IChapter 5EI\ERGY

E,I\ERGYLiving organisms are not in equilibrium. Rather they require a continuous influx of free

energy to maintain order in their internal structures or minimalizing disorder,Metabolism is the overall process though which Iiving organisms acquire and utilize

the free energy they needed to carry out their various functions. They do so by coupling theexothermic reactions of nutrient oxidation to the endothermic processes required to maintain theliving state as:. perforrnance of mechanicalwork;. the active transport of molecules against concentration gradients;. biosynthesis of complex molecules.

Phototrophs (plant and some bacteria) acquire free energy from the sun throughphotosynthesis (light energy powers the reaction of CO, and HrO to form carbohydrates).Chemothrops obtain their energy by oxidizing organic compounds (carbohydrates, lipids,proteins)obtained from other organisms, ultimately phototrophs. Additionally,the nutrients arebroken down in a series of metabolic reactions to common intermediates that are used asprecursors in the synthesis of other biological molecules.

A remarkable property of living organisms is that they maintain a steady state. Forexample, over 40-year time span, a human adult (70 kG average weight)consumes literally tons ofnutrients and drinks over 20 000 L of water. He does so without significant weight change.

Generation of energy in the cell

tn the living cells are carried out the steady reactions of degradation and formation ofcompounds sonnected with consuming and generation of free energy. These processes are calledas metabolism. The metabolism is often divides into two categories: catabolism and anabolism. Thereactants of mentioned processes, their intermediates, and products are referred to as metabolites.

Catabolism is involved in degradation of complex metabolites of into simpler products.Summary free energy, AG, of this process is negative - catabolism is exothermic and it is freeenergy source for anabolism. In humans the final products of catabolism are: C02, H2O, urea, uricacid and creatine, which are secreted mainly by the lungs and kidneys.

Anabolism. lt is a biosynthesis of complex molecules from simpler reactants usingenergy generated in the course of catabolism. Summary free energy, AG, of the anabolism ispositive. This is endoenergetic process.

Simplifying, it can be assumed that the source of energy is the burning of hydrogen inoxygen. The donors of hydrogen mainly are: glucose, and other simple sugars, fatty acids, ketonebodies and hydrocarbon skeleton of amino acids.

The burning of H, in 0, in vitro is explosive and strongly exothermic and its energy isfully converted into heat.

The burning of hydrogen invivo (metabolic process) is slow, multistage process in whichc.a. 40o/o of energy released is stored in the chemical form. lts main carrier of this free energy isATP (adenosine triphosphate).

ATPThe endothermic processes that maintain the life of cell are driven by exothermic reactions of thenutrient oxidation. ln these reactions are synthesized a few types of ,,high-energy" intermediateswhose consumption drives endoenergetic processes. The centra! role in energy metabolismplays ADEN0SINE TRTPHOSPHATE, ATR which consists of one adenosine moiety to which threephosphoryl groups are sequentially linked via phosphoester bond followed by twophosphoanhydride bonds. ADP and AMP are similarly constituted but wrth only two or onophosphorylgroup.

phosphoesterThe structure of ATP indicates itsrelationship to ADP and AMR andadenosine" The phosphoryl grouPS,stafting with that on AMR are referredto as a, p, and y phosphates.

Note the difference betweenphosphoester and phosphoan hyd ridebonds

I I ; rr\_r "tr_JI | +,

L-

\\.

,\t \L\

:]rir-irhtiii*ir.r.,'iir-iril i

,-

l.rr it-itis-., I)--*--. o-*l o-Ai? +li ,l+ +O-p;.()-p.--.rl-* p-Ov

ir 'i itl'i it" Tol "l 'iffil

::;HOOH:rlt-I! , Adenosine ,rrl

ATP

f Transport of chemical energy by ATP in differentmetabolic processes@

-fpyruvate

_;

EXOEi{ER.GtrCPR.OCESSES

NDOENERGETIPROCESSES

ATP- energy carrier - it plays

(neurotransmition )L-/important role in catabolic and anabolic processes,

Adenine nucleosides with high (ATB ADP) and low (AMP) bond energy ofphosphoryl groups

tuvP

Phosphoanhydride bond (red) - Hight energy bondPhosphoester bond (black) - Low energy bond

