Differential effects of glutamate-286 mutations in the aa3-type cytochrome c oxidase from Rhodobacter sphaeroides and the cytochrome bo 3 ubiquinol oxidase from Escherichia coli

Tsuyoshi Egawa, Krithika Ganesan, Myat T. Lin, Michelle A. Yu, Jonathan P. Hosler, Syun-Ru Yeh, Denis L. Rousseau, Robert B. Gennis

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

Both the aa3-type cytochrome c oxidase from Rhodobacter sphaeroides (RsCcOaa3) and the closely related bo3-type ubiquinol oxidase from Escherichia coli (EcQObo3) possess a proton-conducting D-channel that terminates at a glutamic acid, E286, which is critical for controlling proton transfer to the active site for oxygen chemistry and to a proton loading site for proton pumping. E286 mutations in each enzyme block proton flux and, therefore, inhibit oxidase function. In the current work, resonance Raman spectroscopy was used to show that the E286A and E286C mutations in RsCcOaa3 result in long range conformational changes that influence the protein interactions with both heme a and heme a3. Therefore, the severe reduction of the steady-state activity of the E286 mutants in RsCcOaa3 to ~ 0.05% is not simply a result of the direct blockage of the D-channel, but it is also a consequence of the conformational changes induced by the mutations to heme a and to the heme a3-Cu B active site. In contrast, the E286C mutation of EcQObo3 exhibits no evidence of conformational changes at the two heme sites, indicating that its reduced activity (3%) is exclusively a result of the inhibition of proton transfer from the D-channel. We propose that in RsCcOaa3, the E286 mutations severely perturb the active site through a close interaction with F282, which lies between E286 and the heme-copper active site. The local structure around E286 in EcQObo3 is different, providing a rationale for the very different effects of E286 mutations in the two enzymes. This article is part of a Special Issue entitled: Allosteric cooperativity in respiratory proteins.

Original languageEnglish (US)
Pages (from-to)1342-1348
Number of pages7
JournalBiochimica et Biophysica Acta - Bioenergetics
Volume1807
Issue number10
DOIs
StatePublished - Oct 2011

Fingerprint

Rhodobacter sphaeroides
Electron Transport Complex IV
Cytochromes
Escherichia coli
Protons
Glutamic Acid
Mutation
Proton transfer
Catalytic Domain
Heme
Enzymes
Raman spectroscopy
Copper
Oxidoreductases
Proteins
Raman Spectrum Analysis
Oxygen
Fluxes
heme a
ubiquinol oxidase

Keywords

  • Bioenergetics
  • Copper
  • Heme
  • Mutant
  • Proton translocation
  • Raman scattering

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Cell Biology

Cite this

Differential effects of glutamate-286 mutations in the aa3-type cytochrome c oxidase from Rhodobacter sphaeroides and the cytochrome bo 3 ubiquinol oxidase from Escherichia coli. / Egawa, Tsuyoshi; Ganesan, Krithika; Lin, Myat T.; Yu, Michelle A.; Hosler, Jonathan P.; Yeh, Syun-Ru; Rousseau, Denis L.; Gennis, Robert B.

In: Biochimica et Biophysica Acta - Bioenergetics, Vol. 1807, No. 10, 10.2011, p. 1342-1348.

Research output: Contribution to journalArticle

@article{a0d89b49b67a41a1b0dd5cc77fcc8860,
title = "Differential effects of glutamate-286 mutations in the aa3-type cytochrome c oxidase from Rhodobacter sphaeroides and the cytochrome bo 3 ubiquinol oxidase from Escherichia coli",
abstract = "Both the aa3-type cytochrome c oxidase from Rhodobacter sphaeroides (RsCcOaa3) and the closely related bo3-type ubiquinol oxidase from Escherichia coli (EcQObo3) possess a proton-conducting D-channel that terminates at a glutamic acid, E286, which is critical for controlling proton transfer to the active site for oxygen chemistry and to a proton loading site for proton pumping. E286 mutations in each enzyme block proton flux and, therefore, inhibit oxidase function. In the current work, resonance Raman spectroscopy was used to show that the E286A and E286C mutations in RsCcOaa3 result in long range conformational changes that influence the protein interactions with both heme a and heme a3. Therefore, the severe reduction of the steady-state activity of the E286 mutants in RsCcOaa3 to ~ 0.05{\%} is not simply a result of the direct blockage of the D-channel, but it is also a consequence of the conformational changes induced by the mutations to heme a and to the heme a3-Cu B active site. In contrast, the E286C mutation of EcQObo3 exhibits no evidence of conformational changes at the two heme sites, indicating that its reduced activity (3{\%}) is exclusively a result of the inhibition of proton transfer from the D-channel. We propose that in RsCcOaa3, the E286 mutations severely perturb the active site through a close interaction with F282, which lies between E286 and the heme-copper active site. The local structure around E286 in EcQObo3 is different, providing a rationale for the very different effects of E286 mutations in the two enzymes. This article is part of a Special Issue entitled: Allosteric cooperativity in respiratory proteins.",
keywords = "Bioenergetics, Copper, Heme, Mutant, Proton translocation, Raman scattering",
author = "Tsuyoshi Egawa and Krithika Ganesan and Lin, {Myat T.} and Yu, {Michelle A.} and Hosler, {Jonathan P.} and Syun-Ru Yeh and Rousseau, {Denis L.} and Gennis, {Robert B.}",
year = "2011",
month = "10",
doi = "10.1016/j.bbabio.2011.06.001",
language = "English (US)",
volume = "1807",
pages = "1342--1348",
journal = "Biochimica et Biophysica Acta - Bioenergetics",
issn = "0005-2728",
publisher = "Elsevier",
number = "10",

