Voet D., Voet Ju.G. Biochemistry

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Mitochondria + β-hydroxybutyrate added,

NAD

-linked oxidation commences

Rotenone or amytal added,

inhibiting NAD

-linked oxidation

Succinate added, FAD-linked

oxidation begins

Antimycin added, inhibiting

FAD-linked oxidation

TMPD/ascorbate added,

oxidation resumes

–

added,

oxidation terminates

]

Reagents are then injected into the chamber and the O

consumption recorded (Fig. 22-11):

1. Mitochondria and ␤-hydroxybutyrate are injected

into the chamber. Mitochondria mediate the NAD

⫹

-linked

oxidation of ␤-hydroxybutyrate (Section 25-3):

As the resulting NADH is oxidized by the electron-

transport chain with O

as the terminal electron acceptor,

the O

concentration in the reaction mixture decreases.

2. Addition of rotenone or amytal completely stops the

␤-hydroxybutyrate oxidation.

3. Addition of succinate, which undergoes FAD-linked

oxidation, causes the [O

] to resume its decrease. Electrons

from FADH

are therefore still able to reduce O

in the

presence of rotenone; that is, electrons from FADH

enter

the electron-transport chain after the rotenone-blocked step.

4. Addition of antimycin inhibits electron transport

from FADH

5. Although NADH and FADH

are the electron-

transport chain’s two physiological electron donors,

␤-Hydroxybutyrate

Acetoacetate

–

⫹

NAD

⫹

CHCH

␤-hydroxybutyrate

dehydrogenase

NADH

⫹

–

nonphysiological reducing agents can also be used to probe

the flow of electrons. Tetramethyl-p-phenylenediamine

(TMPD) is an ascorbate-reducible redox carrier that

transfers electrons directly to cytochrome c:

Addition of TMPD and ascorbate to the antimycin-

inhibited reaction mixture results in resumption of oxygen

consumption; evidently there is a third point at which elec-

trons can enter the electron-transport chain.

6. The addition of CN

⫺

completely inhibits oxidation

of all three electron donors, indicating that it blocks the

electron-transport chain after the third point of entry of

electrons.

Experiments such as these established the order of elec-

tron flow through the electron-transport chain complexes

and the positions blocked by various electron-transport in-

hibitors (Fig. 22-9).This order was confirmed and extended

by observations that the standard reduction potentials of

the redox carriers forming the electron-transport chain

complexes are very close to the standard reduction poten-

tials of their electron donor substrates (Table 22-1). The

three jumps in reduction potential between NADH, CoQ,

cytochrome c, and O

are each of sufficient magnitude to

drive ATP synthesis. Indeed, these redox potential jumps

correspond to the points of inhibition of rotenone (or amy-

tal), antimycin, and CN

⫺

b. Phosphorylation and Oxidation

Are Rigidly Coupled

The foregoing thermodynamic studies suggest that oxi-

dation of NADH, FADH

, and ascorbate by O

is associ-

ated with the synthesis of about 2.5, 1.5, and 1 ATP, respec-

tively. This stoichiometry, called the P/O ratio [the ratio of

ATP synthesized to O atoms reduced (electron pairs taken

up)], has been confirmed experimentally through measure-

ments of O

uptake by resting and active mitochondria.An

example of a typical experiment used to determine the P/O

ratio is as follows: A suspension of mitochondria (isolated

by differential centrifugation after cell disruption; Section

6-1B) containing an excess of P

but no ADP is incubated in

an oxygen electrode reaction chamber. Oxidation and

Tetramethyl-p-phenylenediamine

(TMPD), oxidized form

Ascorbic acid

TMPD, reduced form Dehydroascorbic acid

Section 22-2. Electron Transport 831

Figure 22-11 Effect of inhibitors on electron transport. This

diagram shows an idealized oxygen electrode trace of a

mitochondrial suspension containing excess ADP and P

.At the

numbered points, the indicated reagents are injected into the

sample chamber and the resulting changes in [O

] are recorded.

The numbers refer to the discussion in the text. [After Nicholls,

D.G., Bioenergetics, p. 110, Academic Press (1982).]

JWCL281_c22_823-870.qxd 3/19/10 10:59 AM Page 831

phosphorylation are closely coupled in well-functioning mi-

tochondria, so electron transport can occur only if ADP is

being phosphorylated (Section 22-3). Indeed, mitochon-

drial metabolism is so tightly regulated that even the appear-

ances of actively respiring and resting mitochondria are

greatly different (Fig. 22-12). Since no ADP is present in

the reaction mixture, the mitochondria are resting and the

consumption rate is minimal (Fig. 22-13; Region 1). The

system is then manipulated as follows:

(a) ADP (75 ␮mol) and an excess of ␤-hydroxybu-

tyrate (an NAD

⫹

-linked substrate) are added. The

mitochondria immediately enter the active state

and the rate of oxygen consumption increases (Fig.

