Cykl Krebsa, łańcuch
oddechowy i elementy
bioenergetyki
A skąd acetylo-CoA?
Glukoza; 6C
Glikoliza
Glukoneogeneza
2 x Pirogronian; 3C
Obecność tlenu
2 x NADH
i 2 x CO2
2 x Acetylo-koenzym A
(Ac-CoA); 2C
Brak tlenu
(Fermentacja)
2 x Mleczan
Dekarboksylacja
oksydacyjna pirogronianu
CoA-SH
Dekarboksylacja
oksydacyjna pirogronianu
Co dalej z Ac-CoA?
szczawiooctan
cytrynian
FADH2
FAD
FADH2
FAD
FADH2
FAD
Cykl Krebsa
inaczej cykl kwasu cytrynowego
cyklu
zachodzi
Krebsa
w mitochondriach
• Efekt
Całkowite
utlenienie
“glukozy”!
•
• 3 x NADH
1
x
FADH
•
• 1 x GTP 2
= 1 x ATP
Przekaźniki elektronów
FADH2
NADH
Glukoza
Glukoneogeneza
Glikoliza
Cykl Krebsa
Pirogronian
CO2
Glukoza
β-oksydacja
Glukoneogeneza
Glikoliza
Cykl Krebsa
Pirogronian
CO2
β-oksydacja
inaczej cykl kwasu cytrynowego
zachodzi w mitochondriach
β-oksydacja
•
• Ostateczny produkt, ac-CoA,
inaczej cykl kwasu cytrynowego
Utlenianie
kwasów
tłuszczowych
zachodzi w mitochondriach
przekazywany jest do cyklu Krebsa
• Przy każdej rundzie β-oksydacji
powstaje:
• 1 x NADH, 1 x FADH • 1 x ac-CoA
2
β-oksydacja
inaczej cykl kwasu cytrynowego
zachodzi w mitochondriach
• A po β-oksydacji 16:0?
7
x
NADH,
7
x
FADH
•
• 7 x ac-CoA (jako produkt każdego
2
“cyklu” β-oksydacji)
• 1 x ac-CoA (jako ostateczna forma
KT)
Ciała ketonowe
• Kwas acetylooctowy
• Produkt pośredni β-oksydacji
• Możliwa synteza z 2 ac-CoA
• Szczególnie przy braku glukozy
• Prowadzi do kwasicy ketonowej
Jakie są dalsze losy
NADH i FADH2?
Potencjał oksydoredukcyjny
NADH
H+
+
H
+ H+
H
H+
+
H
H+ H+
+
H
+
H
H+
e
H+
H+
H+
+
H
H+
H+
H+
NADH = NAD+ + 2e- + H+
H+
H+
H+
H+
H+
+
+
H
H
+
+
H
H
H+
H+
H+
H+
H+
H+
H+
H+
H+
H+
H
+ H+
H+
H+
H+
+
H
H+
H+
H+ H+
H+
H+
H+
+
+
H
H
+
+
H
H
e
NADH = NAD+ + 2e- + H+
H+
H+
H+
H+
H+
H+
H+
H+
H+
H+
H
+ H+
H+
H+
H+
+
H
H+
H+
H+ H+
H+
H+
H+
+
+
H
H
+
+
H
H
e
O2
NADH = NAD+ + 2e- + H+
O2
O2
H+
H+
H+
H+
H+
H+
H+
H+
H+
H+
H
+ H+
H+
H+
H+
+
H
H+
H+
H+ H+
H+
H+
H+
+
+
H
H
+
+
H
H
e
NADH = NAD+ + 2e- + H+
2 x H2O
2 x H2O
2 x H2O
H+
+
H
+
+
+
H
H
+
+
+
+
H
H
H
H
+
H
+
+
H
+
+
H
H
+
H
H
H H+
+
H
+
+
+
H
H
+
+
+
+
+
+
H
H
H
+
+
H
H
H
H
+
+
H
H
H
H+ H+ H
H+
+
H
+
+
+
H
H
+
+
+
H
H
H
H
+
+
+
H
+
H
H
H
H+ H+
+
H
+
+
+
H
H
+
+
+
+
+
H
H H H
+
+
H
H
+
+
+
+
H
H
H
H
H
H
H+
H+
H+
H+
+
H
+
+
+
H
H
+
+
+
H
H
H
H
+
+
+
H
+
H
H
H
H+ H+
+
H
+
+
+
H
H
+
+
+
+
+
H
H H H
+
+
H
H
+
+
+
+
H
H
H
H
H
H
H+
H+
H+
}
Łańcuch transportu
elektronów
Wykorzystanie energii elektronów
do wyrzutu H+ do przestrzeni
międzybłonowej mitochondrium
Chemiosmoza
Wykorzystanie energii potencjalnej
w postaci różnicy stężeń H+ do
syntezy ATP
Fosforylacja
oksydacyjna
Koszt transportu
Rozprzęganie
Trucizny
• Kompleks I: rotenon
• Kompleks III: antymycyna A
• Kompleks IV: cyjanki, azydki, CO?
