Ligand binding to heme proteins: III. FTIR studies of His-E7 and Val-E11 mutants of carbonmonoxymyoglobin

D. P. Braunstein, K. Chu, K. D. Egeberg, H. Frauenfelder, J. R. Mourant, G. U. Nienhaus, P. Ormos, S. G. Sligar, B. A. Springer, R. D. Young

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Abstract

Fourier-transform infrared (FTIR) difference spectra of several His-E7 and Val-E11 mutants of sperm whale carbonmonoxymyoglobin were obtained by photodissociation at cryogenic temperatures. The IR absorption of the CO ligand shows characteristic features for each of the mutants, both in the ligand-bound (A) state and in the photodissociated (B) state. For most of the mutants, a single A substate band is observed, which points to the crucial role of the His-E7 residue in determining the A substate spectrum of the bound CO in the native structure. The fact that some of the mutants show more than one stretch band of the bound CO indicates that the appearance of multiple A substates is not exclusively connected to the presence of His-E7. In all but one mutant, multiple stretch bands of the CO in the photodissociated state are observed; these B substates are thought to arise from discrete positions and/or orientations of the photodissociated ligand in the heme pocket. The red shifts of the B bands with respect to the free-gas frequency indicate weak binding in the heme pocket. The observation of similar red shifts in microperoxidase (MP-8), where there is no residue on the distal side, suggests that the photodissociated ligand is still associated with the heme iron. Photoselection experiments were performed to determine the orientation of the bound ligand with respect to the heme normal by photolyzing small fractions of the sample with linearly polarized light at 540 nm. The resulting linear dichroism in the CO stretch spectrum yielded angles α ≥ 20° between the CO molecular axis and the heme normal for all of the mutants. We conclude that the off-axis position of the CO ligand in the native structure does not arise from steric constraints imposed by the distal histidine. There is no clear correlation between the size of the distal residue and the angle α of the CO ligand.

Original languageEnglish
Pages (from-to)2447-2454
Number of pages8
JournalBiophysical Journal
Volume65
Issue number6
Publication statusPublished - 1993

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Fourier Analysis
Carbon Monoxide
Ligands
Heme
Sperm Whale
carboxymyoglobin
heme-binding protein
Histidine
Iron
Gases
Observation
Light
Temperature

ASJC Scopus subject areas

  • Biophysics

Cite this

Braunstein, D. P., Chu, K., Egeberg, K. D., Frauenfelder, H., Mourant, J. R., Nienhaus, G. U., ... Young, R. D. (1993). Ligand binding to heme proteins: III. FTIR studies of His-E7 and Val-E11 mutants of carbonmonoxymyoglobin. Biophysical Journal, 65(6), 2447-2454.

Ligand binding to heme proteins : III. FTIR studies of His-E7 and Val-E11 mutants of carbonmonoxymyoglobin. / Braunstein, D. P.; Chu, K.; Egeberg, K. D.; Frauenfelder, H.; Mourant, J. R.; Nienhaus, G. U.; Ormos, P.; Sligar, S. G.; Springer, B. A.; Young, R. D.

In: Biophysical Journal, Vol. 65, No. 6, 1993, p. 2447-2454.

