biochemistry - i · pdf filebiochemistry - i mondays and ... • oxidative phosphorylation...

Based on Profs. Kevin Gardner & Reza Khayat 1

Biochemistry - I

Mondays and Wednesdays 9:30-10:45 AM (MR-1307)

SPRING 2017

Lectures 23-24

Outline

• Anatomy of the mitochondrion • Electron transfer • Fractionation of the electron transport chain • The electron transport chain • Proton-motive force • Reactive oxygen species (ROS) • ATP Synthesis • Architecture of ATP synthase (F0F1) complex • The proton-motive-force energizes active

transport • Regulation

Why learn about Oxidative Phosphorylation? This is where the majority of ATP is generated, downstream of

glycolysis, the TCA cycle and fatty acid oxidation

2Chapter 19 - Oxidative Phosphorylation

Anatomy of the Mitochondrion


Anatomy of the Mitochondrion

4

• The cristae (leaflets, inner membrane) of the mitochondria serves as a platform for many copies of different enzymes to assemble in proximity for substrate shuttling (compartmentalization)

• These leaflets are home to tens of thousands of proteins responsible for the electron-transfer system, and ATP synthase

• The mitochondria of heart muscle, a muscle under constant activity, contains >3x the amount of electron-transfer systems than the liver mitochondrion (enzyme regulation)

Chapter 19 - Oxidative Phosphorylation

Electron Transfer

5

In oxidative phosphorylation, electrons are transferred via three mechanisms:

1. Direct transfer, such as reduction of Fe+3 to Fe+2 (e.g. cytochrome) 2. Transfer as proton H+ and electron e- (e.g. flavins, FADH2) 3. Transfer as hydride ion :H–(e.g. dehydrogenases, NADH)


Prosthetic Groups and Ligands involved in Electron Transport: Porphyrin

6

Cytochromes • Porphyrin-carrying

proteins, similar to Hb and Mb

• Tightly, sometimes covalently, associated with flavoprotein

• Fe3+ converted to Fe2+ during reduction, as electrons transferred to oxidizing agent


Prosthetic Groups and Ligands Involved in Electron Transport: Iron-sulfur Centers

7

19.1 Electron-Transfer Reactions in Mitochondria 711

b, and c, which are distinguished by differences in theirlight-absorption spectra. Each type of cytochrome in itsreduced (Fe2!) state has three absorption bands in thevisible range (Fig. 19–4). The longest-wavelength bandis near 600 nm in type a cytochromes, near 560 nm intype b, and near 550 nm in type c. To distinguish amongclosely related cytochromes of one type, the exact ab-sorption maximum is sometimes used in the names, as incytochrome b562.

The heme cofactors of a and b cytochromes aretightly, but not covalently, bound to their associatedproteins; the hemes of c-type cytochromes are cova-lently attached through Cys residues (Fig. 19–3). Aswith the flavoproteins, the standard reduction potentialof the heme iron atom of a cytochrome depends on itsinteraction with protein side chains and is therefore dif-ferent for each cytochrome. The cytochromes of type aand b and some of type c are integral proteins of the in-ner mitochondrial membrane. One striking exception is

the cytochrome c of mitochondria, a soluble proteinthat associates through electrostatic interactions withthe outer surface of the inner membrane.

In iron-sulfur proteins, the iron is present not inheme but in association with inorganic sulfur atoms orwith the sulfur atoms of Cys residues in the protein, orboth. These iron-sulfur (Fe-S) centers range from sim-ple structures with a single Fe atom coordinated to fourCys—SH groups to more complex Fe-S centers withtwo or four Fe atoms (Fig. 19–5). Rieske iron-sulfurproteins (named after their discoverer, John S. Rieske)are a variation on this theme, in which one Fe atomis coordinated to two His residues rather than twoCys residues. All iron-sulfur proteins participate in one-electron transfers in which one iron atom of the iron-sulfur cluster is oxidized or reduced. At least eight Fe-Sproteins function in mitochondrial electron transfer.The reduction potential of Fe-S proteins varies from"0.65 V to !0.45 V, depending on the microenviron-ment of the iron within the protein.

In the overall reaction catalyzed by the mitochond-rial respiratory chain, electrons move from NADH, succi-nate, or some other primary electron donor throughflavoproteins, ubiquinone, iron-sulfur proteins, and cy-tochromes, and finally to O2. A look at the methods usedto determine the sequence in which the carriers act isinstructive, as the same general approaches have beenused to study other electron-transfer chains, such asthose of chloroplasts.

First, the standard reduction potentials of the indi-vidual electron carriers have been determined experi-mentally (Table 19–2). We would expect the carriers tofunction in order of increasing reduction potential, be-cause electrons tend to flow spontaneously fromcarriers of lower E#$ to carriers of higher E#$. The orderof carriers deduced by this method is NADH → Q →cytochrome b → cytochrome c1 → cytochrome c →cytochrome a → cytochrome a3 → O2. Note, however,that the order of standard reduction potentials is notnecessarily the same as the order of actual reductionpotentials under cellular conditions, which depend onthe concentration of reduced and oxidized forms (see

100

Rel

ativ

e lig

ht a

bsor

ptio

n (%

)

50

300 400 500 600

Wavelength (nm)

Oxidizedcyt c

Reducedcyt c

%

&

'

0

FIGURE 19–4 Absorption spectra of cytochrome c (cyt c) in its oxidized(red) and reduced (blue) forms. Also labeled are the characteristic !, ",and # bands of the reduced form.

S CysCys

S CysCys S

S

(a)

Fe

Protein

S

(b)

SFe

SFe

S

SCys S Cys

Cys CysS

(c) Cys

Cys

FeS

S

S

S

S

S S

Fe

Fe Fe CysCys

(d)

FIGURE 19–5 Iron-sulfur centers. The Fe-S centers of iron-sulfur pro-teins may be as simple as (a), with a single Fe ion surrounded by theS atoms of four Cys residues. Other centers include both inorganic andCys S atoms, as in (b) 2Fe-2S or (c) 4Fe-4S centers. (d) The ferredoxinof the cyanobacterium Anabaena 7120 has one 2Fe-2S center (PDB ID1FRD); Fe is red, inorganic S is yellow, and the S of Cys is orange.

(Note that in these designations only the inorganic S atoms arecounted. For example, in the 2Fe-2S center (b), each Fe ion is actuallysurrounded by four S atoms.) The exact standard reduction potential ofthe iron in these centers depends on the type of center and its interac-tion with the associated protein.

