Scheffler Immo E. Mitochondria

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THE ELECTRON TRANSPORT CHAIN 201

ferent organisms have been found in which some of the complex II genes are

found on the mitochondrial genome. In the red algae Chondrus crispus the

sdhB and sdhC genes are found on the mt genome (125, 126) ; in a different

red algae ( Porphyra purpurea ) and in the phylogenetically distant zooﬂ agel-

late Reclinomas americana, the genes for the iron – protein and the two mem-

brane anchor peptides are found in the mitochondria (127) . These data are in

agreement with the endosymbiont hypothesis for the origin of the mitochon-

drial genome, and they have been used to argue in favor of a monophyletic

origin of mitochondria (see Chapter 2 ).

Eukaryotic genes for the Ip peptides (SDH2) in complex II were ﬁ rst

cloned in 1989 (128) , and their number has increased dramatically over the

past few years. Similarly, the gene for the Fp peptide (SDH1) is available from

numerous species. Cloning the SDH3 and SDH4 genes was initially more chal-

lenging, because the sequence conservation across species is poor, in contrast

to the Ip and Fp peptides, and probes form one organism generally do not

hybridize with genes/cDNA from another distantly related organism (102) . An

extensive review of the molecular genetics of complex II with an emphasis on

gene cloning and regulation of gene expression has been published (102) .

Further updates with the progress from the Lemire group were published

susequently (129, 130) . The major conclusions can be found in the chapter on

nuclear genes for mitochondrial proteins (Chapter 4 ).

Gene disruption experiments in yeast have provided yeast mutants with

either the Fp, Ip, or anchor protein genes deleted, and such mutants can be

used as recipients for novel gene constructs with speciﬁ c alterations to inves-

tigate structure - function relationships (131 – 133) . For example, a series of chi-

meric Ip proteins containing yeast and human sequences were investigated

to delineate a sequence between the ﬁ rst and second cysteine cluster which

appears to be species - speciﬁ c and which, when replaced by a heterologous

sequence, makes it impossible to assemble complex II (134) .

An interesting mammalian cell mutant defective in SDH activity and assem-

bly of complex II has been described for Chinese hamster ﬁ broblasts in tissue

culture (135) . The mutant cells are respiration - deﬁ cient and unable to grow in

medium in which glucose is limiting. The mutation has been established to

result from a single nucleotide change converting a tryptophan codon to a stop

codon, thus truncating the C

II - 3

peptide before the third transmembrane

segment (136) . The fate of the truncated peptide has not been determined, but

the result is a complete failure of the assembly of complex II; not even an

active SDH enzyme complex is formed, although the Fp and Ip peptides are

made and imported normally into the mitochondria. This result can be con-

trasted with the ﬁ ndings with the nuo21 Neurospora mutant, where the periph-

eral complex is made in an active form but not attached to the membrane arm.

The implications of this ﬁ nding are that the Fp and Ip are not assembled sepa-

rately before their attachment to the membrane. Instead, the matrix domains

of the integral membrane proteins appear to participate in the folding and

formation of the Fe – S centers in the Ip subunit and the simultaneous associa-

202 MITOCHONDRIAL ELECTRON TRANSFER

tion with the Fp subunit. In this context it is worth noting that the Ip and Fp

peptides cannot be separated without denaturation, and they have never been

successfully reassembled in vitro into an active SDH enzyme.

In the past decade, human patients have been identiﬁ ed with partial complex

II deﬁ ciencies due to mutations in the SDH genes (137) . They will be discussed

later in the context of mitochondrial diseases (Chapter 7 ). While some of these

mutations lead to predictable pathologies from what we now understand

about mitochondrial diseases, there are also mutations in the SDHB, SDHC,

and SDHD genes that give rise to a very speciﬁ c type of tumor: paraganglio-

mas (138 – 140) .