}

TY - JOUR

T1 - Differential effects of glutamate-286 mutations in the aa3-type cytochrome c oxidase from Rhodobacter sphaeroides and the cytochrome bo 3 ubiquinol oxidase from Escherichia coli

AU - Egawa, Tsuyoshi

AU - Ganesan, Krithika

AU - Lin, Myat T.

AU - Yu, Michelle A.

AU - Hosler, Jonathan P.

AU - Yeh, Syun-Ru

AU - Rousseau, Denis L.

AU - Gennis, Robert B.

PY - 2011/10

Y1 - 2011/10

N2 - Both the aa3-type cytochrome c oxidase from Rhodobacter sphaeroides (RsCcOaa3) and the closely related bo3-type ubiquinol oxidase from Escherichia coli (EcQObo3) possess a proton-conducting D-channel that terminates at a glutamic acid, E286, which is critical for controlling proton transfer to the active site for oxygen chemistry and to a proton loading site for proton pumping. E286 mutations in each enzyme block proton flux and, therefore, inhibit oxidase function. In the current work, resonance Raman spectroscopy was used to show that the E286A and E286C mutations in RsCcOaa3 result in long range conformational changes that influence the protein interactions with both heme a and heme a3. Therefore, the severe reduction of the steady-state activity of the E286 mutants in RsCcOaa3 to ~ 0.05% is not simply a result of the direct blockage of the D-channel, but it is also a consequence of the conformational changes induced by the mutations to heme a and to the heme a3-Cu B active site. In contrast, the E286C mutation of EcQObo3 exhibits no evidence of conformational changes at the two heme sites, indicating that its reduced activity (3%) is exclusively a result of the inhibition of proton transfer from the D-channel. We propose that in RsCcOaa3, the E286 mutations severely perturb the active site through a close interaction with F282, which lies between E286 and the heme-copper active site. The local structure around E286 in EcQObo3 is different, providing a rationale for the very different effects of E286 mutations in the two enzymes. This article is part of a Special Issue entitled: Allosteric cooperativity in respiratory proteins.

AB - Both the aa3-type cytochrome c oxidase from Rhodobacter sphaeroides (RsCcOaa3) and the closely related bo3-type ubiquinol oxidase from Escherichia coli (EcQObo3) possess a proton-conducting D-channel that terminates at a glutamic acid, E286, which is critical for controlling proton transfer to the active site for oxygen chemistry and to a proton loading site for proton pumping. E286 mutations in each enzyme block proton flux and, therefore, inhibit oxidase function. In the current work, resonance Raman spectroscopy was used to show that the E286A and E286C mutations in RsCcOaa3 result in long range conformational changes that influence the protein interactions with both heme a and heme a3. Therefore, the severe reduction of the steady-state activity of the E286 mutants in RsCcOaa3 to ~ 0.05% is not simply a result of the direct blockage of the D-channel, but it is also a consequence of the conformational changes induced by the mutations to heme a and to the heme a3-Cu B active site. In contrast, the E286C mutation of EcQObo3 exhibits no evidence of conformational changes at the two heme sites, indicating that its reduced activity (3%) is exclusively a result of the inhibition of proton transfer from the D-channel. We propose that in RsCcOaa3, the E286 mutations severely perturb the active site through a close interaction with F282, which lies between E286 and the heme-copper active site. The local structure around E286 in EcQObo3 is different, providing a rationale for the very different effects of E286 mutations in the two enzymes. This article is part of a Special Issue entitled: Allosteric cooperativity in respiratory proteins.

KW - Bioenergetics

KW - Copper

KW - Heme

KW - Mutant

KW - Proton translocation

KW - Raman scattering

UR - http://www.scopus.com/inward/record.url?scp=80051596768&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=80051596768&partnerID=8YFLogxK

U2 - 10.1016/j.bbabio.2011.06.001

DO - 10.1016/j.bbabio.2011.06.001

M3 - Article

VL - 1807

SP - 1342

EP - 1348

JO - Biochimica et Biophysica Acta - Bioenergetics

JF - Biochimica et Biophysica Acta - Bioenergetics

SN - 0005-2728

IS - 10

ER -