Iron-sulfur proteins • Iron associated with Cys or His AA, and inorganic sulfur • different “clusters” found in different iron-sulfur proteins • Clusters vary in complexity • Some proteins have multiple clusters, can be of different types • The reduction potential of the clusters vary by their environment

(number of sulfurs and irons present, the geometry, and the neighbors) • Rieske iron-sulfur proteins: One Fe atom is coordinated to 2 His rather

than 2 Cys


Additional Prosthetic Groups and Ligands Involved in Electron Transfer

8

Ubiquinone = coenzyme Q = Q • Capable of carrying 2e- and 2H+ • Remember that H+ + e- can also be

written as H•

Water soluble and released into solution by enzyme Carries :H-, H+ released into solution

FAD <=> FADH2Either tightly or covalently bound to flavoproteins (e.g. succinate dehydrogenase in TCA)


• Oxidative phosphorylation uses energy released from oxidation of metabolites to produce ATP

• Electrons flow from more negative reduction potential to more positive electron potential (top to bottom of table)

• Note how all the cytochromes use Fe+3→Fe+2 oxidation yet have different reduction potentials. The ligand environment of the Fe+3 results in different reduction potential


Calculating Free Energy Change from Redox Reactions (Sect 13.4)

10Chapter 13 - Bioenergetics

• Note free energy related by negative of delta E • Remember to pair two half-reactions, and invert sign on oxidation, e.g.:

• oxidization: NADH + H+ -> NAD+ + 2H+ + 2e- = +0.320V • reduction: 0.5(O2) + 2H+ + 2e- -> H2O = +0.817V • net: NADH + H+ 0.5(O2) -> H2O + NAD+ 0.320 + 0.817 = 1.137V :

favorable

• n = number of electrons involved • Faraday’s constant (F) = 96,500 J / (V mol)

Method 1 - Spectroscopic: 1. This requires that you know and can spectroscopically characterize all the players 2. Remove O2 and reduce the entire system by providing reducing agents (NADH + H+) 3. Add O2 and spectroscopically identify which component is oxidized first, second, third… The species that gets oxidized first is the species that is closest to O2 in the electron transport chain.

Method 2 – Chemical inhibitors: 1. Identify inhibitors that kill the electron transport chain 2. Add inhibitor to electron transport chain and identify which components are oxidized and which components are reduced 3. Oxidized components are to the right of the inhibitor and reduced components are to the left of the inhibitor 4. Use many inhibitors to identify the order of components

Determining Order of Electron Transfer

11

Oxidative Phosphorylation and Photophosphorylation712

Eqn 13–5, p. 515). A second method for determining thesequence of electron carriers involves reducing the en-tire chain of carriers experimentally by providing anelectron source but no electron acceptor (no O2). WhenO2 is suddenly introduced into the system, the rate atwhich each electron carrier becomes oxidized (mea-sured spectroscopically) reveals the order in which thecarriers function. The carrier nearest O2 (at the end ofthe chain) gives up its electrons first, the second carrierfrom the end is oxidized next, and so on. Such experi-ments have confirmed the sequence deduced from stan-dard reduction potentials.

In a final confirmation, agents that inhibit the flow ofelectrons through the chain have been used in combina-tion with measurements of the degree of oxidation ofeach carrier. In the presence of O2 and an electrondonor, carriers that function before the inhibited stepbecome fully reduced, and those that function after thisstep are completely oxidized (Fig. 19–6). By usingseveral inhibitors that block different steps in the chain,investigators have determined the entire sequence; it isthe same as deduced in the first two approaches.

Electron Carriers Function in Multienzyme Complexes

The electron carriers of the respiratory chain areorganized into membrane-embedded supramolecularcomplexes that can be physically separated. Gentletreatment of the inner mitochondrial membrane withdetergents allows the resolution of four unique electron-carrier complexes, each capable of catalyzing electrontransfer through a portion of the chain (Table 19–3;Fig. 19–7). Complexes I and II catalyze electron trans-fer to ubiquinone from two different electron donors:NADH (Complex I) and succinate (Complex II). Com-plex III carries electrons from reduced ubiquinone to cy-tochrome c, and Complex IV completes the sequence bytransferring electrons from cytochrome c to O2.

We now look in more detail at the structure andfunction of each complex of the mitochondrial respira-tory chain.

Complex I: NADH to Ubiquinone Figure 19–8 illus-trates the relationship between Complexes I and II andubiquinone. Complex I, also called NADH:ubiquinone

Standard Reduction Potentials of Respiratory Chain and Related Electron Carriers

Redox reaction (half-reaction) E!" (V)

2H# # 2e$ H2 $0.414

NAD# # H# # 2e$ NADH $0.320

NADP# # H# # 2e$ NADPH $0.324

NADH dehydrogenase (FMN) # 2H# # 2e$ NADH dehydrogenase (FMNH2) $0.30

Ubiquinone # 2H# # 2e$ ubiquinol 0.045

Cytochrome b (Fe3#) # e$ cytochrome b (Fe2#) 0.077

Cytochrome ct (Fe3#) # e$ cytochrome ct (Fe2#) 0.22

Cytochrome c (Fe3#) # e$ cytochrome c (Fe2#) 0.254

Cytochrome a (Fe3#) # e$ cytochrome a (Fe2#) 0.29

Cytochrome a3 (Fe3#) # e$ cytochrome a3 (Fe2#) 0.35

0.816612O2 # 2H# # 2e$ ¡ H2O

¡¡¡¡¡

¡¡

¡¡

¡

TABLE 19–2

NADH Q

Cyt c1

Cyt (a # a3) O2

rotenone

antimycin A

CN$ or CO

Cyt cCyt bNADH Q O2

NADH Q O2

Cyt c1 Cyt cCyt b

Cyt c1 Cyt Cyt cCyt b

Cyt (a # a3)

a # a3)(

FIGURE 19–6 Method for determining the sequence of electron carri-ers. This method measures the effects of inhibitors of electron transferon the oxidation state of each carrier. In the presence of an electron

donor and O2, each inhibitor causes a characteristic pattern of oxidized/reduced carriers: those before the block become reduced (blue), andthose after the block become oxidized (pink).