Bilans energetyczny
pełnego utlenienia
1 cząsteczki glukozy
liczba protonów
przeniesionych
do przestrzeni
miêdzyb≥onowej
glukoza
ATP
glikoliza
pirogronian
2
2 NADH
20
2 NADH
20
acetylo-CoA
cykl
Krebsa
2
6 NADH
60
2 innyFADH
przekaŸnik
2
12
-4 na transport
+
H
NADH i ATP
24,94
112
112
108
ok.
ok.29
26
+
Dlaczego 112 H daje
ok. 26 cząsteczek ATP?
+
Dlaczego 112 H daje
ok. 26 cząsteczek ATP?
+
H /ATP
Stosunek
wynosi(ł) ok. 4,33
+
+
Comparison of the H /ATP ratios of the H -ATP
synthases from yeast and from chloroplast
Jan Petersena, Kathrin Försterb, Paola Turinac, and Peter Gräberb,1
a
Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3800, Australia; bDepartment of Physical Chemistry, University of
Freiburg, D-79104 Freiburg, Germany; and cLaboratory of Biochemistry and Biophysics, Department of Biology, University of Bologna, I-40126 Bologna, Italy
Edited by Pierre A. Joliot, Institut de Biologie Physico-Chimique, Paris, France, and approved May 28, 2012 (received for review February 24, 2012)
F0F1-ATP synthases use the free energy derived from a transmembrane proton transport to synthesize ATP from ADP and inorganic
phosphate. The number of protons translocated per ATP (H+/ATP
ratio) is an important parameter for the mechanism of the enzyme
and for energy transduction in cells. Current models of rotational
catalysis predict that the H+/ATP ratio is identical to the stoichiometric ratio of c-subunits to β-subunits. We measured in parallel
the H+/ATP ratios at equilibrium of purified F0F1s from yeast mitochondria (c/β = 3.3) and from spinach chloroplasts (c/β = 4.7). The
isolated enzymes were reconstituted into liposomes and, after
energization of the proteoliposomes with acid–base transitions,
the initial rates of ATP synthesis and hydrolysis were measured
as a function of ΔpH. The equilibrium ΔpH was obtained by interpolation, and from its dependency on the stoichiometric ratio,
[ATP]/([ADP]·[Pi]), finally the thermodynamic H+/ATP ratios were
obtained: 2.9 ± 0.2 for the mitochondrial enzyme and 3.9 ± 0.3
for the chloroplast enzyme. The data show that the thermodynamic H+/ATP ratio depends on the stoichiometry of the c-subunit,
although it is not identical to the c/β ratio.
chemiosmotic theory
C
| protonmotive force | bioenergetics | nanomachine
ells of all life kingdoms use H+-ATP synthases to produce
the cellular energy carrier ATP from the energy of a transmembrane electrochemical potential difference of protons built
up and maintained by proton transport mechanisms, such as the
oxidative electron transport in mitochondria or the photoinduced electron transport in chloroplasts (1). The number of
protons translocated for each synthesized ATP molecule (H+/
stoichiometry of the latter is 14, according to X-ray crystallography
(14, 15) and atomic force microscopy of the isolated c-ring (16).
Hence, their respective H+/ATP ratios, based on the assumptions
mentioned above, should be 3.3 and 4.7. We have isolated the two
complexes and reconstituted them into liposomes, which, if subjected to acid–base transitions to generate a high protonmotive
force, were able to synthesize ATP at physiological rates (17, 18).
The technique of acid–base transitions offers the great advantages
of (i) measuring the imposed transmembrane ΔpH with the accuracy of the pH electrode, thus making high-precision quantitative studies possible (18–20), and (ii) allowing the testing of
ATP synthases from different species with the same method and
under identical experimental conditions.
