THE MINISTRY OF HEALTH OF UKRAINE

ZAPORIZHZHIA STATE MEDICAL UNIVERSITY

Tissue respirationand

Oxidative phosphorylation:

their mechanisms of duration;

factors for the regulation

Produced by ass.professor Krisanova N.V., 2017

Substrates of catabolic phase II

Metabolites of Krebs Cycle, etc)

Aerobic oxidation (+O2)

NADH FADH2 Donors

of electrons

ELECTRON TRANSPORT CHAIN

of the inner membrane of mitochondria

Substrate (SH2) oxidation catalyzed

by NAD+-linked dehydrogenase

SH2 SH

+++

N N

(Z) (E)

C

HH

R R

O

NH2 C

O

NH2

H

++

2 e

NAD+

NADH

-

NAD+-dehydrogenases are found in

cytoplasm and in the matrix of mitochondria

ELECTRON TRANSPORT CHAIN (ETC)

is the sequence of enzymes

and transporters of electrons

(CoQ, Fe-S-Proteins)

promoted the transfer of electrons

from NADH, FADH2

to molecular oxygen O2

During the transport of electrons there is

the duration of reactions with the type:

Reduced A+Oxidized B→Oxidized A + Reduced B

ETC contains four complexes:

Complex I

FMN and Iron-Sulfur (Fe-S)- centers

NADH-coenzyme Q

reductase

(NADH-dehydrogenase)

FAD and Fe-S- centers

Composition

of non-protein parts :

Complex IISuccinate dehydrogenase,

Cytochrome b560

Complex III

Cytochromes b, c1

Fe-S-proteinsHeme containing Fe 2+/ Fe 3+

Complex IV

Cytochrome C oxidase

(Cytochrome aa3)

Heme containing Fe 2+ /Fe 3+

Cu+/Cu 2+

Complex I (FMN and Fe-S-centers) location

NADH-

dehydrogenase

(NADH-coenzyme

Q reductase )

Matrix

Intermembrane

space

Inner membrane

Second name:

Complex I function at the moment

of HADH oxidation

N

N (E)

N

C

NH

C

(E)

R

O

O

N

HN

(Z)

NH

C

NH

C

R

O

O

2e+ +

CH3

CH3

CH3

CH3

2H+

FMN FMNH2

NADH+H+

NADH + H+ + FMN•E → NAD+ + FMNH2•E (1)

FMN• E – NADH-dehydrogenase (oxidized form)

O

O

CH3

OH3C

OH3C

R

OH

OH

CH3

OH3C

O

R

H3C

2H+

2e+ +

FMNH2*E

CoQ CoQH2

Fe-S-proteins

Matrix

Ubuquinone (CoQ) function at the

moment of oxidation of complex I

CoQ + FMNH2•E FMN•E + CoQH2 (2)

Complex III (Cytochromes b, c1,

heme containing Fe 2+ / Fe 3+ )

3+/2+

Heme structure represented

in cytochromes C and C1

The function of complex III

CoQH2 + 2 cyt b (Fe 3+ ) →2 cyt b(Fe 2+) +

+ CoQ + 2H+ (3)

2 cyt b(Fe2+)+2cyt c1(Fe3+)→2cyt b(Fe3+) +

+2 cyt c1(Fe2+) (4)

2 cyt c(Fe3+)+2cyt c1(Fe2+)→2cyt c(Fe2+) +

+ 2 cyt c1(Fe 3+) (5)

Complex IV

or

Cytocrome C

Oxidase

or

Cytochrome

aa3

The Cu-center of cytochrome C oxidase

Linkage mechanism of

Oxygen to Cytochrome oxidase

The function of complex IV

cyt aa3(Fe3+, Cu2+) + 2 cyt c(Fe 2+ )

2 cyt c(Fe3+) + cyt aa3 (Fe 2+, Cu+)

(6)

cyt aa3 (Fe 2+, Cu+) + ½O2

cyt aa3(Fe3+, Cu2+)+ O2-

(7)

or:O

2-+ 2H

+ →H2O

Tasks dissolved due to the function

of Electron Transport Chain (ETC)

1. The transport of electrons from reduced derivatives of vitamins (NADH, FADH2) to molecular oxygen.

2. The release of energy at any step of oxidation in ETC.

3. The pumping of protons from the matrix to intermembrane space

4. The production of active form of oxygen - oxide-anion O2- and then H2O

Some cytotoxic particles may be

formed from molecular oxygen

during the tissue respiration

instead of oxide-anion:

Superoxide anion Ó2-

Superoxide radical Ó2

Hydrogen peroxide radical HOÓ

Hydrogen peroxide H2O2

The change of red-ox-potential of

each red-ox pair of ETC is estimated in

volts. This change becomes more and

more in the direction of electron

transport to oxygen. A part of released

energy is thermal energy, and another

one may be used for ATP synthesis in

mitochondria -

this process is named

oxidative phosphorylation.