Fractionation of the Electron Transport Chain

12

• Outer membrane dissolved and washed away with detergent

• Inner membrane fragmented by osmotic pressure (i.e. water leaking in)

• Inner membrane fragments solubilized with detergents

• Fractionation done by ion-exchange chromatography


The Electron Transport Chain

13

• Electron transport chain serves to: • oxidize the reducing agents (NADH and FADH2) generated during

glycolysis, TCA, and FA oxidation • generate a proton gradient across the mitochondrion membrane

• Proton gradient stores energy that is used to condense ADP and Pi to ATP by ATP synthase

I) NADH dehydrogenase II) Succinate dehydrogenase (TCA, Rx 6)

III) Ubiquinone:Cytochrome c oxidoreductase

IV) Cytochrome oxidase


The Role of Ubiquinone/Coenzyme Q/Q

14

• Electrons from a variety of sources are passed to Q

• Complex 1 uses electrons from NADH+H+

• Complex 2 uses the FADH2 generated in the oxidation of succinate to fumarate (TCA Rx 6)

• Glycerol 3-phosphate dehydrogenase uses FADH2 generated during glycerol oxidation

• Electrons from FADH2 generated in the first step of fatty acid oxidation is passed to Q

• Role of Q is to transport electrons • Q remains in and travels across

membrane bilayer


Coenzyme Q Structure - Membrane association, Redox Sites


Complex I

16

• NADH dehydrogenase • 42 different polypeptide chains • six Fe-S clusters/centers • FMN is the prosthetic group

Coupled processes carried out by Complex I: • Exergonic transfer 2e- from NADH to Q, and

two proton from matrix to Q • Endergonic transfer of 4 protons from matrix

to intermembrane space


departure of protons) to another (the intermembranespace, which becomes positively charged). To empha-size the vectorial nature of the process, the overall reac-tion is often written with subscripts that indicate the

location of the protons: P for the positive side of the in-ner membrane (the intermembrane space), N for thenegative side (the matrix):

(19–2)

Amytal (a barbiturate drug), rotenone (a plant productcommonly used as an insecticide), and piericidin A (anantibiotic) inhibit electron flow from the Fe-S centers ofComplex I to ubiquinone (Table 19–4) and thereforeblock the overall process of oxidative phosphorylation.

Ubiquinol (QH2, the fully reduced form; Fig. 19–2)diffuses in the inner mitochondrial membrane fromComplex I to Complex III, where it is oxidized to Q in aprocess that also involves the outward movement of H!.

NADH ! 5H!N ! Q ¡ NAD! ! QH2 ! 4H!

P

Seriesof Fe-Scenters

Seriesof Fe-Scenters

FMN

FMN

Intermembranespace (P side)

Matrix(N side)

2e–

2H+

2e–N-2

Q

QH2

Complex I

Matrixarm

Membranearm

NADH + H+

NAD+

4H+ FIGURE 19–9 NADH:ubiquinone oxidoreductase (Complex I). Com-plex I catalyzes the transfer of a hydride ion from NADH to FMN, fromwhich two electrons pass through a series of Fe-S centers to the iron-sulfur protein N-2 in the matrix arm of the complex. The domain thatextends into the matrix has been crystallized and its structure solved(PDB ID 2FUG); the structure of the membrane domain of Complex Iis not yet known. Electron transfer from N-2 to ubiquinone on themembrane arm forms QH2, which diffuses into the lipid bilayer. Thiselectron transfer also drives the expulsion from the matrix of four pro-tons per pair of electrons. The detailed mechanism that couples elec-tron and proton transfer in Complex I is not yet known, but probablyinvolves a Q cycle similar to that in Complex III in which QH2 partic-ipates twice per electron pair (see Fig. 19–12). Proton flux produces anelectrochemical potential across the inner mitochondrial membrane (Nside negative, P side positive), which conserves some of the energy re-leased by the electron-transfer reactions. This electrochemical poten-tial drives ATP synthesis.

Agents That Interfere with Oxidative Phosphorylation or Photophosphorylation

Type of interference Compound* Target/mode of action

Inhibition of electron transfer Cyanide Inhibit cytochrome oxidaseCarbon monoxide

Antimycin A Blocks electron transfer from cytochrome b to cytochrome c1

MyxothiazolRotenoneAmytal

Prevent electron transfer from Fe-S center to ubiquinone

Piericidin A

DCMU Competes with QB for binding site in PSII

Inhibition of ATP synthase Aurovertin Inhibits F1

Oligomycin Inhibit Fo and CFoVenturicidin

DCCD Blocks proton flow through Fo and CFo

Uncoupling of phosphorylation FCCP Hydrophobic proton carriersfrom electron transfer DNP

Valinomycin K! ionophore

Thermogenin In brown adipose tissue, forms proton-conducting pores in inner mitochondrial membrane

Inhibition of ATP-ADP exchange Atractyloside Inhibits adenine nucleotide translocase

TABLE 19–4

*DCMU is 3-(3,4-dichlorophenyl)-1,1-dimethylurea; DCCD, dicyclohexylcarbodiimide; FCCP, cyanide-p-trifluoromethoxyphenylhydrazone; DNP, 2,4-dinitrophenol.

⎫⎬⎭

⎫⎬⎭

⎫⎪⎬⎪⎭

⎫⎬⎭

(+)

(-)

similar to FAD


Complex II

17

• Succinate dehydrogenase (enzyme 6 in TCA)

• Four polypeptide chains • 5 prosthetic groups • FAD is tightly bound to enzyme • Transmembrane domains with

cytoplasmic extensions • Transfers electrons to FAD

from oxidation of succinate → fumarate

• Heme b acts to protect against formation of reactive oxygen species by electrons that interact with O2

• Purpose is to generate QH2 • No H+ are transported


Fatty Acid Oxidation

18

• First step in β-oxidation involves generation of FADH2

• FADH2 needs to be oxidized to FAD+ • FAD+ cannot be released into solution • Electrons are passed from one flavoprotein to

another • The final protein ETF:Q passes the electrons to Q to

make QH2.


Complex III

19

• Cytochrome bc1 has 11 polypeptide chains (subunits) • Complex is a homo-dimer of two cytochrome bc1 • Hemes bH, bL, c1, and Q shuttle electrons • Rieske iron-sulfur protein swivels between Heme bL and c1

• 2Fe-2S receives electrons from Qp when it’s close to Qp • 2Fe-2S donates electrons when it’s closer to Heme c1

• Transfer of H+ out of matrix, next slide

(+)

(-)


Complex III

20

• QH2 (on top) passes one electron to 2Fe-2S and another to Heme bL • QH2 passes its two H+ to the intermem. space to increase electro. pot. • 2Fe-2S passes electron to Heme c1 which passes it to Cyt c • The Heme bL electron is passed to bH then to a Q to generate •Q • The same reaction happens again, except that •Q picks up two protons

from the mitochondrial matrix • Thus, 2QH2 pass 4H+ to intermem. space and pull 2H+ from matrix space


Cyt c


Matrix(N side)

Cytochrome b

QH2QH2Q

QQ

Q

Heme c1

Cyt c1

Heme bLHeme bH

2Fe-2S

2H+

2H+

e–

e–

e–

e–

e–

e–

•Q–

Cyt c

QH2QH2

QH2

QH2

Q

Q

Q

2H+e–

e–

e–

e–

e–

e–

•Q–

QH2 cyt c1 (oxidized)! Q !