5.2.6 Complex III

Complex III is ubiquinone – cytochrome c oxidoreductase and is often named

the bc

complex after the two cytochromes found within it; one also ﬁ nds the

simpler name cytochrome c reductase in the literature. The overall reaction

catalyzed by this complex is

QH cyt c 2 H Q cyt c H

3+ 2+

++⇒++

224

Similar to the reaction taking place with complex I, the oxidation of one of

the substrates (QH

) and the transfer of electrons to the mobile carrier (cyt

c) is coupled to the transfer of protons across the inner mitochondrial mem-

brane. The fate of the protons will be the subject of the discussion to follow,

and a more detailed mechanism for proton translocation across the membrane

is also deferred for the moment.

Complex III appears to be quite similar in different species including the

yeast Saccharomyces cerevisiae, Neurospora crassa , animals and plants. A

website devoted to complex III can be consulted for much recent information

( http://www.life.uiuc.edu/crofts/bc - complex_site/ ). One peptide of the complex

is encoded by the mitochondrial genome and incorporated as cytochrome b.

The other peptides (8 in yeast, ∼ 8 in Neurospora , 10 in mammals) are encoded

by the nuclear genome, synthesized in the cytosol, and imported for assembly

in the inner mitochondrial membrane. Both yeast and Neurospora have

become model systems for the study of this complex because genetic manipu-

lations allow detailed questions to be asked about (a) individual peptides and

amino acids within each peptide and (b) their role in assembly, function, and

activity. As described elsewhere in this volume, in yeast even the mitochondrial

gene is now subject to deliberate mutational modiﬁ cation (Chapter 7 ).

Functionally, the most important subunits are the cytochromes b and c

and

the Rieske iron – sulfur protein, since they are the only ones participating in

electron transfer and in the accompanying proton translocation. This fact is

emphasized by the ﬁ nding that the corresponding complex in the bacterial

electron transport chain has only those three proteins. The role of the other

proteins is difﬁ cult to deduce, because they have no prosthetic groups. However,

at least in Neurospora and potato the two large peripheral subunits appear to

THE ELECTRON TRANSPORT CHAIN 203

be processing proteases, and the largest subunit I has been shown to be

involved in the import and processing of proteins from the cytosol (141, 142) .

In the mammalian system, and to a large extent also in Neurospora, the

elegant experimental approaches to studying complex III have primarily relied

on solubilization, partial dissociation into subcomplexes, and reconstitution

studies (141, 143) . The original procedure by Hateﬁ for the isolation from

mammalian mitochondria has been simpliﬁ ed and adapted to a smaller scale

for many purposes. When too much lipid is removed during the puriﬁ cation,

the activity of the complex declines. A most useful technique for isolating OX -

PHOS complexes including complex III directly from homogenized human

tissues is blue - native polyacrylamide gel electrophoresis (BN - PAGE), after

solubilization of the membrane by neutral detergents (27, 144) . It is suitable

for the preparation of complexes in the microgram - to - milligram scale. As iso-

lated from bovine and Neurospora mitochondria, the complex III is a dimer,

a conclusion supported from electron microscopic studies of membrane crys-

tals of the complex from Neurospora . It remains unclear whether dimerization

is absolutely essential for the catalytic activity of the complex, but Covian and

Trumpower have provided strong experimental evidence for the transfer of

electrons between the two b

hemes of the dimer (145) .

Relatively mild detergent can dissociate two subunits from the bovine

complex: the Rieske iron – sulfur protein (ISP) and a small ISP - associated

protein. Further dissociation with 1.5 M guanidine yields a cytochrome c1

subcomplex (cytochrome c1, p9.2, p7.2, a core subcomplex, and several iso-

lated subunits). Cytochrome b appears to remain with the core subcomplex,

but a large fraction is lost in this procedure. When higher detergent concentra-

tions are used to remove neutral lipids from the bovine complex, activity is

lost, but can be restored by slow addition of phosphatidylcholine or phospha-

tidylethanolamine in Triton X - 100. Quantitative measurements have suggested

that a complex III dimer must be surrounded by a complete annulus of phos-

pholipid. Less easily rationalized in terms of a structural model is the obser-

vation that bovine complex III has eight or nine tightly bound cardiolipin

molecules per monomer. Their removal causes irreversible loss of activity.