NH.l"ilT\ ooo=niT ri , ,

I _O= $Hr-O- P-O-P-O-P-ONZ S.A AHoH oH ATP

oocH,-o-$-o-$-o-Ja 8-6-

OH OHADP

ilcHz-o-P-o-

$-

Other "high energy" phosphate

l 13 - b is- p h o s p h o glyc e r ate phosphoenolopyruvate

olto P-O-'!' 8-

I

IH-C-OH OErr-o-#-o-

6.

q ,o-

CIlolilc-o-P-o-ililCHz O-

compounds

oo-'!'9",n\ T-t',N:Q 0ri t-$-o-ti 8-

phosphocreatine

The values of their AGo are given above. They do not provided free energy indirectly,but they are involved in the synthesis of ATP via substral-level phosphorylation. lt is a secondmetabolic path (beside of oxidative phosphorylation) of synthesis of ATR very impoftant for cellsrealizing the anaerobic metabolism (without oxygen). For example, phosphocreatine is ,,stor+house" of energy needed for muscle cells to contract. lndirectly this energy is released byATRwhich is quickly regenerated by transfer of phosphoryl group from phosphocreatine to ADPcatalped by enzyme creatine kinase.

Dhnanha'-r.aa*ina & n nDl rr\ru]rrr\rvrgGrLrrls r nrJr (- ATD r lar.aalina-

nl I r \rt Erllt rg

Other compounds

H3c-ci3_"oo

acetyl-S-CoA

containing ,,rich" energy bonds

'

'-'ttOHsC C:_-S-CoA

palrnitoyl-S-CoA

C-S bond is

,,rich" gngrgybondNHzAo-

o---'o-i:oI

o-carbarnoyl phospate

C-S bond (thioester bond) between atom of S of coenryme A, CoA, and carbonylgroup, >C0, of residue of carboxylic acid is also "rich" in energy. Fatty acids in the active formas acyl-CoA (where "-" represent a rich energy bond)can participate in catabolism (B-oxidation) and anabolism (synthesis esters of glycerol or esters of cholesterol).

Phosphate compounds with low energy bond

.tr,[-o.t/alN+"loH

glucose-6-ph osphate

HIH-C_OHIHO_C_H O

n-8-o-$-o-ll_HO-glycerol-6-phospate

Hse,e T Y ?HIC-N-C-C-O-P-O-H,C ri i, d

phosphocholine

The vatues AGo of their hydrolysis reaction are below 17 kJmol. To thesecompounds belong the phosphate esters of glycerol, inositol, aminoalcohols, and allmonophosphate n ucleotides.

ATP is placed in middle position between phosphate compounds possessingbonds with very high and low energy. ln metabolism ATP plays the role of energytransmifter. In cell there are not other mechanisms, which allow the transfer of phosphategroups from high energy donors to low energy acceptors without the participation of ATP.

MitochondrionThe mitochondrion is the site of eukaryoticoxidative metabolism. lt contains the enzymesthat mediate this process, and enzymes andredox proteins involved in electron transfer andoxidative phosphorylation.It is calied as the cell's "powei' plant".The mitochondrion is bounded by outermembrane and contains an extensivelyinvaginated inner membrane, called cristae.

The inner mitochondrionon compartment, named matrix (gel-like substance), which contains theenzymes of oxidative metabolism (e.9. Krebs cycle enzymes), as well as substrates, nucleotidecofactorc, and inorganic ions. The matrix also contains mitochondria genetic machinery - DNA,RNAand ribosomes.

The first acceptor of H atoms spitted from substrate is mainly NAD*, next FMN followedby coenzyme Q. Allelements of respiratory chain are proteins (except coenzyme Q). Some ofthese are enzymes and other are nonenzymatic proteins, which contain the iron.sulfur clustersknown as prosthetic group of iron-sulfur proteins (nonheme iron proteins). These Fe:S clustersparticipate in etectron-transport process. The most common types, designated as [2Fe'2S] and[aFe- S] clusters consist of equal number of Fe and S ions, are coordinated to four protein Cyssulfhydryl groups. (See the next slide).