QH2 2 cyt c1 (oxidized)!

2HP! Q !!

cyt c1 (reduced)!

Q 2 cyt c1 (reduced)! !

!•Q"

•Q"

!

4HP!

2HP!

2HN!

! 2HN!

QH2

QH2

cyt c1 (oxidized)!

Q! ! cyt c1 (reduced)!

Net equation:

FIGURE 19–12 The Q cycle, shown in two stages. The path of electronsthrough Complex III is shown by blue arrows. In the first stage (left), Qon the N side is reduced to the semiquinone radical, which in the sec-ond stage (right) is converted to QH2. Meanwhile, on the P side of themembrane, two molecules of QH2 are oxidized to Q, releasing two

protons per Q molecule (four protons in all) into the intermembranespace. Each QH2 donates one electron (via the Rieske Fe-S center) tocytochrome c1, and one electron (via cytochrome b) to a molecule ofQ near the N side, reducing it in two steps to QH2. This reduction alsouses two protons per Q, which are taken up from the matrix.

Met His

HisGlu

CysCys

(a)

(b)

180°

Heme a3

Heme a


Matrix(N side)

FIGURE 19–13 Structure of cytochrome oxidase (Complex IV). Thiscomplex from bovine mitochondria has 13 subunits, but only four coreproteins are shown here (PDB ID 1OCC). (a) Complex IV, with foursubunits in each of two identical units of a dimer. Subunit I (yellow) hastwo heme groups, a and a3, near a single copper ion, CuB (greensphere). Heme a3 and CuB form a binuclear Fe-Cu center. Subunit II(purple) contains two Cu ions complexed with the —SH groups of twoCys residues in a binuclear center, CuA, that resembles the 2Fe-2S cen-ters of iron-sulfur proteins. This binuclear center and the cytochrome

c–binding site are located in a domain of subunit II that protrudes fromthe P side of the inner membrane (into the intermembrane space). Sub-unit III (light blue) is essential for rapid proton movement through sub-unit II. The role of subunit IV (green) is not yet known. (b) The binuclearcenter of CuA. The Cu ions share electrons equally. When the center isreduced, the ions have the formal charges Cu1!Cu1!; when oxidized,Cu1.5!Cu1.5!. Six amino acids residues are ligands around the Cu ions:two His, two Cys, Glu, and Met.

electrons transported to complex IV

(+)

(-)


• Uses binuclear Fe-Cu center (Heme a3 and CuB) • Has 13 subunits and the 3 shown in right image are most important • Needs 4 electrons from complex III (each Cyt c carries only 1 e-) • 2e- from 2Cyt c are passed to Heme a3 to reduce O2 to O22-. 2H+ are shuttled from

matrix to balance charge at Heme a3 • This is repeated to make 2H2O molecules • 4H+ are also pumped into the inter membrane space • Thus 8H+ from the matrix are needed to generate 2H2O

Complex IV

21

electrons transported from complex III

(+)

(-)



Much of this energy is used to pump protons out ofthe matrix. For each pair of electrons transferred to O2,four protons are pumped out by Complex I, four byComplex III, and two by Complex IV (Fig. 19–16). Thevectorial equation for the process is therefore

(19–7)

The electrochemical energy inherent in this differencein proton concentration and separation of charge

NADH ! 11H!N ! 1

2O2 ¡ NAD! ! 10H!P ! H2O

NAD!. In actively respiring mitochondria, the actions ofmany dehydrogenases keep the actual [NADH]/[NAD!]ratio well above unity, and the real free-energy change forthe reaction shown in Equation 19–5 is therefore substan-tially greater (more negative) than "220 kJ/mol. A simi-lar calculation for the oxidation of succinate shows thatelectron transfer from succinate (E#$ for fumarate/succi-nate % 0.031 V) to O2 has a smaller, but still negative,standard free-energy change of about "150 kJ/mol.

FIGURE 19–15 A putative respirasome composed of Complexes IIIand IV. (a) Purified supercomplexes containing Complexes III and IV,from yeast, visualized by electron microscopy after staining withuranyl acetate. The electron densities of hundreds of images were av-eraged to yield this composite view. (b) The x-ray structures of one

molecule of Complex III (red; from yeast) and two of Complex IV(green; from bovine heart) could be fitted to the electron-density mapto suggest one possible mode of interaction of these complexes in arespirasome. This view is in the plane of the bilayer (yellow).

(b)(a)

SuccinateFumarate

Cyt c

4H+

2H+4H+

III

Q


Matrix (N side)

III

IVIII

NADH + H+NAD+

O2 H22H++ O21

FIGURE 19–16 Summary of the flow of electrons and protons throughthe four complexes of the respiratory chain. Electrons reach Qthrough Complexes I and II. The reduced Q (QH2) serves as a mobilecarrier of electrons and protons. It passes electrons to Complex III,which passes them to another mobile connecting link, cytochrome c.Complex IV then transfers electrons from reduced cytochrome c to O2.Electron flow through Complexes I, III, and IV is accompanied by pro-

ton flow from the matrix to the intermembrane space. Recall that elec-trons from ! oxidation of fatty acids can also enter the respiratorychain through Q (see Fig. 19–8). The structures shown here are fromseveral sources: Complex I, Thermus thermophilus (PDB ID 2FUG);Complex II, porcine heart (PDB ID 1ZOY); Complex III, bovine heart(PDB ID 1BGY); cytochrome c, equine heart (PDB ID 1HRC); Com-plex IV, bovine heart (PDB ID 1OCC).

The Electron Transport Chain

22

• The electron transport chain serves to oxidize the reducing agents (NADH and FADH2) generated during glycolysis, TCA, and FA with O2.