All 11 mammalian complex III peptides have been puriﬁ ed on SDS - PAGE

and used for partial sequencing and for the production of antibodies that can

be used in the exploration of the topology of the complex in the inner mem-

brane. Another experimental approach to topological studies has employed

EPR techniques. An emerging model places the heme b

562

near the middle of

the lipid bilayer, and the other heme (b

566

) and the Rieske iron sulfur cluster

as well as cytochrome c

are on the “ P - side ” of the membrane (facing the

intermembrane space).

Complex III, like complex I, has two reaction centers for ubiquinone, Q

and Q

. The signiﬁ cance of these multiple sites will become more apparent

when the proton pumping mechanism will be discussed in greater detail.

Studies addressing problems related to the yeast complex III have been

predominantly of a genetic nature. All eight nuclear genes for this complex

have been cloned, and null mutants for each gene have been isolated. With

204 MITOCHONDRIAL ELECTRON TRANSFER

the exception of the mutant affecting the acidic subunit VI, these mutants are

respiration - deﬁ cient. Therefore, even the subunits without prosthetic groups

are necessary for an active complex III, and one can speculate that they are

required for assembly and stabilization of the complex. Among the many pet

mutants analyzed (146) , mutants with a defective ubiquinone – cytochrome c

oxidoreductase activity can be found, although a direct selection or enrich-

ment for such a speciﬁ c defect is not possible. Thus, pet mutants having veriﬁ ed

nuclear mutations must be further screened by biochemical assays to eliminate

mutants affecting mitochondrial protein synthesis or the ubiquinone biosyn-

thetic pathway (147) . Among the mutants with a speciﬁ c lesion in complex III

activity, several classes have been distinguished:

1. Mutants with alterations in the eight nuclear genes for the protein sub-

units of the complex. As discussed above, null mutants are respiration

deﬁ cient. In many of these the cytochrome b is also absent, presumably

because it needs to be stabilized by association with other subunits.

2. A number of mutants are unable to make a cytochrome b detectable by

spectroscopy, even though all structural genes in the nucleus and the

COB gene on the mtDNA are normal. In another chapter on the expres-

sion of mitochondrial genes in yeast, it has been discussed that a variety

of imported proteins are required for cytochrome b mRNA maturation

(group I intron splicing and processing of the 5 ′ terminal) and for mRNA

stabilization. The COB gene encodes a protein that appears to function

both in 5 ′ end processing and mRNA stabilization (translational initia-

tion?). Additional nuclear gene products are necessary for efﬁ cient trans-

lation of the COB mRNA, which in turn also affects the splicing of the

intron, because the COB intron encodes a maturase (see Chapter 4 ).

Another consequence may be a defect in cytochrome oxidase (complex

IV) as a result of defective splicing of the COX1 mRNA.

3. Nuclear gene products are also required for the addition of the pros-

thetic groups to the various subunits. Hemes have to be covalently

attached to apocytochromes, and another gene product, Bcs1p, has been

implicated in the “ maturation ” of the Rieske protein. The precise func-

tion of the BCS1 gene product is not known.

4. Another set of proteins deﬁ ned by nuclear mutations are involved in the

“ late stages of the assembly pathway ” (147) . This is a problem that so far

has been deﬁ ned exclusively by a genetic approach in yeast. Similar

assembly factors have been identiﬁ ed for complexes IV and V. The

mechanisms and pathways of assembly of the complexes in the ETC

represent a major frontier in the quest for understanding mitochondrial

biogenesis.

Cytochrome c reductase (complex III) from potato mitochondria has

received scrutiny, because attention has been attracted more broadly to plant

mitochondria. Similarities to the complex in mammals and yeast may not be

THE ELECTRON TRANSPORT CHAIN 205

surprising, but when sequencing information became available for compara-

tive studies, the so - called core proteins from the potato complex showed the

highest sequence identity with the mitochondrial processing peptidase (MPP)

from mammals and yeast (142) . These enzymes are involved in the cleavage

of matrix targeting sequencing from precursors imported from the cytosol. It

was subsequently veriﬁ ed that a bifunctional protein exists in potato mito-

chondria (all plants?) which plays a role in electron transport as well as in

protein import. The complex is therefore referred to as the cytochrome c

reductase/processing peptidase complex (see Section 4.6 ). It overall composi-

tion (at least 10 subunits) resembles the mammalian and fungal complexes.

