Structural characterization of cytochrome c peroxidase by resonance Raman scattering

S. Dasgupta, Denis L. Rousseau, H. Anni, T. Yonetani

Research output: Contribution to journalArticle

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Abstract

Resonance Raman scattering studies are reported on freshly prepared and aged ferric, ligand-free ferrous, and CO-bound ferrous cytochrome c peroxidase. The ferric form of the fresh enzyme has a heme which is penta-coordinate high spin, independent of buffer over the pH range 4.3-7, as determined by well established Raman marker lines. The aged enzyme displays a mixture of spin and coordination states, but it can be stabilized in the penta-coordinate high spin form in the presence of phosphate. These results can be accounted for by considering the size of the channel (6 Å wide, 11 Å long) between the distal side of the heme and the outer surface of the protein. A phosphate ion may be accommodated in this channel resulting in the stabilization of the distal heme pocket. The ferrous cytochrome c peroxidase in both the ligand-free and CO-bound states has an acidic and an alkaline form. The acidic form has the characteristic spectral features of peroxidases: a high frequency iron-histidine stretching mode (248 cm-1), a high frequency Fe-CO stretching mode (537 cm-1), and a low frequency C-O stretching mode (1922 cm-1). At alkaline pH these frequencies become similar to those of hemoglobin and myoglobin, with the corresponding modes located at 227, 510, and 1948 cm-1, respectively. We attribute the acid/alkaline transition in the ferrous forms of cytochrome c peroxidase to a rearrangement mainly of the proximal side of the heme, culminating in a change of steric interactions between the proximal histidine and the heme or of the hydrogen bonding network involving the proximal histidine. The new data presented here reconcile many inconsistencies reported in the past.

Original languageEnglish (US)
Pages (from-to)654-662
Number of pages9
JournalJournal of Biological Chemistry
Volume264
Issue number1
StatePublished - 1989
Externally publishedYes

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Cytochrome-c Peroxidase
Raman Spectrum Analysis
Heme
Raman scattering
Carbon Monoxide
Histidine
Stretching
Rubiaceae
Phosphates
Ligands
Peroxidases
Myoglobin
Enzymes
Hydrogen Bonding
Hydrogen bonds
Buffers
Membrane Proteins
Hemoglobins
Iron
Stabilization

ASJC Scopus subject areas

  • Biochemistry

Cite this

Structural characterization of cytochrome c peroxidase by resonance Raman scattering. / Dasgupta, S.; Rousseau, Denis L.; Anni, H.; Yonetani, T.

In: Journal of Biological Chemistry, Vol. 264, No. 1, 1989, p. 654-662.

Research output: Contribution to journalArticle

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N2 - Resonance Raman scattering studies are reported on freshly prepared and aged ferric, ligand-free ferrous, and CO-bound ferrous cytochrome c peroxidase. The ferric form of the fresh enzyme has a heme which is penta-coordinate high spin, independent of buffer over the pH range 4.3-7, as determined by well established Raman marker lines. The aged enzyme displays a mixture of spin and coordination states, but it can be stabilized in the penta-coordinate high spin form in the presence of phosphate. These results can be accounted for by considering the size of the channel (6 Å wide, 11 Å long) between the distal side of the heme and the outer surface of the protein. A phosphate ion may be accommodated in this channel resulting in the stabilization of the distal heme pocket. The ferrous cytochrome c peroxidase in both the ligand-free and CO-bound states has an acidic and an alkaline form. The acidic form has the characteristic spectral features of peroxidases: a high frequency iron-histidine stretching mode (248 cm-1), a high frequency Fe-CO stretching mode (537 cm-1), and a low frequency C-O stretching mode (1922 cm-1). At alkaline pH these frequencies become similar to those of hemoglobin and myoglobin, with the corresponding modes located at 227, 510, and 1948 cm-1, respectively. We attribute the acid/alkaline transition in the ferrous forms of cytochrome c peroxidase to a rearrangement mainly of the proximal side of the heme, culminating in a change of steric interactions between the proximal histidine and the heme or of the hydrogen bonding network involving the proximal histidine. The new data presented here reconcile many inconsistencies reported in the past.

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