• These are highly exergonic reactions and the released energies are used to generate a proton gradient across the mitochondrion membrane

• This energy is used to condense ADP and Pi to ATP


Reactive Oxygen Species (ROS)

23


The hydrogen peroxide (H2O2) thus generated is ren-dered harmless by the action of glutathione peroxi-dase (Fig. 19–18). Glutathione reductase recycles theoxidized glutathione to its reduced form, using electronsfrom the NADPH generated by nicotinamide nucleotidetranshydrogenase (in the mitochondrion) or by the pen-tose phosphate pathway (in the cytosol; see Fig. 14–20).Reduced glutathione also serves to keep proteinsulfhydryl groups in their reduced state, preventingsome of the deleterious effects of oxidative stress(Fig. 19–18). Nicotinamide nucleotide transhydroge-nase is critical in this process: it produces the NADPHessential for glutathione reductase activity.

Plant Mitochondria Have Alternative Mechanisms forOxidizing NADH

Plant mitochondria supply the cell with ATP duringperiods of low illumination or darkness by mecha-nisms entirely analogous to those used by nonphoto-synthetic organisms. In the light, the principal sourceof mitochondrial NADH is a reaction in which glycine,

Q

O2

O2

.

OH

.

–

Cyt c

IVI IIIQ

Nicotinamidenucleotidetranshydrogenase

Innermitochondrialmembrane

NADH

NADPH

GSSGH2O2

H2O2 GSH

S

S2 GSHEnz

inactive

active

oxidativestress

protein thiolreduction

GSSG

NADP+glutathionereductase

glutathioneperoxidase

superoxide dismutase

NAD+

NAD+

SH

SH

FIGURE 19–18 ROS formation in mitochondria and mitochondrial de-fenses. When the rate of electron entry into the respiratory chain andthe rate of electron transfer through the chain are mismatched, super-oxide radical (!O2

") production increases at Complexes I and III as thepartially reduced ubiquinone radical (!Q") donates an electron to O2.Superoxide acts on aconitase, a 4Fe-4S protein, to release Fe2#. In thepresence of Fe2#, the Fenton reaction leads to formation of the highlyreactive hydroxyl free radical (!OH). The reactions shown in blue de-fend the cell against the damaging effects of superoxide. Reduced glu-tathione (GSH; see Fig. 22–27) donates electrons for the reduction ofH2O2 and of the oxidized Cys residues (⎯S⎯S⎯) of enzymes andother proteins, and GSH is regenerated from the oxidized form (GSSG)by reduction with NADPH.

produced by a process known as photorespiration, isconverted to serine (see Fig. 20–21):

For reasons discussed in Chapter 20, plants must carryout this reaction even when they do not need NADH forATP production. To regenerate NAD# from unneededNADH, plant mitochondria transfer electrons from NADHdirectly to ubiquinone and from ubiquinone directly toO2, bypassing Complexes III and IV and their protonpumps. In this process the energy in NADH is dissipatedas heat, which can sometimes be of value to the plant(Box 19–1). Unlike cytochrome oxidase (Complex IV),the alternative QH2 oxidase is not inhibited by cyanide.Cyanide-resistant NADH oxidation is therefore the hall-mark of this unique plant electron-transfer pathway.

SUMMARY 19.1 Elec tron-TransferReac tions in Mitochondria

■ Chemiosmotic theory provides the intellectualframework for understanding many biologicalenergy transductions, including oxidativephosphorylation and photophosphorylation. Themechanism of energy coupling is similar in bothcases: the energy of electron flow is conserved bythe concomitant pumping of protons across themembrane, producing an electrochemical gradient,the proton-motive force.

■ In mitochondria, hydride ions removed fromsubstrates by NAD-linked dehydrogenases donateelectrons to the respiratory (electron-transfer)chain, which transfers the electrons to molecularO2, reducing it to H2O.

■ Shuttle systems convey reducing equivalents fromcytosolic NADH to mitochondrial NADH. Reducingequivalents from all NAD-linked dehydrogenationsare transferred to mitochondrial NADHdehydrogenase (Complex I).

■ Reducing equivalents are then passed through aseries of Fe-S centers to ubiquinone, which transfers the electrons to cytochrome b, the firstcarrier in Complex III. In this complex, electronstake two separate paths through two b-type cytochromes and cytochrome c1 to an Fe-S center.The Fe-S center passes electrons, one at a time,through cytochrome c and into Complex IV, cytochrome oxidase. This copper-containing enzyme, which also contains cytochromes a and a3,accumulates electrons, then passes them to O2, reducing it to H2O.

■ Some electrons enter this chain of carriers throughalternative paths. Succinate is oxidized bysuccinate dehydrogenase (Complex II), whichcontains a flavoprotein that passes electrons

2 Glycine # NAD# ¡ serine # CO2 # NH3 # NADH # H#• Flow of e- results in oxide ·O2-

formation (superoxide free radical) • Under normal conditions 0.1-4% of

O2 is converted to O2- (lethal levels) • Superoxide dismutase gets rid of the

superoxides

• Hydrogen peroxide is reduced to water with use of GSH (GSSG is formed, previously discussed)


WORKED EXAMPLE 19–1 Energetics of ElectronTransfer

Calculate the amount of energy conserved in the protongradient across the inner mitochondrial membrane perpair of electrons transferred through the respiratorychain from NADH to oxygen. Assume !! is 0.15 V andthe pH difference is 0.75 units.

Solution: Equation 19–9 gives the free-energy changewhen one mole of protons moves across the inner mem-brane. Substituting the measured values for !pH(0.75 units) and !! (0.15 V) in this equation gives !G "19 kJ/mol (of protons). Because the transfer of two elec-trons from NADH to O2 is accompanied by the outwardpumping of 10 protons (Eqn 19–7), roughly 200 kJ (ofthe 220 kJ released by oxidation of 1 mol of NADH) isconserved in the proton gradient.

When protons flow spontaneously down their elec-trochemical gradient, energy is made available to dowork. In mitochondria, chloroplasts, and aerobic bacte-ria, the electrochemical energy in the proton gradientdrives the synthesis of ATP from ADP and Pi. We returnto the energetics and stoichiometry of ATP synthesisdriven by the electrochemical potential of the protongradient in Section 19.2.

Reactive Oxygen Species Are Generated during Oxidative Phosphorylation

Several steps in the path of oxygen reduction in mito-chondria have the potential to produce highly reactivefree radicals that can damage cells. The passage of elec-trons from QH2 to Complex III, and passage of electronsfrom Complex I to QH2, involve the radical #Q– as an in-termediate. The #Q– can, with a low probability, pass anelectron to O2 in the reaction

The superoxide free radical thus generated is highly re-active; its formation also leads to production of the evenmore reactive hydroxyl free radical, (Fig. 19–18).

