Heme-heme and heme-ligand interactions in the di-heme oxygen-reducing site of cytochrome bd from Escherichia coli revealed by nanosecond absorption spectroscopy

Fabrice Rappaport, Jie Zhang, Marten H. Vos, Robert B. Gennis, Vitaliy B. Borisov

Research output: Contribution to journalArticle

22 Citations (Scopus)

Abstract

Cytochrome bd is a terminal quinol:O2 oxidoreductase of respiratory chains of many bacteria. It contains three hemes, b558, b595, and d. The role of heme b595 remains obscure. A CO photolysis/recombination study of the membranes of Escherichia coli containing either wild type cytochrome bd or inactive E445A mutant was performed using nanosecond absorption spectroscopy. We compared photoinduced changes of heme d-CO complex in one-electron-reduced, two-electron-reduced, and fully reduced states of cytochromes bd. The line shape of spectra of photodissociation of one-electron-reduced and two-electron-reduced enzymes is strikingly different from that of the fully reduced enzyme. The difference demonstrates that in the fully reduced enzyme photolysis of CO from heme d perturbs ferrous heme b595 causing loss of an absorption band centered at 435nm, thus supporting interactions between heme b595 and heme d in the di-heme oxygen-reducing site, in agreement with previous works. Photolyzed CO recombines with the fully reduced enzyme monoexponentially with Τ~12μs, whereas recombination of CO with one-electron-reduced cytochrome bd shows three kinetic phases, with Τ~14ns, 14μs, and 280μs. The spectra of the absorption changes associated with these components are different in line shape. The 14ns phase, absent in the fully reduced enzyme, reflects geminate recombination of CO with part of heme d. The 14-μs component reflects bimolecular recombination of CO with heme d and electron backflow from heme d to hemes b in ~4% of the enzyme population. The final, 280-μs component, reflects return of the electron from hemes b to heme d and bimolecular recombination of CO in that population. The fact that even in the two-electron-reduced enzyme, a nanosecond geminate recombination is observed, suggests that namely the redox state of heme b595, and not that of heme b558, controls the pathway(s) by which CO migrates between heme d and the medium.

Original languageEnglish (US)
Pages (from-to)1657-1664
Number of pages8
JournalBiochimica et Biophysica Acta - Bioenergetics
Volume1797
Issue number9
DOIs
StatePublished - Sep 1 2010
Externally publishedYes

Fingerprint

Carbon Monoxide
Cytochromes
Heme
Absorption spectroscopy
Escherichia coli
Spectrum Analysis
Oxygen
Ligands
Electrons
Genetic Recombination
Enzymes
Photolysis
Hydroquinones
Photodissociation
heme d
Electron Transport
Population
Oxidation-Reduction
Absorption spectra
Bacteria

Keywords

  • Chlorin
  • Cytochrome
  • Gas molecule
  • Ligand binding
  • Photobiology
  • Respiration

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Cell Biology

Cite this

Heme-heme and heme-ligand interactions in the di-heme oxygen-reducing site of cytochrome bd from Escherichia coli revealed by nanosecond absorption spectroscopy. / Rappaport, Fabrice; Zhang, Jie; Vos, Marten H.; Gennis, Robert B.; Borisov, Vitaliy B.

In: Biochimica et Biophysica Acta - Bioenergetics, Vol. 1797, No. 9, 01.09.2010, p. 1657-1664.

Research output: Contribution to journalArticle

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abstract = "Cytochrome bd is a terminal quinol:O2 oxidoreductase of respiratory chains of many bacteria. It contains three hemes, b558, b595, and d. The role of heme b595 remains obscure. A CO photolysis/recombination study of the membranes of Escherichia coli containing either wild type cytochrome bd or inactive E445A mutant was performed using nanosecond absorption spectroscopy. We compared photoinduced changes of heme d-CO complex in one-electron-reduced, two-electron-reduced, and fully reduced states of cytochromes bd. The line shape of spectra of photodissociation of one-electron-reduced and two-electron-reduced enzymes is strikingly different from that of the fully reduced enzyme. The difference demonstrates that in the fully reduced enzyme photolysis of CO from heme d perturbs ferrous heme b595 causing loss of an absorption band centered at 435nm, thus supporting interactions between heme b595 and heme d in the di-heme oxygen-reducing site, in agreement with previous works. Photolyzed CO recombines with the fully reduced enzyme monoexponentially with Τ~12μs, whereas recombination of CO with one-electron-reduced cytochrome bd shows three kinetic phases, with Τ~14ns, 14μs, and 280μs. The spectra of the absorption changes associated with these components are different in line shape. The 14ns phase, absent in the fully reduced enzyme, reflects geminate recombination of CO with part of heme d. The 14-μs component reflects bimolecular recombination of CO with heme d and electron backflow from heme d to hemes b in ~4{\%} of the enzyme population. The final, 280-μs component, reflects return of the electron from hemes b to heme d and bimolecular recombination of CO in that population. The fact that even in the two-electron-reduced enzyme, a nanosecond geminate recombination is observed, suggests that namely the redox state of heme b595, and not that of heme b558, controls the pathway(s) by which CO migrates between heme d and the medium.",
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T1 - Heme-heme and heme-ligand interactions in the di-heme oxygen-reducing site of cytochrome bd from Escherichia coli revealed by nanosecond absorption spectroscopy