22-13, Region 2) and is maintained at this elevated

level until all the ADP is phosphorylated. The mito-

chondria then return to the resting state (Fig. 22-13,

Region 3). Phosphorylation of 75 ␮mol of ADP un-

der these conditions consumes 15 ␮mol of O

. Since

the oxidation of NADH by O

consumes twice as

many moles of NADH (i.e., electron pairs) as of O

the P/O ratio for NADH reoxidation at Region 2 is

75 ␮mol of ADP/(2 ⫻ 15 ␮mol of O

) ⫽ 2.5; that is,

2.5 mol of ADP are phosphorylated per mole of

NADH oxidized.

(b) The experiment is continued by inhibiting electron

transfer from NADH by rotenone and adding an

additional 75 ␮mol of ADP (Fig. 22-13, Region 4),

this time together with an excess of the FAD-linked

substrate succinate. Oxygen consumption again con-

tinues until all the ADP is phosphorylated, and the

system again returns to the resting state (Fig. 22-13,

Region 5). Calculation of the P>O ratio for FADH

oxidation yields the value 1.5; that is, 1.5 mol of ADP

are phosphorylated per mole of FADH

oxidized.

yields a P/O ratio of 1 (Fig. 22-13, Regions 6 and 7).

These conclusions agree with the inhibitor studies indicat-

ing that there are three entry points for electrons into the

electron-transport chain and with the standard reduction

potential measurements exhibiting three potential jumps,

each sufficient to provide the free energy for ATP synthe-

sis (Fig. 22-9).

c. The P/O Ratios Are Difficult to

Measure Accurately

Measurements of P兾O ratios are subject to systematic

experimental errors for which it is difficult to correct, such

as inaccuracies in the measurement of the oxygen concen-

tration, the presence of AMP, and proton leakage through

the inner mitochondrial membrane. In the older literature,

these values were taken to be 3, 2, and 1. However, careful

measurements by Peter Hinkle have yielded values close

to 2.5, 1.5, and 1 for these quantities (we shall see in Section

22-3 that the mechanism of oxidative phosphorylation does

not require that P兾O ratios have integer values). If these

latter values are correct, then the number of ATP molecules

that are synthesized per molecule of glucose oxidized is 2.5

ATP/NADH ⫻ 10 NADH/glucose ⫹ 1.5 ATP/FADH

⫻

2 FADH

/glucose ⫹ 2 ATP/glucose from the citric acid

cycle ⫹ 2 ATP/glucose from glycolysis ⫽ 32 ATP/glucose

(rather than the value of 38 ATP/glucose implied by P/O

ratios of 3, 2, and 1).

The determination of the in vivo yield of ATP/glucose is

even more problematic.The reducing equivalents from the

GAPDH-generated NADH may be imported into the mi-

tochondrion via the glycerophosphate shuttle, which

yields FADH

, or via the malate–aspartate shuttle, which

yields NADH but passes a proton into the matrix (Section

22-1Bc).The mix of these two shuttle systems varies from

tissue to tissue and hence so does the ATP yield. In addi-

tion, the rate of proton leakage back across the inner mi-

tochondrial membrane, which is significant, may vary with

832 Chapter 22. Electron Transport and Oxidative Phosphorylation

Table 22-1 Reduction Potentials of Electron-Transport

Chain Components in Resting Mitochondria

Component e°¿(V)

NADH ⫺0.315

Complex I (NADH:CoQ oxidoreductase; ⬃900 kD,

45 subunits):

FMN ⫺0.380

[2Fe–2S]N1a ⫺0.370

[2Fe–2S]N1b ⫺0.250

[4Fe–4S]N3, 4, 5, 6a, 6b, 7 ⫺0.250

[4Fe–4S]N2 ⫺0.150

Succinate 0.031

Complex II (succinate–CoQ oxidoreductase; ⬃140 kD,

4 subunits):

FAD ⫺0.040

[2Fe–2S] ⫺0.030

[4Fe–4S] ⫺0.245

[3Fe–4S] ⫺0.060

Heme b

560

⫺0.080

Coenzyme Q 0.045

Complex III (CoQ–cytochrome c oxidoreductase; ⬃450 kD,

9–11 subunits):

Heme b

562

) 0.030

Heme b

566

) ⫺0.030

[2Fe–2S] 0.280

Heme c

0.215

Cytochrome c 0.235

Complex IV (cytochrome c oxidase; ⬃410 kD, 8–13 subunits):

Heme a 0.210

0.245

0.340

Heme a

0.385

0.815

Source: Mainly Wilson, D.F., Erecinska, M., and Dutton, P.L., Annu. Rev.