These reactive oxygen species can wreak havoc, re-acting with and damaging enzymes, membrane lipids,and nucleic acids. In actively respiring mitochondria,0.1% to as much as 4% of the O2 used in respirationforms —more than enough to have lethal effectsunless the free radical is quickly disposed of. Factorsthat slow the flow of electrons through the respiratorychain increase the formation of superoxide, perhaps byprolonging the lifetime of generated in the Q cycle.

To prevent oxidative damage by , cells have sev-eral forms of the enzyme superoxide dismutase,which catalyzes the reaction

2 !O$2 % 2H% ¡ H2O2 % O2

!O$2

!O$2

!O$2

!OH

O2 % e$ ¡ !O$2

FIGURE 19–17 Proton-motive force. The inner mitochondrial mem-brane separates two compartments of different [H%], resulting in differ-ences in chemical concentration (!pH) and charge distribution (!!)across the membrane. The net effect is the proton-motive force (!G),which can be calculated as shown here. This is explained more fully inthe text.

N side

[H%]N " C1

OH$

OH$

OH$

OH$

OH$

OH$

OH$

H%

H%

H%

H%

H%

H%

H%

P side

[H%]P " C2

!G " RT ln (C2/C1) % Z !

" 2.3RT !pH % ∆

H%

Protonpump

&

&

represents a temporary conservation of much of theenergy of electron transfer. The energy stored in sucha gradient, termed the proton-motive force, has twocomponents: (1) the chemical potential energy dueto the difference in concentration of a chemicalspecies (H%) in the two regions separated by themembrane, and (2) the electrical potential energythat results from the separation of charge when a pro-ton moves across the membrane without a counterion(Fig. 19–17).

As we showed in Chapter 11, the free-energychange for the creation of an electrochemical gradientby an ion pump is

(19–8)

where C2 and C1 are the concentrations of an ion in tworegions, and C2 > C1; Z is the absolute value of its elec-trical charge (1 for a proton); and !! is the transmem-brane difference in electrical potential, measured involts.

For protons at 25 'C,

and Equation 19–8 reduces to

(19–9)

In actively respiring mitochondria, the measured !! is0.15 to 0.20 V and the pH of the matrix is about 0.75units more alkaline than that of the intermembranespace.

" (5.70 kJ/mol)¢pH % (96.5 kJ/V !mol)¢c ¢G " 2.3RT ¢pH % ¢c

" 2.3(pHN $ pHP) " 2.3 ¢pH

ln aC2

C1b " 2.3(log [H%]P $ log [H%]N)

¢G " RT ln aC2

C1b % Z ¢c


Proton-motive Force

24

• Electron passing/transporting and proton pumping generate an electrochemical gradient

• The gradient is between the mitochondrion matrix and intermembrane space (NOT cytosol!)

• Composed of chemical and electrical potential energy • Energy can be converted to ATP


ATP Synthesis


ATP Synthase (F0F1) Complex

26

• F1 hydrolyzes ATP • F0 destroys electrochemical gradient • F1 and F0 together synthesize ATP • ATP synthase binds ATP (Kd=10-12) stronger than ADP+Pi (10-5) • Binding energy of ATP responsible for condensation (endergonic)

reaction • Protons responsible for releasing ATP from ATP synthase


(!G"# $ %30.5 kJ/mol) for the hydrolysis of ATP free insolution (not on the enzyme surface).

What accounts for the huge difference? ATP syn-thase stabilizes ATP relative to ADP & Pi by binding ATPmore tightly, releasing enough energy to counterbalancethe cost of making ATP. Careful measurements of thebinding constants show that FoF1 binds ATP with veryhigh affinity (Kd ' 10%12 M) and ADP with much loweraffinity (Kd ≈ 10%5 M). The difference in Kd correspondsto a difference of about 40 kJ/mol in binding energy, andthis binding energy drives the equilibrium toward for-mation of the product ATP.

The Proton Gradient Drives the Release of ATP from theEnzyme Surface

Although ATP synthase equilibrates ATP with ADP & Pi,in the absence of a proton gradient the newly synthesizedATP does not leave the surface of the enzyme. It is the

proton gradient that causes the enzyme to release theATP formed on its surface. The reaction coordinate dia-gram of the process (Fig. 19–24) illustrates the differ-ence between the mechanism of ATP synthase and that ofmany other enzymes that catalyze endergonic reactions.

For the continued synthesis of ATP, the enzymemust cycle between a form that binds ATP very tightlyand a form that releases ATP. Chemical and crystallo-graphic studies of the ATP synthase have revealed thestructural basis for this alternation in function.

Each ! Subunit of ATP Synthase Can Assume ThreeDifferent Conformations

Mitochondrial F1 has nine subunits of five differenttypes, with the composition !3"3#$(. Each of the three" subunits has one catalytic site for ATP synthesis. Thecrystallographic determination of the F1 structure byJohn E. Walker and colleagues revealed structural details

FIGURE 19–24 Reaction coordinate diagrams for ATPsynthase and for a more typical enzyme. In a typicalenzyme-catalyzed reaction (left), reaching the transitionstate (‡) between substrate and product is the major en-ergy barrier to overcome. In the reaction catalyzed byATP synthase (right), release of ATP from the enzyme,not formation of ATP, is the major energy barrier. Thefree-energy change for the formation of ATP from ADPand Pi in aqueous solution is large and positive, but onthe enzyme surface, the very tight binding of ATP pro-vides sufficient binding energy to bring the free energyof the enzyme-bound ATP close to that of ADP & Pi, sothe reaction is readily reversible. The equilibrium con-stant is near 1. The free energy required for the release ofATP is provided by the proton-motive force.

18O

18O

ADP

ATP & H218O

&

18OP18O

Enzyme(F1)

(a)

ADP

a-Arg376b-Arg182

b-Glu181

b-Lys155

Mg2+

FIGURE 19–23 Catalytic mechanism of F1. (a) 18O-exchange experiment.F1 solubilized from mitochondrial membranes is incubated with ATP inthe presence of 18O-labeled water. At intervals, a sample of the solution iswithdrawn and analyzed for the incorporation of 18O into the Pi producedfrom ATP hydrolysis. In minutes, the Pi contains three or four 18O atoms,indicating that both ATP hydrolysis and ATP synthesis have occurred sev-eral times during the incubation. (b)The likely transition state complex for

ATP hydrolysis and synthesis by ATP synthase (derived from PDB ID1BMF). The ! subunit is shown in green, " in gray. The positively chargedresidues "-Arg182 and !-Arg376 coordinate two oxygens of the pentavalentphosphate intermediate; "-Lys155 interacts with a third oxygen, and theMg2& ion (green sphere) further stabilizes the intermediate. The bluesphere represents the leaving group (H2O). These interactions result in theready equilibration of ATP and ADP & Pi in the active site.