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AU - Zhang, Jie

AU - Vos, Marten H.

AU - Gennis, Robert B.

AU - Borisov, Vitaliy B.

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N2 - Cytochrome bd is a terminal quinol:O2 oxidoreductase of respiratory chains of many bacteria. It contains three hemes, b558, b595, and d. The role of heme b595 remains obscure. A CO photolysis/recombination study of the membranes of Escherichia coli containing either wild type cytochrome bd or inactive E445A mutant was performed using nanosecond absorption spectroscopy. We compared photoinduced changes of heme d-CO complex in one-electron-reduced, two-electron-reduced, and fully reduced states of cytochromes bd. The line shape of spectra of photodissociation of one-electron-reduced and two-electron-reduced enzymes is strikingly different from that of the fully reduced enzyme. The difference demonstrates that in the fully reduced enzyme photolysis of CO from heme d perturbs ferrous heme b595 causing loss of an absorption band centered at 435nm, thus supporting interactions between heme b595 and heme d in the di-heme oxygen-reducing site, in agreement with previous works. Photolyzed CO recombines with the fully reduced enzyme monoexponentially with Τ~12μs, whereas recombination of CO with one-electron-reduced cytochrome bd shows three kinetic phases, with Τ~14ns, 14μs, and 280μs. The spectra of the absorption changes associated with these components are different in line shape. The 14ns phase, absent in the fully reduced enzyme, reflects geminate recombination of CO with part of heme d. The 14-μs component reflects bimolecular recombination of CO with heme d and electron backflow from heme d to hemes b in ~4% of the enzyme population. The final, 280-μs component, reflects return of the electron from hemes b to heme d and bimolecular recombination of CO in that population. The fact that even in the two-electron-reduced enzyme, a nanosecond geminate recombination is observed, suggests that namely the redox state of heme b595, and not that of heme b558, controls the pathway(s) by which CO migrates between heme d and the medium.

AB - Cytochrome bd is a terminal quinol:O2 oxidoreductase of respiratory chains of many bacteria. It contains three hemes, b558, b595, and d. The role of heme b595 remains obscure. A CO photolysis/recombination study of the membranes of Escherichia coli containing either wild type cytochrome bd or inactive E445A mutant was performed using nanosecond absorption spectroscopy. We compared photoinduced changes of heme d-CO complex in one-electron-reduced, two-electron-reduced, and fully reduced states of cytochromes bd. The line shape of spectra of photodissociation of one-electron-reduced and two-electron-reduced enzymes is strikingly different from that of the fully reduced enzyme. The difference demonstrates that in the fully reduced enzyme photolysis of CO from heme d perturbs ferrous heme b595 causing loss of an absorption band centered at 435nm, thus supporting interactions between heme b595 and heme d in the di-heme oxygen-reducing site, in agreement with previous works. Photolyzed CO recombines with the fully reduced enzyme monoexponentially with Τ~12μs, whereas recombination of CO with one-electron-reduced cytochrome bd shows three kinetic phases, with Τ~14ns, 14μs, and 280μs. The spectra of the absorption changes associated with these components are different in line shape. The 14ns phase, absent in the fully reduced enzyme, reflects geminate recombination of CO with part of heme d. The 14-μs component reflects bimolecular recombination of CO with heme d and electron backflow from heme d to hemes b in ~4% of the enzyme population. The final, 280-μs component, reflects return of the electron from hemes b to heme d and bimolecular recombination of CO in that population. The fact that even in the two-electron-reduced enzyme, a nanosecond geminate recombination is observed, suggests that namely the redox state of heme b595, and not that of heme b558, controls the pathway(s) by which CO migrates between heme d and the medium.

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KW - Photobiology

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