Scheffler Immo E. Mitochondria

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THE ELECTRON TRANSPORT CHAIN 211

Figure 5.17 Different subclasses of inhibitors frequently used in the study of complex

III. The fargets for the different drugs in groups A, B, and C are discussed in the text

(see also Figure 5.14 )

OCH

Myxothiazol

3 CH

OCH

Stigmatellin A

undecylhydroxydioxobenzothiazole

UHDBT

(CH

)

heptadecylmercaptohydroxyquinoline

quinone

HMHQQ

S (CH

)

mark a major milestone in the history of electron transport and respiration.

As always, these papers settled some older arguments and answered a number

of questions quite deﬁ nitively, but a wealth of new questions were raised to

understand at the atomic level how electron transport is intimately coupled

to proton pumping. Only high - resolution computer images with the added

capability of rotating the image and zooming in and out of the structure can

give a true sense of this achievement and its powerful inﬂ uence on future

thinking about these fundamental processes in bioenergetics. As one review

212 MITOCHONDRIAL ELECTRON TRANSFER

summarized the situation at the time: “ The monster is subdued, but far from

tamed ” (159) . In the meantime, more structures have been solved, and existing

structures have been reﬁ ned at higher resolution. The interested reader can

ﬁ nd a wealth of information and links at a website dedicated to cytochrome

oxidase ( http://www - bioc.rice.edu/ ∼ graham/CcO.html ).

The functionally homologous but simpler bacterial structure also became

available in 1995 (160) , by itself a major achievement. A remarkable conserva-

tion of structural features helps to emphasize those aspects most basic and

essential for function. Certain differences will emphasize the requirement for

regulatory mechanisms in higher organisms that may not exist or may be dif-

ferent in bacteria.

A discussion must start with the composition of the complex. The mamma-

lian complex contains 13 subunits, the yeast complex is composed of 9 subunits,

and one may expect to see further variations in the total number of subunits

in different organisms. In all organisms the three largest subunits (I, II, III)

are encoded by the mitochondrial genome and synthesized in the matrix. For

the remaining subunits the nomenclature can be confusing to the uninitiated:

In the mammalian complex we have subunits IV, Va, Vb, VIa, VIb, VIc, VIIa,

VIIb, VIIc, and VIII; in yeast the subunits are IV, V, VI, VII, VIIa, and VIII.

Antimycin A

HO N

(CH

)

Funiculosin

Heptylhydroxyquinoline-N-oxide

CHO

(CH

)

CH(CH

)

Figure 5.17 (Continued)

THE ELECTRON TRANSPORT CHAIN 213

And while a biochemist may refer to subunit VIIa in yeast, a geneticist might

refer to it as the COX9 gene product.

A large number of biochemical and elegant spectroscopic analyses had

established some time ago that the complex contained two hemes (aa

) and

two copper centers. In fact, the cytochromes a and a

were among the ﬁ rst

described by D. Keilin in 1925. The X - ray structure has probably been most

deﬁ nitive in deﬁ ning two additional metal centers containing Mg and Zn.

Studies on the simpler bacterial cytochrome c oxidases and comparisons of

homologous peptides have given clues about the association of these impor-

tant metal centers with speciﬁ c peptides which are now totally conﬁ rmed for

yeast and mammals. Subunit I binds the heme a and heme a

prosthetic groups

and also forms the Cu

redox center. Subunit II binds the Cu

center. Subunits

I – III form the core of the enzyme. While subunits I and II can carry out the

redox reactions and proton pumping, they become irreversibly inactivated

during one cycle, and subunit III has been proposed to maintain the structural

integrity of the complex (see reference 156 for a recent review and listing of

many references). The other subunits may perform regulatory functions or

play a role in insulation, stabilization, or assembly. Such a regulatory function

is indicated by the existence of tissue - speciﬁ c isoforms for the subunits VIa,

VIIa, and VIII in mammals (161, 162) .

In by now standard fashion, the yeast nuclear genes have been disrupted

one at a time, and in the absence of either subunit IV, VI, VII, or VIIa there

is no cytochrome c oxidase activity and cytochromes aa

are missing. A knock-

out of the COX8 gene (subunit VIII) causes only a 20% reduction of activity

at normal aa

levels. There are two isoforms of subunit V, either one is required

for activity, but complexes with either Va or Vb differ slightly in their kinetic

and spectroscopic properties. The yeast complex may have additional subunits,

making it more similar to the mammalian complex, but it has to be clariﬁ ed

whether they are bona ﬁ de subunits, scaffolding proteins required only for

assembly, or simply contaminants.

