ch19a oksidatif fosforilasyon ve fotofosforilasyon yonca duman

8/3/2019 Ch19a Oksidatif Fosforilasyon Ve Fotofosforilasyon Yonca Duman

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OXIDATIVE PHOSPHORYLATION

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Oxidative phosphorylation is the culmination of energy-yielding metabolism in aerobic organisms. All oxidative stepsin the degradation of carbohydrates, fats, and amino acidsconverge at this final stage ofcellular respiration, in whichthe energy of oxidation drives the synthesis ofATP.

Photophosphorylation is the means by which photosyntheticorganismscapture the energy of sunlight -the ultimate sourceof energy in the biosphere- and harness it to make ATP.

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In eukaryotes, oxidative phosphorylation occurs inmitochondria, but photophosphorylation in chloroplasts.

Oxidative phosphorylation involves the reduction of O2 toH2O with electrons donated by NADH and FADH2; it

occurs equally well in light or darkness.Photophosphorylation involves the oxidation of H2O to O2,with NADP+ as ultimate electron acceptor; it is absolutelydependent on the energy oflight.


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Kemiozmotik Teori

Oksidatif fosforilasyon vefotosentetik fosforilasyon mekanizmalar asndan

noktada birbirine benzer:

1.

Her iki sre te membran bal tama zinciri zerinden elektronlarnakn ierir,

2. Elektonlarn yoku aa (down-hill) ya da ekzergonik akn salayanserbest ener i roton e ir en olma an bir membrana kar oku ukar

(up-hill) ya da endergonik proton transportu ile balantldr. Bylelikleyakt molekl oksidasyonunun serbest enerjisi membranlararas

(transmembran) elektokimyasal potansiyeli olarak korunur.

3. Protonlarn zgn proton kanallarndan geerek aaya dorukonsantrasyon gradienti dorultusunda membranlar arasndaki ak ATP

sentezi iin serbest enerji salar. Bu olay yani ATP sentezi; proton akn

ADP nin fosforilasyonu ile balantlandran ATP sentaz tarafndan

katalizienir.lenir.

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+ +

Nikotinamit nkleotid bal dehidrojenazlarn katalizledii tepkimeler:

ndirgenmi substrat + NAD Ykseltgenmi substrat + NADH + H

+ +ndirgenmi substrat + NADP Ykseltgenmi substrat + NADPH + H

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Nikotinamid nkleotid

+ +

NADPH + NAD NADP + NADH

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:H-

NAD+ + 2e- + 2H+ NADH + H+

NADP+ + 2e- + 2H+ NADPH + H+

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FAD + 2e- + 2H+ FADH2

FMN + 2e- + 2H+ FMNH2

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Oksidatif fosforilasyonda tip elektron transferi olur:

1. Elektronlarn hidrid iyonu (:H-) olarak transferi,2.

Elektronlarn hidrojen atomu olarak transferi (H

+

+ e

-

)

3. Fe+3 n Fe+2 ye indirgenmesinde olduu gibi elektronlarn dorudan transferi.

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Fe - 4S

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Three-dimensional structure of rubredoxin fromPyrococcus furiosus


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2Fe - 2S

Ferredoxin of the cyanobacterium Anabaena

30Ferredoxin of the cyanobacterium Anabaena


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Rieske iron-sulphur protein structure (2Fe 2S)

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4Fe - 4S

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Ferredoxin from Peptococcus aerogenes.


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NADH Q Cyt b Cyt c1 Cyt c Cyt a Cyt a3 O2

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2 Cyt b-Fe+2 2 Cyt c1-Fe+3 2 Cyt c-Fe+2 2 Cyt a-Fe+3 2 Cyt a3-Fe

+2 1/2 O2 + 2 H+

2 Cyt b-Fe+3 2 Cyt c1-Fe+2

2 Cyt c-Fe+3 2 Cyt a-Fe+2 2 Cyt a3-Fe+3

H2O

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Complex I : NADH dehydrogenase

Complex II : Succinate dehydrogenase

Fatty acyl-CoA dehaydrogenase

Glycerol-3-phosphate dehydrogenase 48


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ElectronTrans ort

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Chain

Complex I NADH : Ubiquinone oxido reductase


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Complex I NADH : Ubiquinone oxido reductase

NADH + H+

+ Q NAD+

+ QH251


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Kompleks Ideki proton aknailikin toplam reaksiyon

NADH + 5HN+ + Q NAD+ + QH2 + 4HP

+

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Complex II Succinate CoQ oxidoreductase


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Structure of Complex II( i t d h d )


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(succinate dehydrogenase).Porcine heart enzyme

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Structure of Complex II (succinate dehydrogenase) of Escherichia coli


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Succinate

Fumarate

FAD

FADH2

Succinate dehydrogenase

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Glycerol-3-phosphate Shuttle


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Complex III Cytochrome bc1 complex Ubiquinone : cytochrome c oxidoreductase


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Cytochrome bc1 complex Ubiquinone : cytochrome c oxidoreductase


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Q.HL

Q.HL

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Antimycin Ablocks electrontransfer from theheme bH ofcytochrome b to

cytochrome c1.

