Studies of Individual Carbon Sites of Azurin from Pseudomonas aeruginosa by Natural-Abundance Carbon-13 Nuclear Magnetic Resonance Spectroscopy

Kamil Ugurbil, Raymond S. Norton, Adam Allerhand, Richard Bersohn

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74 Citations (Scopus)

Abstract

The environments of the aromatic residues (and of the single arginine residue) of azurin from Pseudomonas aeruginosa are investigated by means of natural-abundance 13C Fourier transform NMR spectroscopy. In the case of the diamagnetic Cu(I) azurin, all 17 nonprotonated aromatic carbons (and Cζ of Arg-79) yield narrow resonances. Furthermore, a single-carbon amide carbonyl resonance with an unusual chemical shift (peak x) is observed. The pH dependence of chemical shifts is used to identify the resonances of Cγ of titrating histidines, and of Cγ and Cζ of the two tyrosines. The resonances of Cγ and Cδ2 of the single tryptophan residue (and Cζ of Arg-79) are also identified. The pKa values of the two tyrosines are different from each other and higher than typical values of “solvent-exposed” tyrosine residues. Two of the four histidine residues do not titrate (in the pH range 4 to 11). The resonance of Cγ of one histidine exhibits a pH titration with fast proton exchange behavior and a pKa of 7.5 ± 0.2. The direction of the titration shift indicates that the imidazole form of this histidine is the Nδ1-H tautomer. The Cγ resonance of the other titrating histidine exhibits slow exchange behavior with a pKa of about 7. The imidazole form of this histidine is the Nϵ2-H tautomer. When going to the paramagnetic Cu(II) protein, only 11 of the 19 carbons mentioned above yield resonances that are narrow enough to be detected. Also, some of the observed resonances exhibit significant paramagnetic broadening. A comparison of spectra of fully reduced azurin, mixtures of reduced and oxidized azurin, and fully oxidized azurin yields the following information, (i) Peak x arises from an amide group that probably is coordinated to the copper, (ii) The two nontitrating histidine residues are probably copper ligands, with Nδ1 coordinated to the metal, (iii) The side chains of Arg-79 and the two tyrosine residues are not coordinated to the copper, and Trp-48 is probably not a ligand either, (iv) The γ carbons of Trp-48, the tyrosine with the lower pKa, the titrating histidine with slow exchange behavior, and three or four of the six phenylalanine residues are sufficiently close to the copper to undergo significant paramagnetic broadening in the spectrum of oxidized azurin.

Original languageEnglish
Pages (from-to)886-894
Number of pages9
JournalBiochemistry
Volume16
Issue number5
DOIs
Publication statusPublished - 1 Mar 1977
Externally publishedYes

Cite this

@article{b2d45d4876b94bb4a947c66888c2cb58,
title = "Studies of Individual Carbon Sites of Azurin from Pseudomonas aeruginosa by Natural-Abundance Carbon-13 Nuclear Magnetic Resonance Spectroscopy",
abstract = "The environments of the aromatic residues (and of the single arginine residue) of azurin from Pseudomonas aeruginosa are investigated by means of natural-abundance 13C Fourier transform NMR spectroscopy. In the case of the diamagnetic Cu(I) azurin, all 17 nonprotonated aromatic carbons (and Cζ of Arg-79) yield narrow resonances. Furthermore, a single-carbon amide carbonyl resonance with an unusual chemical shift (peak x) is observed. The pH dependence of chemical shifts is used to identify the resonances of Cγ of titrating histidines, and of Cγ and Cζ of the two tyrosines. The resonances of Cγ and Cδ2 of the single tryptophan residue (and Cζ of Arg-79) are also identified. The pKa values of the two tyrosines are different from each other and higher than typical values of “solvent-exposed” tyrosine residues. Two of the four histidine residues do not titrate (in the pH range 4 to 11). The resonance of Cγ of one histidine exhibits a pH titration with fast proton exchange behavior and a pKa of 7.5 ± 0.2. The direction of the titration shift indicates that the imidazole form of this histidine is the Nδ1-H tautomer. The Cγ resonance of the other titrating histidine exhibits slow exchange behavior with a pKa of about 7. The imidazole form of this histidine is the Nϵ2-H tautomer. When going to the paramagnetic Cu(II) protein, only 11 of the 19 carbons mentioned above yield resonances that are narrow enough to be detected. Also, some of the observed resonances exhibit significant paramagnetic broadening. A comparison of spectra of fully reduced azurin, mixtures of reduced and oxidized azurin, and fully oxidized azurin yields the following information, (i) Peak x arises from an amide group that probably is coordinated to the copper, (ii) The two nontitrating histidine residues are probably copper ligands, with Nδ1 coordinated to the metal, (iii) The side chains of Arg-79 and the two tyrosine residues are not coordinated to the copper, and Trp-48 is probably not a ligand either, (iv) The γ carbons of Trp-48, the tyrosine with the lower pKa, the titrating histidine with slow exchange behavior, and three or four of the six phenylalanine residues are sufficiently close to the copper to undergo significant paramagnetic broadening in the spectrum of oxidized azurin.",
author = "Kamil Ugurbil and Norton, {Raymond S.} and Adam Allerhand and Richard Bersohn",
year = "1977",
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doi = "10.1021/bi00624a012",
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Studies of Individual Carbon Sites of Azurin from Pseudomonas aeruginosa by Natural-Abundance Carbon-13 Nuclear Magnetic Resonance Spectroscopy. / Ugurbil, Kamil; Norton, Raymond S.; Allerhand, Adam; Bersohn, Richard.