For many years, laboratories had attempted to deﬁ ne the topology of these

subunits within the inner membrane by reactions with membrane imperme-

able reagents, by cross - linking and co - precipitation, and by an exploration of

the location of epitopes with a large variety of monoclonal antibodies (163,

164) . In light of the solution of the crystal structure, these studies need not be

reviewed here, but the development of the technology and reagents will con-

tinue to be useful tools in the examination of the enzyme, particularly in

comparisons between the normal enzyme and enzymes derived from individu-

als with mitochondrial mutations in the genes for the three largest subunits

(164) .

There are 28 transmembrane helices contributed by the various subunits.

Viewed from the top of the membrane, they form an irregular cluster surround-

ing the metal centers, in effect solubilizing the metal sites within the lipid

bilayer and also “ insulating ” the electron path from the surrounding lipid

layers. Many helices at the outside of the cluster probably have no function

214 MITOCHONDRIAL ELECTRON TRANSFER

other than to serve in the assembly and stabilization of the complex. Notably,

most of the transmembrane helices are not parallel to each other, and they are

not vertical to the plane of the membrane. Three subunits (Va, Vb, and VIb)

are not within the membrane, and they are associated as peripheral membrane

proteins with extramembrane domains of the membrane subunits.

In considering the structure in detail, it is proﬁ table to decompose the

problem into separate structure – function relationships. The central issue is the

path of electrons from the substrate ferrocytchrome c to oxygen. Another

problem of fundamental importance is to understand the proton pumping

activity associated with electron transport. Finally, regulatory functions, stabi-

lization of the complex, and the assembly mechanism may be addressed from

a consideration of the high - resolution structure. The emergence of the struc-

ture in two major stages reﬂ ects this order of priority.

The representation of the metal sites within the overall contours of the

complex and in relationship to the membrane was the ﬁ rst major achievement

(157) , followed one year later by the entire structure (158) (Figures 5.18

and 5.19 ).

The two metal ions Mg

and Zn

are not redox centers. The Zn

ion is

quite some distance away from the “ action, ” and its major function appears to

be in the stabilization of the three - dimensional structure of the complex. The

ion, on the other hand, is between Cu

and heme a

, and, although not

directly involved, it may have more than a simple stabilizing role, either in

electron transport (and its direction) or in the coupling with proton transfer.

While the spatial arrangements and precise distances between the other

metal centers were ﬁ nally determined by the crystal structure, numerous inge-

nious spectroscopic studies had contributed to a path for the electrons which

in a simpliﬁ ed form has the following appearance:

ferrocyt c Cu Cu heme a heme a Cu O

AA 3B 2

→−→ → −→

In this scheme the Cu

center (with two copper ions) is located near the

surface of the complex facing the intermembrane space and hence it is able

to be brought in close contact with the reduced cytochrome c. The two heme

groups are buried within the complex as electron carriers with a redox poten-

tial determined by the surrounding protein. The unique feature is the bimetal-

lic heme iron – copper reaction center, which is also the site to which oxygen

approaches for its activation and conversion to water. To understand more

detail, one must understand the nature and redox behavior of the Cu

– Cu

center; and, most importantly, the mechanism of the transfer of electrons to

molecular oxygen from the heme a

– Cu

combination.

The Cu

– Cu

center has the [2Cu – 2S

] cyclic structure in which the two

copper ions are coordinated with two cysteines, two histidines, a methionine,

and a peptide carbonyl of a glutamate of subunit II. These ligands, except for

the carbonyl group, had been proposed previously on the basis of mutagenesis

experiments that were fully conﬁ rmed by the X - ray structures. The presence

THE ELECTRON TRANSPORT CHAIN 215

Figure 5.18 X - ray structure of complex IV. (A) Side view showing the transmembrane

segments of the various subunits; matrix side at the bottom. (B) View from the cytosolic

side. (The photographs were kindly provided by Dr. S. Yoshikawa of the Himeji Insti-

tute of Technology, Hyogo, Japan. From reference 157 with permission.) See color

plates.

216 MITOCHONDRIAL ELECTRON TRANSFER

Figure 5.19 X - ray structure of complex IV. Stereo images of the C

- backbone tracings.

(A) Subunits I, II, and III. (B) Subunits IV, Va, Vb, VIa, Vib, Vic, VIIa, VIIb, and VIII.

(The photographs were kindly provided by Dr. S. Yoshikawa of the Himeji Institute of

Technology, Hyogo, Japan. From reference 157 with permission.) See color plates.