Antifungal agentsm xothiazoland

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stigmatellin

which both blockelectron flow from

QH2 to RieskeFe-S protein atQP site and the

heme bL ofcytochrome b.


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Critical subunits of cytochromeoxidase (Complex IV).


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76The binuclear center of CuA.

Complex IV Cytochrome oxidase


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+2 + +3 +N 2 P 2

+2 + +3 +

N 2 P 2

4 Sit c-Fe + 8 H + O 4 Sit c-Fe + 4 H + 2 H O

2 Sit c-Fe + 8 H + 1 2 O 2 Sit c-Fe + 2 H + H O

4

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Complex IV Cytochrome oxidase


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+2 + +3 +N 2 P 2

+2 + +3 +

N 2 P 2

4 Sit c-Fe + 8 H + O 4 Sit c-Fe + 4 H + 2 H O

2 Sit c-Fe + 8 H + 1 2 O 2 Sit c-Fe + 2 H + H O

4

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+2 + +3 +N 2 P 22 Sit c-Fe + 8 H + 1 2 O 2 Sit c-Fe + 2 H + H O4

2e2 2

o

NADH H 1 2O NAD H O++ ++ + +


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2 2

NAD / NADH

oO / H O

o

o o

o

E 0.320VE 0.816V

E 0.816 ( 0.320) 1.14V

G nF E 2 96.5 kJ/V mol 1.14V

G 220 kJ / mol

+ = =

= =

= =

=

2 2

oFumarat /Sksinat

oO / H O

o

E 0.031V

E 0.816V

E 0.816 0.031

=

=

= o o

o

0.785VG nF E 2 96.5 kJ/V mol 0.785V

G 151.5 kJ / mol

=

= =

= 83


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G = -151.5 kJ mol-1

G = -220 kJ mol-1


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Kompleks I: NADH + 5HN+ + Q NAD+ + QH2 + 4HP

+

Kompleks III: QH2 + 2Sit c1-Fe+3 + 2HN

+ Q + 2Sit c1-Fe+2 + 4HP

+

2 Sit c1-Fe+2 + 2 Sit c-Fe+3 2 Sit c1-Fe

+3 + 2 Sit c-Fe+2

Kompleks IV: 2Sit c-Fe+2 + 4HN+ + O2 + 2Sit c-Fe

+3 +2HP+ + H2O

Toplam: NADH + 11HN+ + O2 NAD

+ + 10HP+ + H2O

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( ) ( )+

+ +2 PN P+ P N

1 N

HCln = 2.303log = 2.303 log H log H = 2.303 pH pH = 2.303pHC H

- -

Bir H+ pompalanmasnda 0.15-0.20 V, pH = 0.75 , G = +20 kJ/mol

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ADP + Pi + n HP+ ATP + H2O + n HN

+90


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Coupling of electron transfer and ATP synthesis in mitochondria. In experiments todemonstrate coupling, mitochondria are suspended in a buffered medium and an O2 electrode

monitors O2 consumption. At intervals, samples are removed and assayed for the presence ofATP. Addition of ADP and Pi alone results in little or no increase in either respiration (O2consumption; black) or ATP synthesis (red). When succinate is added, respiration beginsimmediately and ATP is synthesized. Addition of cyanide (CN), which blocks electron

transfer between cytochrome oxidase and O2, inhibits both respiration and ATP synthesis.

Mitochondria provided with succinate respireand synthesize ATP only when ADP and Pi areadded. Subsequent addition of venturicidin

or oligomycin inhibitors of ATP synthase


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or oligomycin, inhibitors of ATP synthase,blocks both ATP synthesis and respiration. Di-nitrophenol (DNP) is an uncoupler, allowingrespiration to continue without ATP synthesis.