A crowning achievement in the study of complex III was the completion

of the complete crystal structure of the complex at 2.9 - Å resolution by the

groups of Yu and Deisenhofer (148) . Following on the heels of the determina-

tion of the structure of complex IV (see below), it represents another major

landmark in the understanding of the complexes of the mitochondrial electron

transport chain. An atomic model has now become available to relate the

proposed reaction mechanisms of QH

oxidation and the reduction of cyto-

chrome c to the architecture and topology of the complex.

The model conﬁ rms, reﬁ nes, and expands the stuctural features deduced

from biochemical studies. In the crystal, two bc1 monomers interact to form a

dimer with a twofold axis of symmetry perpendicular to the membrane. Each

monomer has 12 transmembrane helices: Eight are from cytochrome b, one is

from cytochrome c1, one is from the iron – sulfur protein, one from subunit 7,

and one is still unassigned (Figure 5.14 ).

On one side of the membrane, more than half of the molecular mass of the

complex extends 75 Å into the matrix. It consists mainly of the so - called core

proteins that have a structural rather than a functional role. The peripheral

core 2 proteins contribute signiﬁ cantly to the stabilization of the dimer, in

addition to the interaction of the cytochrome b helices within the membrane.

The two core proteins of the mammalian bc1 complex have homology to the

α and β subunits of the mitochondrial matrix protein processing peptidase

(MPP), and it is suggested that the soluble mammalian MPP has a similar

structure. As discussed above, in plants the MPP is an integral part of complex

III. A detailed side view of the complex is shown in Figure 5.15 . On the outside

of the membrane (P side), one ﬁ nds the Rieske iron – sulfur cluster and the

domain of cytochrome c

containing the heme. The electron density for this

latter region is relatively poor, and the authors ascribe this problem to mobility

in the crystal, which may in fact reﬂ ect a mobility required for interactions

with, and electron transfer to, cytochrome c. Two additional heme irons could

be located within the membrane domain, corresponding to heme b

and heme

, respectively. The identiﬁ cation and distinction between the two hemes of

cyt b was also aided and conﬁ rmed by the availability of difference density

maps comparing the native crystals with crystals obtained with the inhibitors

antimycin A or myxothiazol. The different targets of these inhibitors had been

assigned from biochemical and spectroscopic studies (see below).

206 MITOCHONDRIAL ELECTRON TRANSFER

Of special interest are the distances between the heme irons in b

, b

, and

and the [2Fe – 2S] cluster, in part to explain and conﬁ rm previous spectro-

scopic studies, and understand the rate of electron transfer. In the dimer the

distances between two b

’ s and two b

’ s are comparable to the the b

– b

dis-

tance within a monomer, raising the possibility of electron transfer between

the monomers.

The overall model with its “ cavities ” and “ pockets ” clearly invites further

speculations about the accessibility of the complex to ubiquinone, antimycin,

and myxothiazol and ultimately about the translocation of protons and the

operation of the Q cycle.

Speciﬁ c inhibitors of electron transport have been valuable as a means of

arresting electron ﬂ ux at known locations for the purpose of spectroscopic

studies, for example, and to “ isolate ” a portion of the chain for detailed study.

They have also been useful and revealing probes of structure, and in some

Figure 5.14 Complex III. Schematic representation of the transmembrane helices

in the dimeric complex seen from the P and N (matrix) side (parts A and B, respec-

tively) (82) . See color plates.

THE ELECTRON TRANSPORT CHAIN 207

cases they have been useful to arrest electron ﬂ ow within a complex to estab-

lish upstream and downstream portions of the complex. In the case of complex

III, the antibiotic antimycin A is the best known of the inhibitors, but a variety

of natural and synthetic inhibitors have been characterized. Initially their

utility was in the deﬁ nition of a speciﬁ c block in the electron transport chain

downstream from complexes I and II and upstream from cytochrome c, as well

as in the localization of the “ coupling sites ” for oxidative phosphorylation.