G (k

J/m

ol)

Reaction coordinate

80

60

40

20

0

‡

P ADP&Pi

ES

E & S

E ADP&Pi

[E ATP]

Typical enzyme ATP synthase

ATP (in solution)

(b)(a)


Architecture of ATP Synthase (F0F1) Complex

27

• Determined using biochemical, biophysical, and structural (X-Ray and cryo-EM) studies

• F1 (alpha, beta, gamma, epsilon) • F0 (c10, a, b2, δ) • The C10, epsilon and gamma rotate

as protons flow from P side to N side

• With each incremental turn, the gamma subunit associates with different alpha-beta pair

• This interaction results in conformational change in beta

• Beta converts ADP+Pi to ATP • Reversible!

F1

F0


Linkage Between Motion & Catalysis: ATP Synthase

28

• 3 non-equivalent binding sites

• ATP • ADP+Pi • empty

• Proton flow causes gamma shaft to turn (arrow)

• ATP is released from beta facing the gamma shaft

• ADP+Pi condenses to generate ATP

• ADP+Pi occupy new empty space

• ~3 H+ are needed per turn


Artist’s Rendition of ATP Synthase in Action

30URL: https://youtu.be/PjdPTY1wHdQ

The Proton-motive Force Energizes Active Transport

31

• The proton force also used to move molecules into and out of the matrix

• Antiporter transports • driven by concentration

gradient • ATP (higher negative charge)

moves into the inter-membrane space (plus charge)

• ADP (lower negative charge) moves into the matrix

• Net change of (-)-charge in the inter-membrane space

• Pi is H2PO4- at physiological pH • Symporter transports

• driven by ∇H+

• H2PO4- and H+ (0 net charge)


Re-oxidizing the NADH Generated via Glycolysis

32

• Oxidize the NADH generated during glycolysis • NADH/NAD+ cannot be transported across mitochondria inner membrane • In liver, kidney and heart: Malate-aspartate shuttle


(e-c ⇄ e-m)

33


malate dehydrogenase. The malate thus formed passesthrough the inner membrane via the malate–!-ketoglu-tarate transporter. Within the matrix the reducing equiva-lents are passed to NAD! by the action of matrix malatedehydrogenase, forming NADH; this NADH can pass elec-trons directly to the respiratory chain. About 2.5 mole-cules of ATP are generated as this pair of electrons passesto O2. Cytosolic oxaloacetate must be regenerated bytransamination reactions and the activity of membranetransporters to start another cycle of the shuttle.

Skeletal muscle and brain use a different NADH shut-tle, the glycerol 3-phosphate shuttle (Fig. 19–30). Itdiffers from the malate-aspartate shuttle in that it deliv-ers the reducing equivalents from NADH to ubiquinoneand thus into Complex III, not Complex I (Fig. 19–8), pro-viding only enough energy to synthesize 1.5 ATP mole-cules per pair of electrons.

The mitochondria of plants have an externally ori-ented NADH dehydrogenase that can transfer electronsdirectly from cytosolic NADH into the respiratory chainat the level of ubiquinone. Because this pathwaybypasses the NADH dehydrogenase of Complex I andthe associated proton movement, the yield of ATP fromcytosolic NADH is less than that from NADH generatedin the matrix (Box 19–1).

Matrix (N side)


QH2

NADH + H+NAD+

Glycolysis

Dihydroxyacetonephosphate

Glycerol 3-phosphate

FADFADH2

III

CH2OH

CH2

CHOH

O P

P

––

– –

CH2OH

CH2

C

O

––

–

––

–

O

mitochondrialglycerol 3-phosphate

dehydrogenase

cytosolicglycerol 3-phosphate

dehydrogenase

FIGURE 19–30 Glycerol 3-phosphate shuttle. This alternative means ofmoving reducing equivalents from the cytosol to the mitochondrial ma-trix operates in skeletal muscle and the brain. In the cytosol, dihydrox-yacetone phosphate accepts two reducing equivalents from NADH in areaction catalyzed by cytosolic glycerol 3-phosphate dehydrogenase.An isozyme of glycerol 3-phosphate dehydrogenase bound to the outerface of the inner membrane then transfers two reducing equivalentsfrom glycerol 3-phosphate in the intermembrane space to ubiquinone.Note that this shuttle does not involve membrane transport systems.

S U M M A R Y 1 9 . 2 AT P S y n t h e s i s■ The flow of electrons through Complexes I, III, and

IV results in pumping of protons across the innermitochondrial membrane, making the matrixalkaline relative to the intermembrane space. Thisproton gradient provides the energy (in the form ofthe proton-motive force) for ATP synthesis fromADP and Pi by ATP synthase (FoF1 complex) in theinner membrane.

■ ATP synthase carries out “rotational catalysis,” inwhich the flow of protons through Fo causes eachof three nucleotide-binding sites in F1 to cyclefrom (ADP ! Pi)–bound to ATP-bound to emptyconformations.

■ ATP formation on the enzyme requires littleenergy; the role of the proton-motive force is topush ATP from its binding site on the synthase.

■ The ratio of ATP synthesized per reduced toH2O (the P/O ratio) is about 2.5 when electronsenter the respiratory chain at Complex I, and 1.5when electrons enter at ubiquinone.

■ Energy conserved in a proton gradient can drivesolute transport uphill across a membrane.

■ The inner mitochondrial membrane is impermeableto NADH and NAD!, but NADH equivalents aremoved from the cytosol to the matrix by either oftwo shuttles. NADH equivalents moved in by themalate-aspartate shuttle enter the respiratory chainat Complex I and yield a P/O ratio of 2.5; thosemoved in by the glycerol 3-phosphate shuttle enterat ubiquinone and give a P/O ratio of 1.5.