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Evidence for the role of a proton gradient

in ATP synthesis. An artificially imposedelectrochemical gradient can drive ATP

synthesis in the absence of an oxidizableb t t l t d I thi t t


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sy t es s t e abse ce o a o d ab esubstrate as electron donor. In this two-stepexperiment, (a) isolated mitochondria arefirst incubated in a pH 9 buffer containing 0.1M KCl. Slow leakage of buffer and KCI intothe mitochondria eventually brings the matrix

into equilibrium with the surroundingmedium. No oxidizable substrates arepresent. (b) Mitochondria are now separatedfrom the pH 9 buffer and resuspended in pH

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u er conta n ng valinomycin ut no .The change in buffer creates a difference of

two pH units across the inner mitochondrialmembrane. The outward flow of K+, carried(by valinomycin) down its concentrationgradient without a counterion, creates acharge imbalance across the membrane(matrix negative). The sum of the chemical

potential provided by the pH difference andthe electrical potential provided by theseparation of charges is a proton motive forcelarge enough to support ATP synthesis in theabsence of an oxidizable substrate.


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ATP SynthaseIn the laboratory, small membranevesicles formed from inner mitochon-

drial membranes carry out ATP synthe-sis coupled to electron transfer When


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y ysis coupled to electron transfer. WhenF1is gently extracted, the "stripped"vesicles still contain intact respiratorychains and the Fo portion of ATP syn-thase. The vesicles can catalyze elec-

tron transfer from NADH to O2 butcan not produce a proton gradient: Fohas a proton pore through which pro-

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electron transfer, and without a proton

gradient the F1-depleted vesicles cannot make ATP. Isolated F1 catalyzesATP hydrolysis (the reversal of syn-thesis) and was therefore originallycalled FiATPase. When purified F1 isadded back to the depleted vesicles, it

reassociates with Fo, plugging itsproton pore and restoring the mem-brane's capacity to couple electrontransfer and ATP synthesis.

Catalytic mechanism of

F1. 18O-exchange experi-ment F solubilized from


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g pment. F1 solubilized frommitochondrial membranesis incubated with ATP inthe presence of18O-labe-

led water. At intervals, asample of the solution iswithdrawn and analyzed

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18O into the Pi produced

from ATP hydrolysis. Inminutes, the Pi containsthree or four 18O atoms,indicating that both ATPhydrolysis and ATP syn-

thesis have occurredseveral times during theincubation.

The likely transition statecomplex for ATP hydro-lysis and synthesis in ATP


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synthase (derived fromPDB ID 1BMF). The subunit is shown in green, in gray. The positively

charged residues -Arg182a2 and -Arg376 coordinatetwo oxygens of the penta-valent phosphate inter-

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mediate; -Lys155 interactswith a third oxygen, andthe Mg2+ ion (greensphere) further stabilizesthe intermediate. The bluesphere represents theleaving group (H2O).

These interactions result inthe ready equilibration ofATP and ADP + Pi in theactive site.


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Reaction coordinate diagrams for ATP synthase and for a more typical enzyme. In a typical enzyme-catalyzed reaction (left), reaching the transition state () between substrate and product is the majorenergy barrier to overcome. In the reaction catalyzed by ATP synthase (right), release of ATP from theenzyme, not formation of ATP, is the major energy barrier. The free energy change for the formation of

ATP from ADP and Pi in aqueous solution is large and positive, but on the enzyme surface, the very tightbinding of ATP provides sufficient binding energy to bring the free energy of the enzyme bound ATPclose to that of ADP + Pi, so the reaction is readily reversible. The equilibrium constant is near 1. Thefree energy required for the release of ATP is provided by the proton-motive force.

Mitochondrial ATP Synthase Complex


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When researchers crystallized the proteinin the presence of ADP and App(NH)p, a

close structural analog of ATP that cannotbe hydrolyzed by the ATPase activity ofF1, the binding site of one of the three subunits was filled with App(NH)p, thesecond was filled with ADP, and the thirdwas empty. The corresponding subunitconformations are designated -ATP, -ADP, and -empty.

http://www.iubmb-nicholson.org/swf/ATPSynthase.swf


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Oksidatif fosforilasyon iin kemiosmotik model kabul edilmeden

nce ATP oluumunun varsaylan toplam denklemi:


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x ADP +x Pi + O2 + H+ + NADH x ATP + H2O + NAD

+

x : P/O oran ya da P/2e oran.

NADH elektron donr olduunda P/O (ATP/ O2) 2-3 arasnda,

Sksinat elektron donr olduunda P/O 1-2 arasnda.