Biophys. Bioeng. 3, 205 and 208 (1974); and Wilson, D.F., In Bittar, E.E.

(Ed.), Membrane Structure and Function, Vol. 1, p. 160, Wiley (1980).

JWCL281_c22_823-870.qxd 3/19/10 10:59 AM Page 832

Time

Δ[O

]

(P/O = 1.5)

Δ[O

]

(P/O = 2.5)

Δ[O

]

(P/O = 1)

]

(a) ADP + β-Hydroxybutyrate

(b) ADP + Succinate + rotenone

+ antimycin

conditions and cellular identity. Hence, the in vivo yield of

ATP per glucose is likely to be significantly less than the

above figures of 32 ATP/glucose.

How the free energy of electron transport is actually

coupled to ATP synthesis, a subject of active research, is

discussed in Section 22-3.We first examine the structures of

the four respiratory complexes in order to understand how

they are related to the function of the electron-transport

chain. Keep in mind, however, that as in most areas of bio-

chemistry, this field is under intense scrutiny and much of

the information we need for a complete understanding of

these relationships has yet to be elucidated.

C. Components of the Electron-Transport Chain

Many of the proteins embedded in the inner mitochondrial

membrane are organized into the four respiratory com-

plexes of the electron-transport chain. Each complex con-

sists of several protein components that are associated with

a variety of redox-active prosthetic groups with successively

Section 22-2. Electron Transport 833

Figure 22-13 Determination of the stoichiometry of coupled

oxidation and phosphorylation (the P/O ratio) with different

electron donors. Mitochondria are incubated in excess phosphate

buffer in the sample chamber of an oxygen electrode. (a) Next,

75 ␮mol of ADP and excess ␤-hydroxybutyrate are added.

Respiration continues until all the ADP is phosphorylated. ⌬O

in Region 2 is 15 ␮mol, corresponding to 30 ␮mol of NADH

oxidized; thus, P>O ⫽ 75>30 ⫽ 2.5. (b) Then 75 ␮mol of ADP and

excess succinate are added together with rotenone to inhibit

electron transfer from NADH. ⌬O

in Region 4 is 25 ␮mol,

corresponding to 50 ␮mol of FADH

oxidized; P>O ⫽ 75>50 ⫽ 1.5.

added with antimycin to inhibit electron transfer from FADH

⌬O

in Region 6 is 37.5 ␮mol, corresponding to 75 ␮mol of

ascorbate oxidized; P>O ⫽ 75>75 ⫽ 1.

Figure 22-12 Electron micrographs of mouse liver

mitochondria. (a) In the actively respiring state and (b) in the

resting state. The cristae in actively respiring mitochondria are

far more condensed than they are in resting mitochondria.

[Courtesy of Charles Hackenbrock, University of North Carolina

Medical School.]

(a)

(b)

JWCL281_c22_823-870.qxd 6/8/10 9:19 AM Page 833

Cys

[2Fe –2S]

[4Fe – 4S]

2–

Cys

2–

[Fe–S]

Cys

(a)

(b)

(c)

Cys

increasing reduction potentials (Table 22-1). In the follow-

ing paragraphs, we examine their structures and the agents

that transfer electrons between them. Their relationships

are summarized in Fig. 22-14.

1. Complex I (NADH Dehydrogenase or

NADH–Coenzyme Q Oxidoreductase)

Complex I (also called NADH dehydrogenase) passes

electrons from NADH to CoQ. This probably largest pro-

tein component of the inner mitochondrial membrane

(⬃980 kD in mammals and ⬃700 kD in Neurospora crassa)

contains one molecule of flavin mononucleotide (FMN; a

redox-active prosthetic group that differs from FAD only

by the absence of the AMP group) and eight or nine

iron–sulfur clusters that participate in the electron-

transport process (Table 22-1). In mammals, 7 of its 45 sub-

units, its most hydrophobic subunits, which form the core of

its transmembrane region, are encoded by mitochondrial

genes, with the remainder encoded by nuclear genes. Most

of its subunits are homologous to soluble redox center-

containing proteins, which strongly suggests that Complex

I arose through the evolutionary aggregation of these pre-

viously existing proteins.

a. Iron–Sulfur Clusters Are Redox Active

Iron–sulfur clusters, first discovered by Helmut Beinert,

commonly occur as prosthetic groups in iron–sulfur pro-

teins. There are four common types of iron–sulfur clusters

(Fig. 22-15). Those designated [2Fe–2S] and [4Fe–4S]

834 Chapter 22. Electron Transport and Oxidative Phosphorylation

Figure 22-14 The mitochondrial electron-transport chain. The

pathways of electron transfer (black) and proton pumping (red)

are indicated. Electrons are transferred between Complexes I

and III by membrane-soluble CoQ (Q) and between Complexes

FeS

Intermembrane

space

Inner

mitochondrial

membrane

Matrix

FeS

FMN

NADH

Cyt c

Cyt b

FeS

Cyt b

Cyt c

Cyt a

Complex IIIComplex I Complex IV

Cyt a

– Cu

–

+ NAD

III and IV by the peripheral membrane protein cytochrome c

(Cyt c). Complex II (not shown) transfers electrons from

succinate to CoQ.

