Direct calorimetric analysis of the enzymatic activity of yeast cytochrome c oxidase

Paul E. Morin, Ernesto I Freire

Research output: Contribution to journalArticle

Abstract

The kinetic and thermodynamic parameters associated with the enzymatic reaction of yeast cytochrome c oxidase with its biological substrate, ferrocytochrome c, have been measured by using a titration microcalorimeter to monitor directly the rate of heat production or absorption as a function of time. This technique has allowed determination of both the energetics and the kinetics of the reaction under a variety of conditions within a single experiment. Experiments performed in buffer systems of varying ionization enthalpies allow determination of the net number of protons absorbed or released during the course of the reaction. For cytochrome c oxidase the intrinsic enthalpy of reaction was determined to be -16.5 kcal/mol with one (0.96) proton consumed for each ferrocytochrome c molecule oxidized. Activity measurements at salt concentrations ranging from 0 to 200 mM KCl in the presence of 10 mM potassium phosphate, pH 7.40, and 0.5 mM EDTA display a biphasic dependence of the electron transferase activity upon ionic strength with a peak activity observed near 50 mM KCl. The ionic strength dependence was similar for both detergent-solubilized and membrane-reconstituted cytochrome c oxidase. Despite the large ionic strength dependence of the kinetic parameters, the enthalpy measured for the reaction was found to be independent of ionic strength. Additional experiments involving direct transfer of the enzyme from low to high salt conditions produced negligible enthalpy changes that remained constant within experimental error throughout the salt concentrations studied (0-200 mM KCl). These results indicate that the salt effect on the enzyme activity is of entropic origin and further suggest the absence of a major conformational change in the enzyme due to changes in ionic strength. High-sensitivity differential scanning calorimetric experiments performed at very low ionic strength show a calorimetric transition profile characterized by two well-defined peaks centered at 50.5 and 60°C. At high ionic strength, the high-temperature peak shifts to lower temperatures (57.4°C), giving rise to a single but slightly asymetric transition profile. No shift in the transition temperature was observed for the low-temperature peak under the experimental conditions studied. These results suggest that variation of ionic strength primarily affects the structural stability of the protein subunits giving rise to the main transition peak, primarily subunits I and II.

Original languageEnglish (US)
Pages (from-to)8494-8500
Number of pages7
JournalBiochemistry®
Volume30
Issue number34
StatePublished - 1991

Fingerprint

Electron Transport Complex IV
Ionic strength
Yeast
Osmolar Concentration
Yeasts
Enthalpy
Salts
Cytochromes c
Temperature
Protons
Enzymes
Experiments
Kinetics
Thermogenesis
Transition Temperature
Protein Subunits
Enzyme activity
Transferases
Titration
Thermodynamics

ASJC Scopus subject areas

  • Biochemistry

Cite this

Direct calorimetric analysis of the enzymatic activity of yeast cytochrome c oxidase. / Morin, Paul E.; Freire, Ernesto I.

In: Biochemistry®, Vol. 30, No. 34, 1991, p. 8494-8500.

Research output: Contribution to journalArticle

@article{8abfc50d280c43e49ddd0f0fe888d108,
title = "Direct calorimetric analysis of the enzymatic activity of yeast cytochrome c oxidase",
abstract = "The kinetic and thermodynamic parameters associated with the enzymatic reaction of yeast cytochrome c oxidase with its biological substrate, ferrocytochrome c, have been measured by using a titration microcalorimeter to monitor directly the rate of heat production or absorption as a function of time. This technique has allowed determination of both the energetics and the kinetics of the reaction under a variety of conditions within a single experiment. Experiments performed in buffer systems of varying ionization enthalpies allow determination of the net number of protons absorbed or released during the course of the reaction. For cytochrome c oxidase the intrinsic enthalpy of reaction was determined to be -16.5 kcal/mol with one (0.96) proton consumed for each ferrocytochrome c molecule oxidized. Activity measurements at salt concentrations ranging from 0 to 200 mM KCl in the presence of 10 mM potassium phosphate, pH 7.40, and 0.5 mM EDTA display a biphasic dependence of the electron transferase activity upon ionic strength with a peak activity observed near 50 mM KCl. The ionic strength dependence was similar for both detergent-solubilized and membrane-reconstituted cytochrome c oxidase. Despite the large ionic strength dependence of the kinetic parameters, the enthalpy measured for the reaction was found to be independent of ionic strength. Additional experiments involving direct transfer of the enzyme from low to high salt conditions produced negligible enthalpy changes that remained constant within experimental error throughout the salt concentrations studied (0-200 mM KCl). These results indicate that the salt effect on the enzyme activity is of entropic origin and further suggest the absence of a major conformational change in the enzyme due to changes in ionic strength. High-sensitivity differential scanning calorimetric experiments performed at very low ionic strength show a calorimetric transition profile characterized by two well-defined peaks centered at 50.5 and 60°C. At high ionic strength, the high-temperature peak shifts to lower temperatures (57.4°C), giving rise to a single but slightly asymetric transition profile. No shift in the transition temperature was observed for the low-temperature peak under the experimental conditions studied. These results suggest that variation of ionic strength primarily affects the structural stability of the protein subunits giving rise to the main transition peak, primarily subunits I and II.",
author = "Morin, {Paul E.} and Freire, {Ernesto I}",
year = "1991",
language = "English (US)",
volume = "30",
pages = "8494--8500",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "34",

}

TY - JOUR

T1 - Direct calorimetric analysis of the enzymatic activity of yeast cytochrome c oxidase

AU - Morin, Paul E.