Research output: Contribution to journalArticle

Braunstein, DP, Chu, K, Egeberg, KD, Frauenfelder, H, Mourant, JR, Nienhaus, GU, Ormos, P, Sligar, SG, Springer, BA & Young, RD 1993, 'Ligand binding to heme proteins: III. FTIR studies of His-E7 and Val-E11 mutants of carbonmonoxymyoglobin', Biophysical Journal, vol. 65, no. 6, pp. 2447-2454.
Braunstein DP, Chu K, Egeberg KD, Frauenfelder H, Mourant JR, Nienhaus GU et al. Ligand binding to heme proteins: III. FTIR studies of His-E7 and Val-E11 mutants of carbonmonoxymyoglobin. Biophysical Journal. 1993;65(6):2447-2454.
Braunstein, D. P. ; Chu, K. ; Egeberg, K. D. ; Frauenfelder, H. ; Mourant, J. R. ; Nienhaus, G. U. ; Ormos, P. ; Sligar, S. G. ; Springer, B. A. ; Young, R. D. / Ligand binding to heme proteins : III. FTIR studies of His-E7 and Val-E11 mutants of carbonmonoxymyoglobin. In: Biophysical Journal. 1993 ; Vol. 65, No. 6. pp. 2447-2454.
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abstract = "Fourier-transform infrared (FTIR) difference spectra of several His-E7 and Val-E11 mutants of sperm whale carbonmonoxymyoglobin were obtained by photodissociation at cryogenic temperatures. The IR absorption of the CO ligand shows characteristic features for each of the mutants, both in the ligand-bound (A) state and in the photodissociated (B) state. For most of the mutants, a single A substate band is observed, which points to the crucial role of the His-E7 residue in determining the A substate spectrum of the bound CO in the native structure. The fact that some of the mutants show more than one stretch band of the bound CO indicates that the appearance of multiple A substates is not exclusively connected to the presence of His-E7. In all but one mutant, multiple stretch bands of the CO in the photodissociated state are observed; these B substates are thought to arise from discrete positions and/or orientations of the photodissociated ligand in the heme pocket. The red shifts of the B bands with respect to the free-gas frequency indicate weak binding in the heme pocket. The observation of similar red shifts in microperoxidase (MP-8), where there is no residue on the distal side, suggests that the photodissociated ligand is still associated with the heme iron. Photoselection experiments were performed to determine the orientation of the bound ligand with respect to the heme normal by photolyzing small fractions of the sample with linearly polarized light at 540 nm. The resulting linear dichroism in the CO stretch spectrum yielded angles α ≥ 20° between the CO molecular axis and the heme normal for all of the mutants. We conclude that the off-axis position of the CO ligand in the native structure does not arise from steric constraints imposed by the distal histidine. There is no clear correlation between the size of the distal residue and the angle α of the CO ligand.",
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T2 - III. FTIR studies of His-E7 and Val-E11 mutants of carbonmonoxymyoglobin

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AU - Egeberg, K. D.

AU - Frauenfelder, H.

AU - Mourant, J. R.

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N2 - Fourier-transform infrared (FTIR) difference spectra of several His-E7 and Val-E11 mutants of sperm whale carbonmonoxymyoglobin were obtained by photodissociation at cryogenic temperatures. The IR absorption of the CO ligand shows characteristic features for each of the mutants, both in the ligand-bound (A) state and in the photodissociated (B) state. For most of the mutants, a single A substate band is observed, which points to the crucial role of the His-E7 residue in determining the A substate spectrum of the bound CO in the native structure. The fact that some of the mutants show more than one stretch band of the bound CO indicates that the appearance of multiple A substates is not exclusively connected to the presence of His-E7. In all but one mutant, multiple stretch bands of the CO in the photodissociated state are observed; these B substates are thought to arise from discrete positions and/or orientations of the photodissociated ligand in the heme pocket. The red shifts of the B bands with respect to the free-gas frequency indicate weak binding in the heme pocket. The observation of similar red shifts in microperoxidase (MP-8), where there is no residue on the distal side, suggests that the photodissociated ligand is still associated with the heme iron. Photoselection experiments were performed to determine the orientation of the bound ligand with respect to the heme normal by photolyzing small fractions of the sample with linearly polarized light at 540 nm. The resulting linear dichroism in the CO stretch spectrum yielded angles α ≥ 20° between the CO molecular axis and the heme normal for all of the mutants. We conclude that the off-axis position of the CO ligand in the native structure does not arise from steric constraints imposed by the distal histidine. There is no clear correlation between the size of the distal residue and the angle α of the CO ligand.

AB - Fourier-transform infrared (FTIR) difference spectra of several His-E7 and Val-E11 mutants of sperm whale carbonmonoxymyoglobin were obtained by photodissociation at cryogenic temperatures. The IR absorption of the CO ligand shows characteristic features for each of the mutants, both in the ligand-bound (A) state and in the photodissociated (B) state. For most of the mutants, a single A substate band is observed, which points to the crucial role of the His-E7 residue in determining the A substate spectrum of the bound CO in the native structure. The fact that some of the mutants show more than one stretch band of the bound CO indicates that the appearance of multiple A substates is not exclusively connected to the presence of His-E7. In all but one mutant, multiple stretch bands of the CO in the photodissociated state are observed; these B substates are thought to arise from discrete positions and/or orientations of the photodissociated ligand in the heme pocket. The red shifts of the B bands with respect to the free-gas frequency indicate weak binding in the heme pocket. The observation of similar red shifts in microperoxidase (MP-8), where there is no residue on the distal side, suggests that the photodissociated ligand is still associated with the heme iron. Photoselection experiments were performed to determine the orientation of the bound ligand with respect to the heme normal by photolyzing small fractions of the sample with linearly polarized light at 540 nm. The resulting linear dichroism in the CO stretch spectrum yielded angles α ≥ 20° between the CO molecular axis and the heme normal for all of the mutants. We conclude that the off-axis position of the CO ligand in the native structure does not arise from steric constraints imposed by the distal histidine. There is no clear correlation between the size of the distal residue and the angle α of the CO ligand.

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