Notions of chemiosmotic

coupling hypothesis proposed

by Peter Mitchell in 1961

1. The inner membrane is not

permeable for protons in any

site of membrane in the

direction from the

intermembrane space to the

matrix.

2.Some electron carriers act as

pumps, which cause directional

pumping of protons across the

inner membrane from the matrix to

the intermembrane space. As the

electrons move down the chain

protons are expelled, penetrating

from the matrix to the

intermembrane space.

3. Because proton is a charged

particle the flow of free energy

across the inner membrane is due

to the combination of

a concentration gradient (ΔpH),

and a charge gradient (ΔΨ).

The sum of them is electrochemical

potential of the inner membrane:

ΔμH+ = ΔpH + ΔΨ

4. The chemical gradient of protons

across the inner membrane of

mitochondria serves as the means of

coupling the energy flow of electron

transport to the formation of ATP

5. Protons pass back into the matrix at

a special site

where ATP synthetase resides

(proton channel - factor Fo).

The pumps for protons movement

from the matrix to the

intermembrane space

Complex I

Complex III

Complex IV

(proved in 1988)

Inhibitors for ATP synthetase (Factor F0):

Oligomycin and Dicyclohexylcarbodiimide (DCCD)

Fo

F1

ADP

H3PO4

*ATPATP

2H+

2H+

Intr

am

em

bra

ne

sp

ac

e

2H+

Inner membrane Matrix

ATP-synthetase

Matrix

Inner membrane

Intermembrane

space

6. Oxidative phosphorylation is the

synthesis of ATP in the inner membrane of

mitochondria catalyzed by ATP synthetase

due to energy produced by ETC enzymes

7. ATP formation from ADP and phosphoric

acid does not require energy.

ATP is obtained in the linked form with F1.

Energy is required for ATP removal

from F1. This action is due to the moment of

protons movement through the inner

membrane across the Fo as a channel for

protons.

8.The moment of protons movement

through the inner membrane across

the Fo as a channel is the moment

for coupling of phosphorylation (ATP

synthesis) with the change of

electrochemical potential of the

inner membrane (or the use of

energy produced by the ETC).

The P/O ratio signs how many molecules of

ATP is synthesized per 2 electrons

transferred across the ETC to one atom of

molecular oxygen

The P/O ratio

Three sites of coupling in ETC:

1 – site where electrons are transferred from

complex I to CoQ;

2 – site where electrons are transferred from

cytochrome b to cytochrome c1 ;

3 - site where electrons are transferred from

cytochrome c oxidase to molecular oxygen.

P/O ratio values

• Substrates that donate electrons to NAD+ yield P/O ratio of 3

• Substrates that donate electrons to FAD or FMN yield P/O ratio of 2

P/O ratio is changed:

1) under the influence of substances-uncouplers;

2) at the presence of inhibitors of respiratory chain complexes, etc.

Five states of respiratory control have been

defined in which the rate of respiration is

limited by different factors:State 1 is limited by availability of ADP and a sub-

strate (source of electrons).

State 2 is limited by the availability of substrate.

State 3 is limited by the capacity of the electron

chain itself, when ADP, oxygen and substrate are

saturating.

State 4 is limited by the availability of ADP.

State 5 is limited by availability of oxygen.

ADP/ATP transport is due to ATP/ADP translocase

system. Inhibitors of this system are:

1) Atractyloside; 2) Bongkrekic acid.

Inhibitors for complex I:

•Rotenone, an insecticide;

•Barbiturates (drugs for

sleepless treatment);

•Piericidin A, an antibiotic.

Result: NADH is accumulated in

the matrix!

P/O=0 for all NAD+-dependent

oxidation reactions

Inhibitors for complex II:

Malonic acid (1) – the competitive

inhibitor for succinate

dehydrogenase (SDH ase);

Carboxin (2); Thenoyl trifluoride

acetone (TTFA, 3).

Substances (2) and (3) block the

electron transport

from reduced SDHase to CoQ.

P/O = 0 for succinate oxidation.

Inhibitors for complex III:

•Dimercaprol (1);

•Antimycin A, an antibiotic (2)

P/O may be lower then 3 or 2

(respectively oxidized

substrate)

Inhibitors for complex IV:

Carbon monooxide which competes

with oxygen for its binding site on

Cytochrome aa3;

Cyanides block the heme-centers of

cytochrome aa3 binding by covalent

bonds;

Hydrogen sulfide;

Azides .

The death of biological system!

Uncouplersallow the transport of protons

across the inner membrane (they are

very lipophylic compounds), thus

collapsing the proton gradient

before it may be used for ATP

synthesis.

P/O = 0 in all cases !

The energy produced by ETC is

thermal energy, only!

•2,4 - Dinitrophenol (weight-loss drug

used in 1970s but was discontinued

because of its toxicity);

• Dicumarol that is anticoagulant;

•Thyroxin in high abnormal concen-

tration;

•Valinomycin, an antibiotic.

Uncouplers

39

THANK YOUFor

ATTENTION!