane distortion of the low-potential heme, the proposed peroxidatic heme. Most of the increase in out-of-plane distortion occurs when the high affinity site I is occupied, but a small further increase in distortion occurs when site II is also occupied by Ca{sup 2+}or Mg{sup 2+}. Thismore » increase in the heme distortion also explains the red shift in the Soret absorption band that occurs upon Ca{sup 2+} binding. Changes also occur in the low frequency substituent modes of the heme, indicating that a structural change in the covalently attached fingerprint pentapeptide of the LP heme occurs upon CM{sup 2+} binding to site I. These structural changes, possibly enhanced in the semi-reduced form of the enzyme, may lead to loss of the sixth ligand at the peroxidatic heme and activation of the enzyme.« less
  • A combination of multinuclear two-dimensional NMR experiments served to identify and assign the combined {sup 1}H, {sup 13}C, and {sup 15}N spin systems of the single tryptophan, three phenylalanines, three histidines, and seven tyrosines of staphylococcal nuclease H124L in its ternary complex with calcium and thymidine 3{prime},5{prime}-bisphosphate at pH 5.1 (H{sub 2}O) or pH* 5.5 ({sup 2}H{sub 2}O). Samples of recombinant nuclease were labeled with {sup 13}C or {sup 15}N as appropriate to individual NMR experiments: uniformly with {sup 15}N (all sites to >95%), uniformly with {sup 13}C (all sites to 26%), selectively with {sup 13}C (single amino acids uniformlymore » labeled to >26%), or selectively with {sup 15}N (single amino acids uniformly labeled to >95%). NMR data used in the analysis included single-bond and multiple-bond {sup 1}H-{sup 13}C and multiple-bond {sup 1}H-{sup 15}N correlations, {sup 1}H-{sup 13}C single-bond correlations with Hartmann-Hahn relay ({sup 1}H({sup 13}C)SBC-HH), and {sup 1}H-{sup 13}C single-bond correlation with NOE relay ({sup 1}H({sup 13}C)SBC-NOE). The aromatic protons of the spin systems were identified from {sup 1}H({sup 13}C)SBC-HH data, and the nonprotonated aromatic ring carbons were identified from {sup 1}H-{sup 13}C multiple-bond correlations. Measurement of NOE buildup rates between protons on different aromatic residues of the major ternary complex species yielded a number of interproton distances that could be compared with those from X-ray structures of the wild-type nuclease ternary complex with calcium and thymidine 3{prime},5{prime}-bisphosphate.« less
  • Hyperfine {sup 1}H NMR signals of the 2 Fe-2S* vegetative ferredoxin from Anabaena 7,120 have been studied by two-dimensional (2D) magnetization exchange spectroscopy. The rapid longitudinal relaxation rates of these signals required the use of very short nuclear Overhauser effect (NOE) mixing times (0.5-20 ms). The resulting pattern of NOE cross-relaxation peaks when combined with previous 1D NOE results led to elucidation of the carbon-bound proton spin systems from each of the four cysteines ligated to the 2Fe-2S* cluster in the reduced ferredoxin. Additional NOE cross peaks were observed that provide information about other amino acid residues that interact withmore » the iron-sulfur cluster. NOE cross peaks were assigned tentatively to Leu{sup 27}, Arg{sup 42}, and Ala{sup 43} on the basis of the X-ray coordinates of oxidized Anabaena 7,120 ferredoxin. Three chemical exchange cross peaks were detected in magnetization exchange spectra of half-reduced ferredoxin and assigned to the {sup 1}H{sup {alpha}} protons of Cys{sup 49} and Cys{sup 79} (both of whose sulfur atoms are ligated to Fe(III)) and Arg{sup 42} (whose amide nitrogen is hydrogen-bonded to one of the inorganic sulfurs of the 2Fe-2S* cluster). The chemical exchange cross peaks provide a means of extending assignments in the spectrum of reduced ferredoxin to assignments in the spectrum of the oxidized protein. The results suggest that 2D magnetization exchange spectroscopy employing short mixing times will be useful for the assignment and characterization of hyperfine {sup 1}H peaks in a variety of paramagnetic proteins.« less
  • The key to protein structure determination by NMR lies in the identification of as many {sup 1}H-{sup 1}H nuclear Overhauser effects (NOEs) as possible in order to obtain a large set of approximate interproton distance restraints. With the advent of a range of heteronuclear three-dimensional (3D) NMR experiments, it has now become possible to obtain complete {sup 1}H, {sup 15}N, and {sup 13}C assignments and to determine the 3D structures of proteins in the 15-25-kDa molecular weight range. Despite these advances, it has remained impossible to observe NOEs between protons with degenerate chemical shifts. Such interactions occur repeatedly, both amongmore » aliphatic or aromatic protons and between sequential amide protons in helical proteins. Here the authors describe a 3D heteronuclear experiment that allows the observation of these NOEs and demonstrate its applicability for calmodulin, a protein of 148 residues and molecular weight 16.7 kDa.« less
  • Abstract not provided.