In: Biochemistry, Vol. 16, No. 5, 01.03.1977, p. 886-894.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Studies of Individual Carbon Sites of Azurin from Pseudomonas aeruginosa by Natural-Abundance Carbon-13 Nuclear Magnetic Resonance Spectroscopy

AU - Ugurbil, Kamil

AU - Norton, Raymond S.

AU - Allerhand, Adam

AU - Bersohn, Richard

PY - 1977/3/1

Y1 - 1977/3/1

N2 - The environments of the aromatic residues (and of the single arginine residue) of azurin from Pseudomonas aeruginosa are investigated by means of natural-abundance 13C Fourier transform NMR spectroscopy. In the case of the diamagnetic Cu(I) azurin, all 17 nonprotonated aromatic carbons (and Cζ of Arg-79) yield narrow resonances. Furthermore, a single-carbon amide carbonyl resonance with an unusual chemical shift (peak x) is observed. The pH dependence of chemical shifts is used to identify the resonances of Cγ of titrating histidines, and of Cγ and Cζ of the two tyrosines. The resonances of Cγ and Cδ2 of the single tryptophan residue (and Cζ of Arg-79) are also identified. The pKa values of the two tyrosines are different from each other and higher than typical values of “solvent-exposed” tyrosine residues. Two of the four histidine residues do not titrate (in the pH range 4 to 11). The resonance of Cγ of one histidine exhibits a pH titration with fast proton exchange behavior and a pKa of 7.5 ± 0.2. The direction of the titration shift indicates that the imidazole form of this histidine is the Nδ1-H tautomer. The Cγ resonance of the other titrating histidine exhibits slow exchange behavior with a pKa of about 7. The imidazole form of this histidine is the Nϵ2-H tautomer. When going to the paramagnetic Cu(II) protein, only 11 of the 19 carbons mentioned above yield resonances that are narrow enough to be detected. Also, some of the observed resonances exhibit significant paramagnetic broadening. A comparison of spectra of fully reduced azurin, mixtures of reduced and oxidized azurin, and fully oxidized azurin yields the following information, (i) Peak x arises from an amide group that probably is coordinated to the copper, (ii) The two nontitrating histidine residues are probably copper ligands, with Nδ1 coordinated to the metal, (iii) The side chains of Arg-79 and the two tyrosine residues are not coordinated to the copper, and Trp-48 is probably not a ligand either, (iv) The γ carbons of Trp-48, the tyrosine with the lower pKa, the titrating histidine with slow exchange behavior, and three or four of the six phenylalanine residues are sufficiently close to the copper to undergo significant paramagnetic broadening in the spectrum of oxidized azurin.

AB - The environments of the aromatic residues (and of the single arginine residue) of azurin from Pseudomonas aeruginosa are investigated by means of natural-abundance 13C Fourier transform NMR spectroscopy. In the case of the diamagnetic Cu(I) azurin, all 17 nonprotonated aromatic carbons (and Cζ of Arg-79) yield narrow resonances. Furthermore, a single-carbon amide carbonyl resonance with an unusual chemical shift (peak x) is observed. The pH dependence of chemical shifts is used to identify the resonances of Cγ of titrating histidines, and of Cγ and Cζ of the two tyrosines. The resonances of Cγ and Cδ2 of the single tryptophan residue (and Cζ of Arg-79) are also identified. The pKa values of the two tyrosines are different from each other and higher than typical values of “solvent-exposed” tyrosine residues. Two of the four histidine residues do not titrate (in the pH range 4 to 11). The resonance of Cγ of one histidine exhibits a pH titration with fast proton exchange behavior and a pKa of 7.5 ± 0.2. The direction of the titration shift indicates that the imidazole form of this histidine is the Nδ1-H tautomer. The Cγ resonance of the other titrating histidine exhibits slow exchange behavior with a pKa of about 7. The imidazole form of this histidine is the Nϵ2-H tautomer. When going to the paramagnetic Cu(II) protein, only 11 of the 19 carbons mentioned above yield resonances that are narrow enough to be detected. Also, some of the observed resonances exhibit significant paramagnetic broadening. A comparison of spectra of fully reduced azurin, mixtures of reduced and oxidized azurin, and fully oxidized azurin yields the following information, (i) Peak x arises from an amide group that probably is coordinated to the copper, (ii) The two nontitrating histidine residues are probably copper ligands, with Nδ1 coordinated to the metal, (iii) The side chains of Arg-79 and the two tyrosine residues are not coordinated to the copper, and Trp-48 is probably not a ligand either, (iv) The γ carbons of Trp-48, the tyrosine with the lower pKa, the titrating histidine with slow exchange behavior, and three or four of the six phenylalanine residues are sufficiently close to the copper to undergo significant paramagnetic broadening in the spectrum of oxidized azurin.

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U2 - 10.1021/bi00624a012

DO - 10.1021/bi00624a012

M3 - Article

VL - 16

SP - 886

EP - 894

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 5

ER -