10

lron-sulfur cluster [4Fe4S] coordinated with NADH-dehydrogenaseNADH dehydrogenase protein

fg2+r3+ ions placed in Fe:S clusterstransfer the electrons between FMN andcoenzyme Q, and between cytochromeand cytochrorne c,.The oxidized and reduced states of Fe:Sclusters differ by one formal charge. Thisis because the Fe atoms in each clusterform a conjugated system and thus canhave oxidation stages between tlte +2and +3 values possible for individual Featoms. E.g. the Fe:S cluster can containtwo Fe(ll) and two Fe(ll[.

-S \FE

:cy,s

:

i

+,lo

v\n M Gp .nnnn^

The change of UV absorption spectrumderived from change of structure ofnicotinic acld ring. A new maximum at340 nm is formed.

:S--Cys

:

l1

The change of adsorption spectrum of nicotinic acid in course of oxidation

zH* + 2e'(

{zH* + ze-

340wavelength (nm)

Reduction of NAD* to NADH leadsto change of structure ofnicotinic acid ring (part of NAD* ),which can be detected by UVspectroscopy. Pair of electrons(2e') is transferred to nextelements of respiratory chain.

o

IQ^NHZI

NADH + H*

?

-{\NHz$rI

NAD*

.L

H

.S-Cys(i? .;H

\2

Abs

oH,cnz*\Ao*HscM,'nA*-\o

I

dimethy lisoalloxasi rre(oxidized)

The changes oi absorption in UV specirum.rnaxirnum at 450 nm is shrinking.

?oHsc rrH,cMT^TAo

H

dimethyisoalloxasine(reduced)

13

Koenzyme 0 (ubichinon)n :6 to 9

o

o

Hsc -o./

HgC-O\ FH,CH:C--CHzhH

Conversion of structure of dimethylisoalloxasine ring as a result of reduction ofFMN into FMNH2 or FAD into FADH,

FMN lub F.AD

Di methylisoalloxasine isa pafi of FMN and FADas well as FMNH, andFADH2.

zH* + ze'(

atzH* + ze'

The resuit of ring reriuciion ihe

FVINH, lub FADH,

H3c o-A-cH:t lt ?H,H: C

-oT(CFI2 -cH { -CHz) nH

cxidized fonn

\ -r2H*+ 2e

zH'+ fr" \

OH

reduced form

T4

Heme C in cyt*chroffie c bonded to protein

CHsI

,r" ,&-s-cy'to

H,cr "7{*rLT.*-o,ln" L ^1 ll

-ooc-crrz -*,X"-1t -=H;ll

-

S-cy,r o

l_-l CII3

r' cHr

ffi;Cytochrome c is soluble in water. Fe atoms in cytochrome are easy oxidized and

reduced (Fez+ 5 Fe3*), and it is reversible process. Thus, the cytochromes can be donorsand acceptors of electronS in respiratory chain. ln cytochrome C heme in covalently bondedwith enrymatic protein by two S atoms from residues of cysteine contained in amino acidschain.

Cytochrome C

The Cytochrome c is a small heme protein foundloosely associated with the inner membrane of themitochondrion, lt is a highly soluble protein,unlike other cytochromes, lt is an essentialcomponent of the electron transport chain, whereit carries one electron. lt is capable of undergoingoxidation and reductionn but does not bindoxygen.

Three.dimensional structure of cytochrome c(green) with a heme molecule coordinating acentral lron atom (orange).

15

f'T

fHeme Afound in cytochrome a + as

QHCH-CHz

Metso

Cytochrome a + a, is the last unit in electron transport chain. lt transfer electrons to molecularoxygen. ln this place electrons, molecular orygen and free protons bond together and moleculeof water is formed. Heme A is not covalently bonded with enzymatic protein, but it forms onlycoordinative bonds with histidine(18) residue and methionine(80) residue.