See the Animated Figures

Figure 22-15 Structures of the common iron–sulfur clusters. (a) An [Fe–S] cluster,

(b) a [2Fe–2S] cluster, and (c) a [4Fe–4S] cluster. The [3Fe–4S] cluster resembles the

[4Fe–4S] cluster with one of its Fe ions removed. Note that, whereas the Fe and

2⫺

ions of [4Fe–4S] clusters form what appears to be a distorted cube, the structure

is really two interpenetrating tetrahedra of Fe ions and S

2⫺

ions.

JWCL281_c22_823-870.qxd 7/2/10 11:09 AM Page 834

clusters consist of equal numbers of iron and sulfide ions

and are both coordinated to four protein Cys sulfhydryl

groups. The [3Fe–4S] cluster is essentially a [4Fe–4S] clus-

ter that lacks one Fe. One means of identifying these clus-

ters utilizes the fact that their sulfide ions are acid labile:

They are released as H

S near pH 1. The [Fe–S] cluster,

which occurs only in bacteria, consists of a single Fe atom

liganded to four Cys residues. Note that the Fe ions in all

four types of clusters are each coordinated by four S atoms,

which are more or less tetrahedrally disposed around the

Fe. However, in Rieske iron–sulfur proteins (named after

their discoverer, John Rieske), one of the Fe atoms in a

[2Fe–2S] cluster is coordinated by two His residues rather

than two Cys residues.

The oxidized and reduced states of all iron–sulfur clus-

ters differ by one formal charge regardless of their number

of Fe ions. This is because the Fe ions in each cluster form a

conjugated system and thus can have oxidation states be-

tween the ⫹2 and ⫹3 values possible for individual Fe ions.

For example, each of the two [4Fe–4S] clusters in the pro-

tein ferredoxin (Fig. 22-16) contains one Fe(II) and three

Fe(III) in its oxidized form and two Fe(II) and two Fe(III)

in its reduced form. The standard reduction potential of a

given type of iron–sulfur cluster depends on its interaction

with its associated protein as well as on its oxidation state.

Iron–sulfur proteins also occur in the photosynthetic

electron-transport chains of plants and bacteria (Section

24-2); indeed, photosynthetic electron-transport chains are

thought to be the evolutionary precursors of oxidative

electron-transport chains (Section 1-5Cb).

b. The Coenzymes of Complex I

FMN and ubiquinone (CoQ), the coenzymes of Com-

plex I, can each adopt three oxidation states (Fig. 22-17).

Although NADH can only participate in a two-electron

transfer, both FMN and CoQ are capable of accepting and

donating either one or two electrons because their semi-

quinone forms are stable. In contrast, the cytochromes of

Complex III (see below), to which reduced CoQ passes its

electrons, are only capable of one-electron reductions.

FMN and CoQ therefore provide an electron conduit be-

tween the two-electron donor NADH and the one-electron

acceptors, the cytochromes.

CoQ’s hydrophobic tail makes it soluble in the inner mi-

tochondrial membrane’s lipid bilayer. In mammals, this tail

consists of ten C

isoprenoid units and hence the coenzyme

is designated Q

10.

In other organisms, CoQ may have only

six (Q

) or eight (Q

) isoprenoid units.

c. Electrons Follow a Multistep Path

through Complex I

Complex I from prokaryotes consists of 13 to 15 sub-

units with an aggregate molecular mass of ⬃550 kD. Ho-

mologs of all of its conserved subunits occur in the mito-

chondrial enzyme and they contain equivalent redox

components. Evidently, the prokaryotic enzyme is a

stripped down version of mitochondrial Complex I. The

functions of the 30 to 32 additional “accessory” subunits

that are components of mitochondrial Complex I are

largely unknown.

The low resolution X-ray structure of the 15-subunit

Complex I from the thermophilic bacterium Thermus ther-

mophilus (Fig. 22-18), determined by Leonid Sazanov,

reveals an L-shaped protein with one arm of the L, the

transmembrane arm, immersed in the plasma membrane

(the inner mitochondrial membrane for eukaryotes) and

the other, the peripheral arm, extending into the cytosol

(the matrix for eukaryotes).The transmembrane arm, con-

sists of seven subunits that collectively have 63 transmem-

brane helices. The three largest transmembrane subunits,

Nqo12, 13, and 14, are structurally similar to each other and

to previously determined structures of Na

⫹

antiporters.