P/O bir tam say olmak durumunda:

NADH elektron donr olduunda (P/O) = 3Sksinat elektron donr olduunda (P/O) = 2

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Kemiozmotik model sz konusu olduktan sonraATP sentazreaksiyonunun stokiometrisi ile ilgili olarak u sorular

sorulmaldr:

1. Bir NADH ten molekler O ne bir elektron iftinin transferi lemitokondriyel matriks dna ka tane proton pompalanr?

2. Bir ATP sentezi iin F0F1 kompleksinden mitokondriyel matrikseka tane proton geer?

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KONSENSS

Elektron ifti bana darya pompalanan proton says:


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NADH iin :10

Sksinat iin : 6

1 ATP retimi i in matriks i ine iren roton sa s : 4

(1 tanesi mitokondriyel membrandan adenin nkleotid ve fosfat translokazlarla Pi ,

ATP ve ADP tanmasnda kullanlr)

Proton tabanl P/O oran:

NADH iin : (10/4) = 2.5

Sksinat iin : (6/4) = 1.5120


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The Proton-Motive Force EnergizesActive Transport


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Active Transport

Although the primary role of the protonradient in mitochondria is to furnish

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energy for the synthesis of ATP, theproton-motive force also drives severaltransport processes essential to

oxidative phosphorylation.

The adenine nucleotide

translocase,antiporter movesfour negative charges out forevery three moved in, its

i i i f d b h


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33 H+ +

activity is favored by thetransmembrane electroche-mical gradient, which gives

the matrix a net negativecharge; the proton-motiveforce drives ATP-ADP

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.

The phosphate translocase,which promotes symport ofone H2PO4 and one H

+ intothe matrix. This transportprocess, too, is favored bythe transmembrane protongradient.

Shuttle Systems Indirectly Convey Cytosolic

NADH into Mitochondria for Oxidation

The NADH dehydrogenase of the innermitochondrial membrane of animal cells can


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mitochondrial membrane of animal cells canaccept electrons only from NADH in the matrix.

Given that the inner membrane is not permeableto NADH, how can the NADH generated by

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glycolysis in the cytosol be reoxidized to NAD+

by O2 via the respiratory chain?

Yet the inner mitochondrial membrane lack anNADH transport protein. Only the electrons from

cytosolic NADH are transported into themitochondrion by one of theseveral ingenious

shuttle systems.

The most active NADH shuttle, which functions in liver, kidney, and heart mitochondria, is the malate-aspartate shuttle which is mediated by two membrane carriers and four enzyme.

This process occurs in two phases of thteereactions each:

Phase A (transport of electrons into thematrix)

1 In the cytosol NADH reduces oxaloacetate


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1. In the cytosol, NADH reduces oxaloacetateto yield NAD+ and malate in a reactioncatalysed by cytosolic malat dehydrogenase.

2. The malate--ketoglutarate carriertransports malate from the cytosol to themitochondriyel matrix in exchange for -ketoglutarate from the matrix.

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3. In t e matrix, NAD+ reoxi izes malate toyield NADH and oxaloacetate in a reactioncatalyzed by mitochondrial malat

dehydrogenase.

Phase B (regeneration of cytosolicoxaloacetate)

4. In the matrix, a tansaminase convertsoxaloacetate to aspartate with the concomitantconversion of glutamate to -ketoglutarate.

5. The glutamate-aspartate carrier transportsaspartate from the matrix to the cytosol inexchange for cytosolic glutamate.

6. In the cytosol, a tansaminase convertsaspartate to oxaloacetate with the concomitantconversion of-ketoglutarate to glutamate.


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Skeletal muscle and brain use adifferent NADH shuttle, theglycerol-3-phosphate shuttle.It differs from the malate-as-partate shuttle in that it delivers


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the reducing equivalents fromNADH to ubiquinone and thus

into Complex III, not ComplexI, providing only enough ener-gy to synthesize 1.5 ATP mole-cules er air of electrons.

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Malate-aspartate shuttle Glycerol-3-phosphate shuttle


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Reversible Irreversible


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Regulation of Oxidative Phosphorylation

The rate of respiration (02 consumption) in mitochondria is tightly regulated; it

is generally limited by the availability of ADP as a substrate forphosphorylation. Dependence of the rate of 02 consumption on the availability

of the Pi acceptor ADP, the acceptor control of respiration, can be remarkable.


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The intracellular concentration of ADP ([ADP])is one measure of the energy

status of cells. Another, related measure is the mass-action ratio of the ATP-

ADP system, [ATP]/([ADP][Pi]).

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Normally this ratio is very high, so the ATP-ADP system is almost fully

phosphorylated. When the rate of some energy-requiring process (protein

synthesis, for example) increases, the rate of breakdown of ATP to ADP and P i

increases, lowering the mass-action ratio. With more ADP available for

oxidative phosphorylation, the rate of respiration increases, causing

regeneration of ATP. This continues until the mass-action ratio returns to its

normal high level, at which point respiration slows again.

In short, ATP is formed only as fast as it is used in energy-requiring cellular

activities.


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New born mammals, hibernating mammals containbrown fat in their neck and upper back that functions in

nonshivering thermogenesis, that is, as a biologicalheating pad. (The ATP hydrolysis that occurs during themuscle contractions or shiveringor any other move-ment also produce heat.

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