THE ELECTRON TRANSPORT CHAIN 217

of two copper ions per site A had long been uncertain and controversial. An

insightful account of the history of the Cu

site in cytochrome oxidase has been

written by H. Beinert (165) . It is highly instructive in tracing the development

of ideas about Cu in complex IV, in emphasizing the development of methodol-

ogy and techniques (puriﬁ cation, spectroscopy, molecular genetics). Many

general concepts in protein structure with special emphasis on metal – protein

(ligand) interactions ultimately had to be applied to the understanding of a

complex with seven metal ions, three of them Cu. It now appears that there are

major similarities to the [2Fe – 2S] centers, where one - electron transfers can be

facilitated by delocalizing the charge between the two metal ions.

The most unusual and interesting structure is the binuclear heme a

– Cu

center, and major interest in this center arises from the nature of the reaction

occurring there. It is usually presented as a simple addition of electrons and

protons to oxygen:

442e

−

++ →OH HO

However, details of this process are more complicated (156, 166, 167) . With

20% oxygen in the atmosphere, the organic material on the earth ’ s surface

would spontaneously burst into ﬂ ame if the activation of molecular oxygen

were simpler and energetically more favorable. The low normal reactivity of

arises from the triplet electronic ground state of oxygen, with two unpaired

electrons, which makes both single - and two - electron transfers from singlet -

state donors kinetically slow. Thus, the thermodynamically highly favorable

reduction of oxygen to water can be kept in check, except at high temperatures

or at reaction centers such as the one under discussion. The latest scheme of

oxygen activation proposed by Branden and colleagues is shown in Figure

5.20 . The cycle starts with R (Fe(II)/Cu(I)). Oxygen binds to the heme iron

(A), but the proximity of the copper ion allows two electrons from within the

center to be transferred to oxygen, converting it to the peroxy ion (O

2 −

) and

resulting in the oxidation of the metal ions to P

(Fe(IV)/Cu(II), including a

tyrosine radical at the catalytic site. The ﬁ rst of the four electrons from the

upstream heme a enters together with a proton to form the intermediate F. In

the F → OH transition another electron and a proton are added and the

oxygen molecule is split. The proton redistribution leaves Fe(III) and Cu(II),

each coordinated with a hydroxyl ion. The hydroxyl ions are released one at

a time as water in two more steps (O

→ E and E → R) with the addition of

one proton and one electron in each to return to the starting position Fe(II)/

Cu(I). Many of these steps have been resolved by optical measurements at

low temperatures, or by time - resolved Raman spectroscopy (168) . For the

more intricate biophysical, thermodynamic, and kinetic aspects of this problem

the reader is referred to the specialized literature (e.g., references 156 and 167

and references therein). Not all aspects of the mechanism are resolved to

everyone ’ s satisfaction, especially with regard to the steps that drive the proton

pumping. What makes the pump unidirectional? Recent expert reviewers have

218 MITOCHONDRIAL ELECTRON TRANSFER

referred to this enzyme as a “ hysteric ” enzyme; that is, its properties are depen-

dent on its history, which depends (at least under experimental, in vitro condi-

tions) on the rate of delivery of the electrons from cyt c.

A most important consequence arises when this overall process is per-

turbed by the premature departure of partially reduced oxygen. Partial reduc-

tion yields the superoxide radical (O

−

), from which hydrogen peroxide (H

)

or the hydroxyl radical (OH

) can be derived. These species are often referred

to collectively as the “ reactive oxygen species ” (ROS), and their production

is strongly implicated in oxygen toxicity and mutagenesis of mitochondrial

DNA (see Chapter 7 ). A more elaborate discussion of the ROS and their

reactions will be presented in Section 5.7 . In the present context it is important

to clarify whether reactive oxygen species produced in mitochondria arise

from incomplete reduction of oxygen in complex IV. It has been estimated

that a signiﬁ cant fraction (a few percent??) of oxygen entering mitochondria

is reduced only partially, and this fraction may increase under abnormal condi-

tions or as a result of electron transport complexes impaired by mutations.

Normal individuals suffer from various afﬂ ictions when exposed to greater

than 21% oxygen, and prolonged exposure can lead to lung damage. Prema-

ture babies exposed to higher - than - normal oxygen concentrations in incuba-

tors can develop retrolental ﬁ broblasia (ocular damage) unless carefully

monitored and given α - tocopherol as a free radical scavenger. It is generally

accepted that mitochondrial ROS production does not occur at the site

designed for reduction of oxygen in complex IV, but rather from the reaction

of the highly soluble and diffusible oxygen with reduced upstream sites of the

Figure 5.20 Proposed sequence of reactions for transferring four electrons from

cytochrome c to an oxygen molecule to form two molecules of water. (Adapted from

reference 155 .)

YOH

O O

H O

YOH

H O

THE ELECTRON TRANSPORT CHAIN 219

ETC. In other words, a leakage of electrons can occur from high potential sites,

especially in damaged or abnormal mitochondria .