However, the most unusual feature of the transmembrane

portion is a 110-Å long amphipathic ␣ helix that extends

from Nqo12 parallel to the plane of the membrane so as to

span Nqo13 and 14.

The high resolution X-ray structure of the 8-subunit,

hydrophilic, peripheral arm of Complex I from the ther-

mophilic bacterium. Thermophilus, also determined by

Sazanov, reveals a Y-shaped assembly that is 140 Å high

(Fig. 22-19a).This subcomplex contains all of the enzyme’s

redox centers: an FMN, seven [4Fe–4S] clusters, and two

[2Fe–2S] clusters. The FMN is located at the end of a

solvent-exposed cavity that presumably forms the NADH

Section 22-2. Electron Transport 835

Figure 22-16 X-ray structure of ferredoxin from Peptostrepto-

coccus asaccharolyticus. This monomeric 55-residue protein is

drawn in ribbon form colored in rainbow order from its N-terminus

(blue) to its C-terminus (red). Its two [4Fe–4S] clusters are drawn

in ball-and-stick form with S yellow and Fe red-brown. The Cys

side chains that ligand each Fe atom are drawn in stick form with

C green and S yellow. [Based on an X-ray structure by Elinor

Adman, Larry Sieker, and Lyle Jensen, University of Washington.

PDBid 1DUR.]

See Interactive Exercise 16

JWCL281_c22_823-870.qxd 10/19/10 8:07 AM Page 835

(a)

HCHO

CHO

OPO

2–

HCHO

(CH

)

HCCH

Isoprenoid units

Coenzyme Q (CoQ) or ubiquinone

(oxidized or quinone form)

Flavin mononucleotide (FMN)

(oxidized or quinone form)

[H•]

[H•] [H•]

[H•]

Coenzyme QH• or ubisemiquinone

(radical or semiquinone form)

FMNH• (radical or semiquinone form)

O•

•

Coenzyme QH

or ubiquinol

(reduced or hydroquinone form)

FMNH

(reduced or hydroquinone form)

R H

(b)

836 Chapter 22. Electron Transport and Oxidative Phosphorylation

Figure 22-17 Oxidation states of the organic coenzymes of complex I. (a) FMN and (b) CoQ.

Both coenzymes form stable semiquinone free radical states.

Figure 22-18 Low (4.5-Å) resolution X-ray structure of

Complex I from T. thermophilus. The structure is viewed parallel

to the plane of the plasma membrane with the cytoplasm above

and the periplasm below. Its 15 subunits are drawn in ribbon

form in different colors with the 110-Å-long horizontal helix of

Nqo12 purple. The nine Fe–S clusters in the peripheral arm are

drawn in space-filling form with S yellow and Fe red-brown. At

the structure’s low resolution, only the transmembrane helices of

the transmembrane arm can be seen; their connecting loops are

not visible, which accounts for the apparent gap between the

transmembrane and peripheral arms. However, the loops of the

peripheral arm are visible because that part of the structure was

based on the higher resolution X-ray structure of the peripheral

arm alone (see Fig. 22-19). [Courtesy of Leonid Sazanov, Medical

Research Council, Cambridge, U.K. PDBid 3M9S.]

JWCL281_c22_823-870.qxd 7/8/10 8:31 AM Page 836

binding site. Model building suggests that bound NADH

protects the FMN from solvent. The CoQ binding site

appears to be in a cavity below the [4Fe–4S] cluster N2

that is located at the interface with Complex I’s transmem-

brane arm.

The transit of electrons from NADH to CoQ in Com-

plex I begins with a two-electron reduction in the form of a

hydride ion (H

⫺

) transfer from NADH to FMN to yield

NAD

⫹

and FMNH

⫺

(similar to the reverse of the final step

of the dihydrolipoyl dehydrogenase reaction; Section 21-

2Bb). The electrons are then transferred, one at a time, to

Fe–S cluster N3 and then between Fe–S clusters along a

gradient of increasing redox potential (Table 22-1) until

they reach Fe–S cluster N2. This “downhill” process in-

volves the transient reduction of each Fe–S cluster as it

binds electrons and its reoxidation when it passes the elec-

trons to the next cluster. Their spatial arrangement indi-

cates the probable path of the electrons (Fig. 22-19b). Note

that redox centers need not be in contact in order to trans-

fer electrons. Finally, the electrons are passed from N2 to

CoQ, which is sequentially reduced to CoQH⋅ and then to

CoQH

, which is released into the membrane and replaced

by CoQ, thereby completing the catalytic cycle.

