Product-controlled steady-state kinetics between cytochrome aa3 from Rhodobacter sphaeroides and equine ferrocytochrome c analyzed by a novel spectrophotometric approach

Myat T. Lin, Robert B. Gennis

Research output: Contribution to journalArticle

Abstract

Cytochrome c oxidase (CcO) catalyzes the reduction of molecular oxygen to water using ferrocytochrome c (cyt c2 +) as the electron donor. In this study, the oxidation of horse cyt c2 + by CcO from Rhodobacter sphaeroides, was monitored using stopped-flow spectrophotometry. A novel analytic procedure was applied in which the spectra were deconvoluted into the reduced and oxidized forms of cyt c by a least-squares fitting method, yielding the reaction rates at various concentrations of cyt c2 + and cyt c3 +. This allowed an analysis of the effects of cyt c3 + on the steady-state kinetics between CcO and cyt c2 +. The results show that cyt c3 + exhibits product inhibition by two mechanisms: competition with cyt c2 + at the catalytic site and, in addition, an interaction at a second site which further modulates the reaction of cyt c 2 + at the catalytic site. These results are generally consistent with previous reports, indicating the reliability of the new procedure. We also find that a 6 × His-tag at the C-terminus of the subunit II of CcO affects the binding of cyt c at both sites. The approach presented here should be generally useful in spectrophotometric studies of complex enzyme kinetics.

Original languageEnglish (US)
Pages (from-to)1894-1900
Number of pages7
JournalBiochimica et Biophysica Acta - Bioenergetics
Volume1817
Issue number10
DOIs
StatePublished - Oct 1 2012

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Rhodobacter sphaeroides
Electron Transport Complex IV
Cytochromes c
Horses
His-His-His-His-His-His
Catalytic Domain
Enzyme kinetics
Molecular oxygen
Spectrophotometry
Least-Squares Analysis
Reaction rates
Electrons
Oxygen
Oxidation
Water
Enzymes

Keywords

  • Cytochrome c
  • Cytochrome c oxidase
  • Kinetics

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Cell Biology

Cite this

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title = "Product-controlled steady-state kinetics between cytochrome aa3 from Rhodobacter sphaeroides and equine ferrocytochrome c analyzed by a novel spectrophotometric approach",
abstract = "Cytochrome c oxidase (CcO) catalyzes the reduction of molecular oxygen to water using ferrocytochrome c (cyt c2 +) as the electron donor. In this study, the oxidation of horse cyt c2 + by CcO from Rhodobacter sphaeroides, was monitored using stopped-flow spectrophotometry. A novel analytic procedure was applied in which the spectra were deconvoluted into the reduced and oxidized forms of cyt c by a least-squares fitting method, yielding the reaction rates at various concentrations of cyt c2 + and cyt c3 +. This allowed an analysis of the effects of cyt c3 + on the steady-state kinetics between CcO and cyt c2 +. The results show that cyt c3 + exhibits product inhibition by two mechanisms: competition with cyt c2 + at the catalytic site and, in addition, an interaction at a second site which further modulates the reaction of cyt c 2 + at the catalytic site. These results are generally consistent with previous reports, indicating the reliability of the new procedure. We also find that a 6 × His-tag at the C-terminus of the subunit II of CcO affects the binding of cyt c at both sites. The approach presented here should be generally useful in spectrophotometric studies of complex enzyme kinetics.",
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AU - Lin, Myat T.

AU - Gennis, Robert B.

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Y1 - 2012/10/1

N2 - Cytochrome c oxidase (CcO) catalyzes the reduction of molecular oxygen to water using ferrocytochrome c (cyt c2 +) as the electron donor. In this study, the oxidation of horse cyt c2 + by CcO from Rhodobacter sphaeroides, was monitored using stopped-flow spectrophotometry. A novel analytic procedure was applied in which the spectra were deconvoluted into the reduced and oxidized forms of cyt c by a least-squares fitting method, yielding the reaction rates at various concentrations of cyt c2 + and cyt c3 +. This allowed an analysis of the effects of cyt c3 + on the steady-state kinetics between CcO and cyt c2 +. The results show that cyt c3 + exhibits product inhibition by two mechanisms: competition with cyt c2 + at the catalytic site and, in addition, an interaction at a second site which further modulates the reaction of cyt c 2 + at the catalytic site. These results are generally consistent with previous reports, indicating the reliability of the new procedure. We also find that a 6 × His-tag at the C-terminus of the subunit II of CcO affects the binding of cyt c at both sites. The approach presented here should be generally useful in spectrophotometric studies of complex enzyme kinetics.

AB - Cytochrome c oxidase (CcO) catalyzes the reduction of molecular oxygen to water using ferrocytochrome c (cyt c2 +) as the electron donor. In this study, the oxidation of horse cyt c2 + by CcO from Rhodobacter sphaeroides, was monitored using stopped-flow spectrophotometry. A novel analytic procedure was applied in which the spectra were deconvoluted into the reduced and oxidized forms of cyt c by a least-squares fitting method, yielding the reaction rates at various concentrations of cyt c2 + and cyt c3 +. This allowed an analysis of the effects of cyt c3 + on the steady-state kinetics between CcO and cyt c2 +. The results show that cyt c3 + exhibits product inhibition by two mechanisms: competition with cyt c2 + at the catalytic site and, in addition, an interaction at a second site which further modulates the reaction of cyt c 2 + at the catalytic site. These results are generally consistent with previous reports, indicating the reliability of the new procedure. We also find that a 6 × His-tag at the C-terminus of the subunit II of CcO affects the binding of cyt c at both sites. The approach presented here should be generally useful in spectrophotometric studies of complex enzyme kinetics.

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