The complete solution of the structure of complex IV in mammals and of

the homologous simpler complex in bacteria has greatly stimulated the discus-

sion of the mechanism of proton pumping coupled to electron transport (156,

158, 160, 167) , but here even a structure at 1.8 - Å resolution cannot give an

absolutely deﬁ nitive answer. Previous approaches employing mutagenesis had

suggested the existence of two proton channels: one for protons that become

associated with oxygen to form water, and another for the protons pumped

across the membrane. From theoretical considerations, one would expect the

proton channels to consist of a network of hydrogen - bonded side chains whose

’ s would be controlled by conformational changes associated with redox

reactions. Acidic groups on either side of the membrane must be alternately

accessible, and their p K must vary depending on the oxidation state of the

enzyme. Protons are shuttled through the protein complex along a string of

residues that have been referred to as a “ proton wire ” (169) . The relevant

residues were identiﬁ ed primarily with the help of mutagenesis experiments

on the bacterial enzyme(s). Several such networks were found, and included

in these localized structures were cavities likely to contain water molecules

that can participate in proton conduction. Since the original X - ray structures

were reported in 1995, additional structures have now been examined of the

enzyme in the fully reduced, fully oxidized, azide - bound, and carbon monox-

ide - bound states (170) . These studies greatly reﬁ ne the conformational changes

associated with the redox reaction and contribute to the formulation of more

speciﬁ c proton pathways and models (see Figure 5.21 ). In fact, the power of

modern computers is such that the behavior of the entire enzyme can nowa-

days be simulated by molecular dynamics, with various restraints imposed

depending on the cost of the calculations (167) . The proton pathways from the

matrix to the a

– Cu

center appear to be resolved, but the subsequent path to

the IMS remains to be further elucidated. The D pathway (Figure 5.21 ) appears

to be involved in the pumping of all four protons. Although the pathways may

be identiﬁ ed, there is still the challenge to understand how the rate and direc-

tion of movement are controlled by the electron ﬂ ow. Finally, one must also

identify a water channel for the escape of the product, H

O, and possibly a

pathway through which the substrate O

can approach the heme a

– Cu

reac-

tion site. Potential pathways have been suggested from a consideration of the

crystal structure, but none of them are wide enough in the static crystal struc-

ture to accommodate these molecules. Thus, rapid, reversible conformational

changes have been postulated to operate in the opening and closing of such

channels. Expert, comprehensive, and up - to - date reviews (156, 167, 171 – 173)

should be consulted for further details.

Finally it should be noted that most of the more sophisticated biophysical

studies have been performed with the prokaryotic complex from Rhodobacter

sphaeroidis . In the eukaryotic enzymes the basic mechanisms of electron

transport, oxygen reduction and proton pumping are likely to be similar, but

superimposed on these will be regulatory mechanisms from ligand binding and

220 MITOCHONDRIAL ELECTRON TRANSFER

protein phosphorylation (see Section 5.6 ). Cytochrome oxidase may have the

capacity to regulate its own H

/ e

–

ratio — that is, the efﬁ ciency with which

proton pumping is coupled to electron ﬂ ow; this may act as a protective mecha-

nism to prevent the membrane potential to become too high (167) . The speciﬁ c

role of the “ supernumerary ” subunits remains a challenge for the future.

In closing this section, one should not dwell on the outstanding issues and

unsolved problems. Instead it should be recognized that since the discovery of

the Atmungsferment by O. Warburg almost seven decades ago, this enzyme has

been one of the most important objects of study in the area of bioenergetics. As

such, it has attracted the attention of some of the greatest minds in the history

of biochemistry. The whole structure at high resolution (1.8 Å ) represents a

crowning achievement. Words fail to describe it fully, but most institutions and

many individual laboratories can now obtain the structure via the internet, or

on computer disks for display on a workstation. When combined with three

dimensional vision achieved with special goggles, the inspection and explora-

tion of this structure becomes an awsome spectacle even for the nonspecialist.

5.2.8 The Assembly of the Electron Transport Chain Complexes

While the compositions, structures, and functions of the electron transport

complexes are now understood in impressive detail, a remaining challenge is

Figure 5.21 (A) Schematic view of complex IV with the four essential subunits (155) .

(B) The proposed D and K pathways for the entry of protons to form water and to be

pumped across the membrane. The path from the region of the hemes to the outside

is still poorly deﬁ ned. (From reference 155 with permission.) See color plates.

SU II

SU I

SU III

SU IV

P side

side

heme a

Y288

T359

S299

K362

E101

E286

D pathway

K pathway

D132

N139

N121

S201

−