In translocating an electron pair from NADH to CoQ,

Complex I pumps four protons out of the mitochondrial

Section 22-2. Electron Transport 837

matrix (the bacterial cell). How complex I accomplishes

this task is discussed in Section 22-3Bj.

2. Complex II [Succinate Dehydrogenase or

Succinate–Coenzyme Q Oxidoreductase (SQR)]

Complex II, which is also the citric acid cycle enzyme

succinate dehydrogenase (Section 21-3F), passes electrons

from succinate to CoQ to yield fumarate and CoQH

. It

does so with the participation of a covalently bound FAD,

a [2Fe–2S] cluster, a [4Fe–4S] cluster, a [3Fe–4S] cluster,

and one heme b

560

(Table 22-1). All of its four subunits are

encoded by nuclear genes.

The standard redox potential difference for electron

transfer from succinate to CoQ (Fig. 22-9) is insufficient to

provide the free energy necessary to drive ATP synthesis.

Complex II is, nevertheless, important because it injects

these relatively high-potential electrons into the electron-

transport chain. Two other enzymes, in addition to Com-

plexes I and II, synthesize and release CoQH

in the inner

mitochondrial membrane and thereby power oxidative

phosphorylation via the actions of Complexes III and IV.

They are glycerol-3-phosphate dehydrogenase of the glyc-

erophosphate shuttle (Fig. 22-8) and ETF–ubiquinone oxi-

doreductase, which participates in fatty acid oxidation (Sec-

tion 25-2Ca; ETF stands for electron-transfer flavoprotein).

Figure 22-19 X-ray structure of the peripheral arm of

Complex I from T. thermophilus. (a) The protein is viewed

parallel to the plane of the plasma membrane but oriented

differently to that in Fig. 22-18. Its eight subunits are drawn in

semitransparent ribbon form in different colors (which differ

from those in Fig. 22-18). The FMN (upper left) and the nine

Fe–S clusters are shown in space-filling form with C green,

N blue, O red, P orange, S yellow, and Fe red-brown.The likely

CoQ binding site (Q) is indicated by a gray arrow. (b) The

arrangement of redox groups viewed similarly to Part a.The

FMN (stick model with C yellow and the two [Fe–S] and seven

[4Fe–4S] clusters (space-filling models) are shown together with

their center-to-center distances in angstroms (shortest

edge-to-edge distances are indicated in parentheses). Blue

arrows represent the ⬃94-Å-long main path of electrons after

their transfer from NADH to FMN. Fe–S clusters N1a and N7 lie

too far off this path for electrons to pass through them but

perhaps they function to fine-tune its electronic properties.

Spectroscopic measurements suggest that the CoQ binding site is

⬃12 Å distant from N2. [Part a based on an X-ray structure by

and Part b courtesy of Leonid Sazanov, Medical Research

Council, Cambridge, U.K. PDBid 2FUG.]

(a)

(b)

JWCL281_c22_823-870.qxd 7/20/10 6:14 PM Page 837

a. Cytochromes Are Heme Proteins That

Transport Electrons

Cytochromes, whose function was elucidated in 1925 by

David Keilin, are redox-active proteins that occur in all or-

ganisms except a few types of obligate anaerobes. These

proteins contain heme groups that reversibly alternate be-

tween their Fe(II) and Fe(III) oxidation states during elec-

tron transport.

The heme groups of the reduced [Fe(II)] cytochromes

have prominent visible absorption spectra consisting of

three peaks: the ␣, ␤, and ␥ (Soret) bands (Fig. 22-20a).The

wavelength of the ␣ peak, which varies characteristically

with the particular reduced cytochrome species (it is absent

in oxidized cytochromes), is useful for differentiating the

various cytochromes.Accordingly, the spectra of mitochon-

drial membranes (Fig. 22-20b) indicate that they contain

three cytochrome types, cytochromes a, b, and c.

Within each type of cytochrome, different heme group

environments may be characterized by slightly different ␣

peak wavelengths. For example, Complex III (see below)

has two b-type hemes:That absorbing maximally at 562 nm

is referred to as heme b

562

or b

(for high potential),

whereas that absorbing maximally at 566 nm is referred to

as heme b

566

or b

(for low potential).

Each type of cytochrome contains a differently substi-

tuted porphyrin ring (Fig. 22-21a) coordinated with the

redox-active iron atom.A b-type cytochrome contains pro-

toporphyrin IX, which also occurs in hemoglobin and

myoglobin (Section 10-1A).The heme group of a c-type cy-

tochrome differs from protoporphyrin IX in that its vinyl

groups have added the Cys sulfhydryls in the sequence

Cys-X-Y-Cys-His across their double bonds to form

thioether linkages to the protein (Fig. 9-39). Heme a con-

tains a long hydrophobic tail of three isoprene units (a far-

nesyl group) linked to the porphyrin via a hydroxyethyl

group, as well as a formyl group in place of a methyl

substituent.