Standard red uction potentials

*ir"

-OOC-CHz-CHz

o).H

Cytochrome a + os

is a synonim forcytochrame oxidaseassociated with thepumping of protons andthe resultantphosphorylation of ADPto ATP. The reaction isthe terminal event in theelectro transport schemeby which oxygen is useclfor fuel combustion. lt isa part of the respiratorychain.

f",CHzA*-

ltLI

18

Oxidtzer Reductant n Eo' tVI

Acetate + CO, +2I{+ Pyruvate +FI?O 2 - 0.74Succinate* CO, +2F{- u-ketoglutarate * HrO 2 - a.67Acetic acid + ZIj{+ Acetaldehl,de 2 - 0,60

o, or- I - 0.15

zIJ+ H, 2 - 4,42Pyrurrate* CO, +H+ malate 2 - 0,33IYAD+ + H+ I{ADH 2 - 0^32NADP+ + H+ I\ADPI{ 2 - 432FMI{ (bonded w'ith enzyme + 2H.) FMl\Hr(bonded n'ith

enryme)I/a -0'3 0

Dehydnoliponate + Z}I" dihS'droliponate 2 -0.291 r3-&is-phosphoglycerate + 7I{+ 3-phosphoglycerol

alcteh3,de + Pi) a.29

Glutatione (oxidizecl) +7}J+ 2 glutatione reducecl 2 -4,23EAD + 2Ftr+ Fi\BH, 2 -0.22

Standard reduction potentials (cont 1.)

Standard reduction potentlals (cont 2.1

Oxidant Reductant n E6' tvlAcetaldehyde + 2H* ethanol 2 -0,24Pyruvate + 2[J* Iactate 2 -0,19Oxaloacetate + 2H+ malate 2 -0,17cr-ketoglutarate + NHo*+ 2H* glutaminian + HrO 2 -al,4Fumarate* 2H* siccin ate 2 -0,03CoQ+ZIJ- CoQII2 2 4,0+Cytochrome b (F*'*) cytochrom e b (Fe2*) 1 0,47Dehydroascorbinate askorbininate 2 0,08Cytochrome cr (Fe3*) cytochrome ct (Fe3*) 1 0,23Cytochrome c (Fe'*) cytochrorn e c (Fe3*) 1 0,25Cytochrome a (Fe'*) cytochrom e fi (F*3*) 1 4,29%or+ Hro HrO, 2 0,3 0Cytochrome 0, (Fe3*) cytochrom frs (Fe'*) 1t 0^5 5

19

Oxidant Reductant n Es' tv]Fe3* Fe2- 1 4,77

HrO 2 0,92

20

Mitochondrial respiratory chain complexesThe proteins embedded into inner mitochondrial membrane are organized into 5 respiratorycomplexes of electron-transfer chain. Each complex consists of several proteins componentsthat are associated with the redox-active prosthetic groups with successively increasingreduction potentials.

succinate f umarate 3ADP +or

2ADP +

3Pi

21

noo()()()()oo

(]ffifl(XXX)oooo

zPi ^. .*6Hor4H*

6H* (or 4H*)

Respiratory complexes

Components of respiratory chain called as complexes are designated by Latinnumbers from I to V. The complexes Ito lV are composed with proteins,allosteric groups, coenzymes and they are the parts of mitochondriarespiratory chain. Complex V last in respiratory chain, is the enzyme calledATP synthase.

22

Complex

Complex I composed with oryreductase NADH: (dehydrogenase NADFI) and coenzyme Q,CoQ (ubiquinone). lt contains one molecule of molecule of FMN (flavin mononucleotide), whichis a redox-active prosthetic group, and 6 to 7 iron-sulfur clusters (Fe:S) . FMN accepts 2 hydrogenatoms (2H* + 2e) forming FMNHT. Complex ltransfers hydrogen atoms to ubquinone.

Cornplex I is coupled with phosphorylation process.

CoQ- coenryme Q (CoQ or ubiquinone (oxidized or quinone form).CoQH, - coenzyme GoH, or ubiquinol (reduced or hydroquinone form).Fe2+S (Fe s+)

- Fe : S (iron- sulfur clusters)

23

Cornplex ll

Complex llcomposed with enzymatic protein: succinate-coenzyme Q reductase and FAD , andalso Fe:S clusters and cytochrome b* . This complex participates in oxidation of succinate tofumarate . lt converts ubiquinone into ubiquinol (hydroquinone form).

-!- -a-SlJt-LrlllClLE

Complex I-^:::

X::-X:: X::-

X::X::X::-fu rnarateCornplex

24

Complex lll

Complex lll is composed with enzymatic protein: cytochrome c-coenzyme Q reductase.Complex lll contains also Fe:S clusters and two cytochromes b and one cytochrome c . Thiscomplex participate in oxidation of succinate to fumarate . lt converts ubiquinone into ubiquinol(hydroquinone form). tt is capable to one-electron reductions. Therefore, it provides an electronconduit between the two-electrons donor NADH and the one-electron acceptors, thecytochromes.