19.3 Regulation of OxidativePhosphorylationOxidative phosphorylation produces most of the ATPmade in aerobic cells. Complete oxidation of a moleculeof glucose to CO2 yields 30 or 32 ATP (Table 19–5). Bycomparison, glycolysis under anaerobic conditions (lac-tate fermentation) yields only 2 ATP per glucose.Clearly, the evolution of oxidative phosphorylation pro-vided a tremendous increase in the energy efficiency ofcatabolism. Complete oxidation to CO2 of the coenzymeA derivative of palmitate (16:0), which also occurs in themitochondrial matrix, yields 108 ATP per palmitoyl-CoA(see Table 17–1). A similar calculation can be made forthe ATP yield from oxidation of each of the amino acids(Chapter 18). Aerobic oxidative pathways that result inelectron transfer to O2 accompanied by oxidative phos-phorylation therefore account for the vast majority ofthe ATP produced in catabolism, so the regulationof ATP production by oxidative phosphorylation tomatch the cell’s fluctuating needs for ATP is absolutelyessential.

12O2

• In skeletal muscle and brain: glycerol 3-phosphate shuttle

• GPDH isozymes:cytosolic, mitochondrial intermembrane space, and mitochondrial, associated with intermembrane

• NADH+H+ diffuse through outer membrane (<5000 Da)

• GPDHc reduces DHAP to G3P with NADH

• GPDHm oxidizes G3P to DHAP to create FADH2

• FADH2 passes electrons to Q


The NAD+/NADH Pool

34

• The pool of NAD+/NADH in the mitochondria is distinct from the cytoplasm

• Mitochondria responsible for oxidizing both pools of NADH to NAD+

• NAD+ and NADH can cross the outer but not inner mitochondrial membrane

• Shuttle systems act to oxidize cytoplasmic NADH using oxygen present in mitochondria

• Mitochondria can synthesize its own NAD+

• NAD+ precursors can be transported into and out of mitochondria


Regulation

35

19.3 Regulation of Oxidative Phosphorylation 733

FIGURE 19–31 Structure of bovine F1-ATPase in a complex with itsregulatory protein IF1. (Derived from PDB ID 1OHH) Two F1 mole-cules are viewed here as in Figure 19–25c. The inhibitor IF1 (red) bindsto the !" interface of the subunits in the diphosphate (ADP) conforma-tion (!-ADP and "-ADP), freezing the two F1 complexes and therebyblocking ATP hydrolysis (and synthesis). (Parts of IF1 that failed to re-solve in the crystals of F1 are shown in white outline as they occur incrystals of isolated IF1.) This complex is stable only at the low cytoso-lic pH characteristic of cells that are producing ATP by glycolysis;when aerobic metabolism resumes, the cytosolic pH rises, the in-hibitor is destabilized, and ATP synthase becomes active.

ATP Yield from Complete Oxidation of Glucose

Process Direct product Final ATP

Glycolysis 2 NADH (cytosolic) 3 or 5*2 ATP 2

Pyruvate oxidation (two per glucose) 2 NADH (mitochondrial matrix) 5

Acetyl-CoA oxidation in citric acid cycle 6 NADH (mitochondrial matrix) 15(two per glucose) 2 FADH2 3

2 ATP or 2 GTP 2

Total yield per glucose 30 or 32

TABLE 19–5

*The number depends on which shuttle system transfers reducing equivalents into the mitochondrion.

Oxidative Phosphorylation Is Regulated by Cellular Energy Needs

The rate of respiration (O2 consumption) in mitochon-dria is tightly regulated; it is generally limited by theavailability of ADP as a substrate for phosphorylation.Dependence of the rate of O2 consumption on the avail-ability of the Pi acceptor ADP (Fig. 19–20b), the accep-tor control of respiration, can be remarkable. In someanimal tissues, the acceptor control ratio, the ratio ofthe maximal rate of ADP-induced O2 consumption tothe basal rate in the absence of ADP, is at least 10.

The intracellular concentration of ADP is one measureof the energy status of cells. Another, related measure isthe mass-action ratio of the ATP-ADP system,[ATP]!([ADP][Pi]). Normally this ratio is very high, sothe ATP-ADP system is almost fully phosphorylated.When the rate of some energy-requiring process (pro-tein synthesis, for example) increases, the rate of break-down of ATP to ADP and Pi increases, lowering themass-action ratio. With more ADP available for oxidativephosphorylation, the rate of respiration increases, caus-ing regeneration of ATP. This continues until the mass-action ratio returns to its normal high level, at whichpoint respiration slows again. The rate of oxidation ofcellular fuels is regulated with such sensitivity and pre-cision that the [ATP]"([ADP][Pi]) ratio fluctuates onlyslightly in most tissues, even during extreme variationsin energy demand. In short, ATP is formed only as fast asit is used in energy-requiring cellular activities.

An Inhibitory Protein Prevents ATP Hydrolysis during Hypoxia

We have already encountered ATP synthase as an ATP-driven proton pump (see Fig. 11–39), catalyzing the re-verse of ATP synthesis. When a cell is hypoxic (deprivedof oxygen), as in a heart attack or stroke, electron trans-fer to oxygen slows, and so does the pumping of protons.The proton-motive force soon collapses. Under theseconditions, the ATP synthase could operate in reverse,hydrolyzing ATP to pump protons outward and causing adisastrous drop in ATP levels. This is prevented by a small

(84 amino acids) protein inhibitor, IF1, which simultane-ously binds to two ATP synthase molecules, inhibitingtheir ATPase activity (Fig. 19–31). IF1 is inhibitory onlyin its dimeric form, which is favored at pH lower than 6.5.In a cell starved for oxygen, the main source of ATP be-comes glycolysis, and the pyruvic or lactic acid thusformed lowers the pH in the cytosol and the mitochondr-ial matrix. This favors IF1 dimerization, leading to inhibi-tion of the ATPase activity of ATP synthase and therebypreventing wasteful hydrolysis of ATP. When aerobic me-tabolism resumes, production of pyruvic acid slows, thepH of the cytosol rises, the IF1 dimer is destabilized, andthe inhibition of ATP synthase is lifted.

Hypoxia Leads to ROS Production and Several AdaptiveResponses

In hypoxic cells there is an imbalance between the inputof electrons from fuel oxidation in the mitochondrialmatrix and transfer of electrons to molecular oxygen,leading to increased formation of reactive oxygen

• Oxidative phosphorylation works best with O2 • +O2 = 32ATP/glucose • -O2 = 2ATP/glucose

• O2 consumption also regulated by [ADP+Pi] • Under low O2 (hypoxia) proton-motive force collapses,

leading to drop of pH. ATP synthase runs in reverse • IF1 dimerizes at pH ≤ 6.5 • Dimer of IF1 inhibits reverse ATP synthase

--why does the pH drop?


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