The axial ligands of the heme iron also vary with the cy-

tochrome type. In cytochromes a and b, both ligands are

His residues, whereas in cytochrome c, one is the His in the

Cys-X-Y-Cys-His sequence and the other is Met (Fig.

22-21b). Note that the designation of a given cytochrome

type refers only to the identity of the cytochrome’s heme

prosthetic group(s); a given cytochrome type may have any

of several unrelated protein folds.

b. Complex II Contains a Linear Chain

of Redox Cofactors

Both bacterial and mitochondrial Complex II’s consist

of four subunits: two water-soluble subunits, a flavoprotein

(Fp, ⬃665 residues) that binds the enzyme’s substrate suc-

cinate and FAD (which is covalently linked to a specific

His side chain via a bond to FAD atom C8a; Section

21-3F), and an iron–sulfur protein (Ip, ⬃290 residues) that

binds the three Fe–S clusters; and two transmembrane sub-

units, CybL (⬃170 residues) and CybS (⬃160 residues)

that collectively bind the heme b

560

and the CoQ. Fp and Ip

are highly conserved, whereas the sequences of CybS and

CybL vary among different organisms.

The X-ray structures of porcine, chicken, and E. coli

Complex II were independently determined by Zihe Rao,

Edward Berry, and So Iwata. In all cases, it has the shape of

the letter “q,” with its top lobe containing Fp and Ip and its

tail composed of the transmembrane subunits (Fig. 22-22a).

The complex is oriented in the inner mitochondrial mem-

brane such that Fp and Ip extend into the matrix. The

838 Chapter 22. Electron Transport and Oxidative Phosphorylation

Figure 22-20 Visible absorption spectra of cytochromes.

(a) Absorption spectrum of reduced cytochrome c showing its

characteristic ␣, ␤, and ␥ (Soret) absorption bands. The

absorption maxima for cytochromes a, b, c, and c

are listed.

(b) The three separate ␣ bands in the visible absorption

Absorbance

Wavelength (nm)

400300 500 600

Cytochrome a

Cytochrome b

Cytochrome c

439

429

415

418

600

563

550

554

532

521

524

(a)(b)

Cytochrome c

αβγ

Absorbance

Wavelength (nm)

500 550450 600 650

Cytochrome c

Beef heart

mitochondrial

membranes

c α

b α

a α

spectrum of beef heart mitochondrial membranes (below)

indicate the presence of cytochromes a, b, and c.The spectrum of

purified cytochrome c (above) is provided for reference. [After

Nicholls, D.G. and Ferguson, S.J., Bioenergetics 3, p. 96,Academic

Press (2002).]

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Section 22-2. Electron Transport 839

( CH

)

COO

–

COO

–

Heme a

(a)

⫹

COO

–

COO

–

Heme b

(iron–protoporphyrin IX)

⫹

COO

–

COO

–

Heme c

Cys

Protein

⫹

Figure 22-21 Porphyrin rings in cytochromes. The (a) chemical structures and

(b) axial liganding of the heme groups contained in cytochromes a, b, and c are shown.

Figure 22-22 X-ray structure of chicken Complex II in complex

with its inhibitor oxaloacetate and CoQ. (a) A semitransparent

ribbon diagram viewed parallel to the inner mitochondrial

membrane with the matrix above and in which the enzyme’s four

subunits are drawn in different colors.The oxaloacetate (OAA),

(b)

His

Hemes a and b

Heme c

⫹

Met His

His

FAD, three Fe–S clusters, CoQ, and heme b are shown in

space-filling form with OAA C magenta, FAD and heme b C

green, CoQ C cyan, N blue, O red, P orange, S yellow, and Fe

red-brown.The inferred position of the inner mitochondrial

membrane is indicated. (b) The ligands and redox cofactors

viewed and colored as in Part a but drawn in stick form.Their

closest edge-to-edge distances are indicated. [Based on an X-ray

structure by Edward Berry, Lawrence Berkeley National

Laboratory, Berkeley, California. PDBid 1YQ3.]