AU - Freire, Ernesto I

PY - 1991

Y1 - 1991

N2 - The kinetic and thermodynamic parameters associated with the enzymatic reaction of yeast cytochrome c oxidase with its biological substrate, ferrocytochrome c, have been measured by using a titration microcalorimeter to monitor directly the rate of heat production or absorption as a function of time. This technique has allowed determination of both the energetics and the kinetics of the reaction under a variety of conditions within a single experiment. Experiments performed in buffer systems of varying ionization enthalpies allow determination of the net number of protons absorbed or released during the course of the reaction. For cytochrome c oxidase the intrinsic enthalpy of reaction was determined to be -16.5 kcal/mol with one (0.96) proton consumed for each ferrocytochrome c molecule oxidized. Activity measurements at salt concentrations ranging from 0 to 200 mM KCl in the presence of 10 mM potassium phosphate, pH 7.40, and 0.5 mM EDTA display a biphasic dependence of the electron transferase activity upon ionic strength with a peak activity observed near 50 mM KCl. The ionic strength dependence was similar for both detergent-solubilized and membrane-reconstituted cytochrome c oxidase. Despite the large ionic strength dependence of the kinetic parameters, the enthalpy measured for the reaction was found to be independent of ionic strength. Additional experiments involving direct transfer of the enzyme from low to high salt conditions produced negligible enthalpy changes that remained constant within experimental error throughout the salt concentrations studied (0-200 mM KCl). These results indicate that the salt effect on the enzyme activity is of entropic origin and further suggest the absence of a major conformational change in the enzyme due to changes in ionic strength. High-sensitivity differential scanning calorimetric experiments performed at very low ionic strength show a calorimetric transition profile characterized by two well-defined peaks centered at 50.5 and 60°C. At high ionic strength, the high-temperature peak shifts to lower temperatures (57.4°C), giving rise to a single but slightly asymetric transition profile. No shift in the transition temperature was observed for the low-temperature peak under the experimental conditions studied. These results suggest that variation of ionic strength primarily affects the structural stability of the protein subunits giving rise to the main transition peak, primarily subunits I and II.

AB - The kinetic and thermodynamic parameters associated with the enzymatic reaction of yeast cytochrome c oxidase with its biological substrate, ferrocytochrome c, have been measured by using a titration microcalorimeter to monitor directly the rate of heat production or absorption as a function of time. This technique has allowed determination of both the energetics and the kinetics of the reaction under a variety of conditions within a single experiment. Experiments performed in buffer systems of varying ionization enthalpies allow determination of the net number of protons absorbed or released during the course of the reaction. For cytochrome c oxidase the intrinsic enthalpy of reaction was determined to be -16.5 kcal/mol with one (0.96) proton consumed for each ferrocytochrome c molecule oxidized. Activity measurements at salt concentrations ranging from 0 to 200 mM KCl in the presence of 10 mM potassium phosphate, pH 7.40, and 0.5 mM EDTA display a biphasic dependence of the electron transferase activity upon ionic strength with a peak activity observed near 50 mM KCl. The ionic strength dependence was similar for both detergent-solubilized and membrane-reconstituted cytochrome c oxidase. Despite the large ionic strength dependence of the kinetic parameters, the enthalpy measured for the reaction was found to be independent of ionic strength. Additional experiments involving direct transfer of the enzyme from low to high salt conditions produced negligible enthalpy changes that remained constant within experimental error throughout the salt concentrations studied (0-200 mM KCl). These results indicate that the salt effect on the enzyme activity is of entropic origin and further suggest the absence of a major conformational change in the enzyme due to changes in ionic strength. High-sensitivity differential scanning calorimetric experiments performed at very low ionic strength show a calorimetric transition profile characterized by two well-defined peaks centered at 50.5 and 60°C. At high ionic strength, the high-temperature peak shifts to lower temperatures (57.4°C), giving rise to a single but slightly asymetric transition profile. No shift in the transition temperature was observed for the low-temperature peak under the experimental conditions studied. These results suggest that variation of ionic strength primarily affects the structural stability of the protein subunits giving rise to the main transition peak, primarily subunits I and II.

UR - http://www.scopus.com/inward/record.url?scp=0026052762&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0026052762&partnerID=8YFLogxK

M3 - Article

C2 - 1653014

AN - SCOPUS:0026052762

VL - 30

SP - 8494

EP - 8500

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 34

ER -