Later they were exploited in deﬁ ning the distinction between center Q

center Q

and in elucidating the protonmotive Q cycle (Figure 5.16 ).

The formulation of the Q cycle (149 – 152) required the postulate of two

quinone reaction sites (see below). The Q

or quinone reduction center is

located on the matix side (N - side) of the inner membrane is associated with

the recycling of half of the electrons back into the quinone pool and the uptake

of protons from the matrix. It is inhibited by antimycin A, funiculosin, and

Figure 5.14 (Continued)

208 MITOCHONDRIAL ELECTRON TRANSFER

Figure 5.15 (A) Side view of complex III. The matrix side is at the bottom. Cyto-

chrome c1 extends into the intermembrane space. The membrane - spanning helices of

the various subunits are in the region indicated by the middle arrow (42 Å ). (B) Ste-

reoview of the transmembrane helices of a monomeric complex III. (C) Stereoview

from the outside showing the myxothiazol binding pocket. (D) Stereoview from the

matrix side showing the antimycin binding pocket. (From reference 147 with permis-

sion. The ﬁ gure was generously provided by Dr. Yu.) See color plates.

THE ELECTRON TRANSPORT CHAIN 209

hydroquinoline - N - oxides, inhibitors that interfere with the electron transfer

from heme b

to Q or QH

−

. At the Q

center, electrons from reduced ubiqui-

none are accepted and divided into two pathways: half for recycling, and half

for transfer via the iron – sulfur center and cyt c

to cytochrome c. It is located

near the outer face of the inner membrane, and protons are released into the

intermembrane space. A number of different compounds can inhibit at the Q

center: 2 - hydroxy - 1,4 - benzoquinone drivatives, stigmatellins, and MOA inhibi-

tors containing the E - b - methoxyacrylate group (MOA - stilbene, myxothiazol).

Some further discussion of the Q cycle can be found in Section 5.4.2 .

A general review of the approaches and methodologies used with these

inhibitors (Figure 5.17 ) has been written by Link et al. (153) . Having estab-

lished the speciﬁ city and efﬁ cacy of each inhibitor in arresting electron trans-

port, additional studies have included light and EPR spectroscopy, the isolation

of resistant mutants of a variety of species, the identiﬁ cation of mutated amino

acid side chains in speciﬁ c peptides from such mutants, characterization of

Figure 5.15 (Continued)

210 MITOCHONDRIAL ELECTRON TRANSFER

mutations induced by site - directed mutagenesis (154) , and the synthesis of

additional analogues — for example, antimycin A analogues (155) .

5.2.7 Complex IV

Complex IV is generally referred to as cytochrome c oxidase. The overall reac-

tion catalyzed is the following:

44 42cyt c H 4 H O 4 cyt c H H O

(s)in

(p)in

(p)out

+++→ + +

Molecular oxygen is the terminal electron acceptor, the mobile carrier cyt c is

reoxidized, and four protons are transferred to the intermembrane space.

There are two processes involved. First, four electrons are donated on the IMS

side, while four protons are taken up on the matrix side, resulting in a transfer

of four positive charges across the membrane. Second, an average of one

proton is pumped through the enzyme for each electron transferred to oxygen

(156) .

After three decades of study and analysis, this complex had the distinction

of being the ﬁ rst complex of the ETC to have its high - resolution crystal struc-

ture determined at ∼ 2.8 - Å resolution. Two landmark papers on the bovine

complex IV presented the largest and most complex membrane protein to be

solved at this resolution in 1996 (157, 158) . A monumental achievement, they

Figure 5.16 The Qcycle and electron ﬂ ow through complex III. QH

is oxidized, and

electrons are transferred to the mobile carrier cytochrome c (upper right in the ﬁ gure).

For a detailed explanation see the text. (After Trumpower (152) .)

2QH

Q -

Pool

[2Fe-2S]

center