(a)

(b)

JWCL281_c22_823-870.qxd 3/19/10 11:00 AM Page 839

substrate and CoQ binding sites are connected by a nearly

linear ⬃40-Å-long chain of redox centers with the sequence

substrate–FAD—[2Fe–2S]—[4Fe–4S]—[3Fe–4S]—CoQ

(top to bottom in Fig. 22-22b). Thus, despite the measured

–0.245 V reduction potential of the [4Fe–4S] cluster (Table

22-1), which would appear to be too low for it to accept

electrons from succinate in the succinate S fumarate reac-

tion, the [4Fe–4S] cluster probably participates in the

electron-transfer process.

c. Complex II Suppresses the Formation

of Reactive Oxygen Species

The bacterial respiratory complex quinol–fumarate re-

ductase (QFR) is a homolog of Complex II that functions

in anaerobic organisms that use fumarate as a terminal

electron acceptor. There it catalyzes the same reaction as

does Complex II but in the opposite direction, that is, it

uses a quinol to reduce fumarate to succinate.

Even though QFR and Complex II have similar struc-

tures, organisms such as E. coli that are capable of both

aerobic and anaerobic metabolism employ the two differ-

ent complexes for these different purposes. This appears

to be because under aerobic conditions, QFR produces 25

times as much superoxide radical ( ) as does E. coli

Complex II as well as H

, which Complex II does not

produce, presumably by leaking electrons to O

.As we dis-

cuss in Section 22-4Cg,these reactive oxygen species (ROS)

are highly destructive. Comparison of the X-ray structure

of QFR with that of the closely similar E. coli Complex II

indicates that the electron distributions about their various

redox centers greatly favors the ROS-generating side re-

actions of O

with the flavin ring of QFR relative to that

of Complex II. Indeed, the heme b

560

of Complex II,

which QFR lacks and which is not in Complex II’s direct

electron transfer pathway (Fig. 22-22b), appears to fine-

tune its electronic properties so as to suppress the side

reactions that generate the ROS.This suggests that muta-

tions of the genes encoding human Complex II, which

cause a wide variety of disorders including tumor forma-

tion, neurological defects, and premature aging, result

from ROS generation.

3. Complex III (Coenzyme Q–Cytochrome

c Oxidoreductase or Cytochrome bc

Complex)

Complex III passes electrons from reduced CoQ to cy-

tochrome c. It contains four redox cofactors: two b-type

hemes, a c-type heme, and one [2Fe–2S] cluster (Table 22-1).

a. X-Ray Structure of the Cytochrome bc

Complex

All known cytochrome bc

complexes contain three com-

mon subunits: cytochrome b, which binds both the b

and b

hemes, cytochrome c

, which contains a single c-type heme,

and a Rieske iron–sulfur protein (ISP), which contains a

[2Fe–2S] cluster. The bovine bc

complex contains 8 addi-

tional subunits for a total of 11 different subunits that

combine to form a 2166-residue (243-kD) protomer that

dimerizes. Of these,only cytochrome b is encoded by a mito-

chondrial gene.

⫺

ⴢ

The X-ray structures of bovine, chicken, and yeast cy-

tochrome bc

complexes were independently determined

by Johann Deisenhofer, by Iwata and Bing Jap, by Berry,

Antony Crofts, and Sung-Hou Kim, and by Hartmut

Michel. All of these structures reveal a 2-fold symmetric

pear-shaped molecule of maximum diameter ⬃130 Å and

height ⬃150 Å, whose wide end extends ⬃75 Å into the

matrix space and whose narrow end extends ⬃35 Å into

the intermembrane space (Fig. 22-23). Its membrane-

spanning region is ⬃40 Å thick and consists of 13 trans-

membrane helices in each protomer (12 in yeast, which

consists of 9 different subunits). Eight of these transmem-

brane helices are contributed by the cytochrome b sub-

unit, which binds both heme b

and heme b

within its

transmembrane region, with heme b

being closest to the

intermembrane space. One of the remaining transmem-

brane helices is the membrane anchor of cytochrome c

the rest of which is a globular domain that extends into the

intermembrane space. This is the portion of the complex

840 Chapter 22. Electron Transport and Oxidative Phosphorylation

Figure 22-23 X-ray structure of the yeast Complex III

homodimer in complex with cytochrome c and the inhibitor

stigmatellin. The view is perpendicular to its 2-fold axis and

parallel to the membrane with the matrix below. The nine

different subunits in each protomer of Complex II, which

collectively have 12 transmembrane helices, are drawn in

semitransparent ribbon form and are differently colored with

cytochrome b pale yellow, cytochrome c

light purple, the ISP

tan, Core 1 cyan, and Core 2 orange. The bound cytochrome c is

pink.The four different heme groups, the [2Fe–2S] cluster, and

stigmatellin are drawn in space-filling form with heme C green,

stigmatellin C magenta, N blue, O red, S yellow, and Fe red-

brown.The horizontal lines delineate the inferred position of the

membrane. Note that only one cytochrome c is bound to the

homodimeric Complex III. [Based on an X-ray structure by

Carola Hunte, Max Planck Institute for Biophysics, Frankfurt am

Main, Germany. PDBid 1KYO.]

See Interactive Exercise 17.

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