Complex lll is coupled with phosphorylation process.

Complex III

CoQHz cYt bo*. Cyt c red.

CoQ cYt c o*.

cyt - cytochrome, ox. - oxidizcd, red- - reduced 25

Complex lV is composed with enzymatic protein: Oxidoreductase and reduced cytochrome c:and molecular oxygen.

Complex lV contains cytochromes a, and a, and transfer the electrons from reducedcytochrome c to molekule of orygen.

Comptex lV is coupled with phosphorylation reaction.Sometime it is called cytochromeoxidase.

cYtcreo \f

cYtao*

1z- clta3red

)a a2

cytco*. -\ cytsreo" A clts3ox */ \ z,zo

=e2*s )f

cytcTox.

Fe3*S -^-- cYt cr rea.

Complex lV

Complex

cyt - cytochrome, oX. - oxidized, red. - reduced

26

Gomplex V

Synthase ATP. Each carrier takes electrons from donor and transfers it to acceptor (next inrespiratory chain). Orygen atom is the final acceptor of electron pair. ln this end step 9z-ion isformed, to which 2 protons (H+) are added and the molecule of water (HrO) is formed. ln thisprocess the organism uses the most oxygen taken from atmosphere .

Complex V (ATP synthase)

ADP + Pi

-ATPPi = inorganic phosphate

27

Scheme of electrons transport from substrate to oxygen molecule

XHz 2"' , NAD '"- -

amytal retenone

I

I

*

2e'

I

Y

ATP

antimycin

I

I

3- 2cyt b 1.* 2cyt ci 1,.'I

ATP V2cyt a + a3

CN.-LI\"?. I ,"- ArPaztae Y

ttzOz

The blue narrows show the inhibition sites of electrons transport.lnhibitors: amytat retenone, antimycin, CN- ion, caron oxide (CO)and azide.

XH, - reduced substrate

28

Oxidation of NADH by FMN

ox idative-red uctive pa irs

Eo--0,32V

NADH + H* \ /

FNIVI +2e' + zH*

\r/\

NAD* + 2e- + 2H* / \ FMNH, Eo = -0,30 v

Componets of ned-ox reaction wEth trr-'o oxidatlve-reductlve pairs

Elimination of HrO, (and (ROS) by coupled action of glutathione reductase andgl utath ione peroxidase

MDPH + H*

*/N1qpp*

G-S.S.G/ (oxidixed)

xG-S-H

(reduced)

G-SH- reduced glutathioneG-S-S.G

- oxidized glutathione

29

2HzO

Some substrates are oxidized orreduced without participation ofrespiratory chain. ln theseprocesses the ATP is not released.Glutathione red uctase catalyzesthe NDPH-dependent reduction ofglutathione disulfide (GSSG) toglutathione (GSH).

HzOz

The major function of GSH is to eliminate HrO, and other organic peroxides, the toxic products ofvarious oxidative processes damaging the cell's structures. Peroxides are etiminated ihrough theaction of glutathione peroxidase yielding glutathione GSH. The coupled action of these twoenzymes defense cell structures from attacks of reactive oxygen species (RoS)

30

General scheme of reaction

thionetase

qfut\Loil

Formation of peroxide anion radical

+o2- + H*peroxideanion radical

semiquinoneradical

+o1- + H*peroxideanion radical

semiquinoneradical

Oxidation of ubiquinone leads to formation of peroxide anion radical (ROS).For simplifying,in the scheme a ring of ubiquinone is presented only

- ring with hydroxyl groups-

(rino-side substituents),

Formation and elimination of oxygen reactive species (ROS)

OH

oOH

ubilrydo+rincx'te

o'

+OH

o'

+OH

31

2e' 2e'

Oz !. - o) Hzozpero 2'OH '*__- HzO +ltzOz.

hydroxyl radical

:)2oH --'- Hjo' ttzOz

G-SS-Gglutathione oxidized2 G-SH

glutathione reduced

32