Results
According to the chemiosmotic theory (1), the synthesis of ATP
catalyzed by the H+-ATP synthase is coupled to the translocation of n protons from the internal to the external
compartment:
ADP þ Pi þ nH þ in ↔ ATP þ H2 O þ nH þ out :
[1]
The factor n is the number of protons translocated per ATP, and
it is called the thermodynamic H+/ATP ratio. The Gibbs free
energy of this coupled reaction can be expressed as:
μHþ
ΔG′ ¼ ΔG′p − nΔ~
¼ ΔG8′p þ 2:3 RT logðQÞ − nð2:3 RT ΔpH þ FΔφÞ;
[2]
+
+
Comparison of the H /ATP ratios of the H -ATP
synthases from yeast and from chloroplast
Jan Petersena, Kathrin Försterb, Paola Turinac, and Peter Gräberb,1
a
Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3800, Australia; bDepartment of Physical Chemistry, University of
Freiburg, D-79104 Freiburg, Germany; and cLaboratory of Biochemistry and Biophysics, Department of Biology, University of Bologna, I-40126 Bologna, Italy
Edited by Pierre A. Joliot, Institut de Biologie Physico-Chimique, Paris, France, and approved May 28, 2012 (received for review February 24, 2012)
F0F1-ATP synthases use the free energy derived from a transmembrane proton transport to synthesize ATP from ADP and inorganic
phosphate. The number of protons translocated per ATP (H+/ATP
ratio) is an important parameter for the mechanism of the enzyme
and for energy transduction in cells. Current models of rotational
catalysis predict that the H+/ATP ratio is identical to the stoichiometric ratio of c-subunits to β-subunits. We measured in parallel
the H+/ATP ratios at equilibrium of purified F0F1s from yeast mitochondria (c/β = 3.3) and from spinach chloroplasts (c/β = 4.7). The
isolated enzymes were reconstituted into liposomes and, after
energization of the proteoliposomes with acid–base transitions,
the initial rates of ATP synthesis and hydrolysis were measured
as a function of ΔpH. The equilibrium ΔpH was obtained by interpolation, and from its dependency on the stoichiometric ratio,
[ATP]/([ADP]·[Pi]), finally the thermodynamic H+/ATP ratios were
obtained: 2.9 ± 0.2 for the mitochondrial enzyme and 3.9 ± 0.3
for the chloroplast enzyme. The data show that the thermodynamic H+/ATP ratio depends on the stoichiometry of the c-subunit,
although it is not identical to the c/β ratio.
chemiosmotic theory
C
| protonmotive force | bioenergetics | nanomachine
ells of all life kingdoms use H+-ATP synthases to produce
the cellular energy carrier ATP from the energy of a transmembrane electrochemical potential difference of protons built
up and maintained by proton transport mechanisms, such as the
oxidative electron transport in mitochondria or the photoinduced electron transport in chloroplasts (1). The number of
protons translocated for each synthesized ATP molecule (H+/
stoichiometry of the latter is 14, according to X-ray crystallography
(14, 15) and atomic force microscopy of the isolated c-ring (16).
Hence, their respective H+/ATP ratios, based on the assumptions
mentioned above, should be 3.3 and 4.7. We have isolated the two
complexes and reconstituted them into liposomes, which, if subjected to acid–base transitions to generate a high protonmotive
force, were able to synthesize ATP at physiological rates (17, 18).
The technique of acid–base transitions offers the great advantages
of (i) measuring the imposed transmembrane ΔpH with the accuracy of the pH electrode, thus making high-precision quantitative studies possible (18–20), and (ii) allowing the testing of
ATP synthases from different species with the same method and
under identical experimental conditions.
Results
According to the chemiosmotic theory (1), the synthesis of ATP
catalyzed by the H+-ATP synthase is coupled to the translocation of n protons from the internal to the external
compartment:
ADP þ Pi þ nH þ in ↔ ATP þ H2 O þ nH þ out :
[1]
The factor n is the number of protons translocated per ATP, and
it is called the thermodynamic H+/ATP ratio. The Gibbs free
energy of this coupled reaction can be expressed as:
μHþ
ΔG′ ¼ ΔG′p − nΔ~
¼ ΔG8′p þ 2:3 RT logðQÞ − nð2:3 RT ΔpH þ FΔφÞ;
[2]
Oddychanie tlenowe
glukoza
dro¿d¿e
2 ATP
alkohol
etylowy
CO2
CO2
glikoliza
pirogronian
komórki miêœniowe
bakterie mlekowe
mleczan
reakcja pomostowa
acetylo-CoA
2 ATP
cykl
Krebsa
NADH
O2
ok. 26
25 ATP
≥añcuch oddechowy
H2O
Porównanie
Glukoza
KT 16:0
NADH
10
7 + 8 x 3 = 31
FADH2
2
7 + 8 x 1 = 15
Protony
10 x 10 + 2 x 6 = 112
31 x 10 + 15 x 6 = 400
ATP
ok. 26
ok. 92
Ostatecznie
ok. 30
ok. 99 (= 92 - 1 + 8)
A jak nie ma tlenu?
Fermentacja
glukoza
glikoliza
pirogronian
O2
du¿o
ATP
ma≥o
O2
mleczan
ATP
Glukoza; 6C
Glikoliza
Glukoneogeneza
2 x Pirogronian; 3C
Obecność tlenu
2 x NADH
i 2 x CO2
2 x Acetylo-koenzym A
(Ac-CoA); 2C
Dużo ATP = ok. 30 cząsteczek
Brak tlenu
(Fermentacja)
2 x Mleczan
Mało ATP =
2 cząsteczki
FADH2