Figure 14-5The relationship between mitochondria and microtubules

(A) A light micrograph of chains of elongated mitochondria in a living mammalian cell in

culture. The cell was stained with a fluorescent dye (rhodamine 123) that specifically
labels mitochondria in living cells. (B) An immunofluorescence micrograph of the
same cell stained (after fixation) with fluorescent antibodies that bind to
microtubules. Note that the mitochondria tend to be aligned along microtubules.
(Courtesy of Lan Bo Chen.)
(B) (A) Mikrograf cahaya rantai mitokondria memanjang dalam sel mamalia
hidup dalam kultur. Sel diwarnai dengan pewarna fluorescent
(rhodamine 123) yang secara khusus melabeli mitokondria dalam sel
hidup. (B) Sebuah mikrograf imunofluoresensi dari sel yang sama
bernoda (setelah fiksasi) dengan antibodi fluoresen yang berikatan
dengan mikrotubulus. Perhatikan bahwa mitokondria cenderung
disejajarkan di sepanjang mikrotubulus. (Atas perkenan Lan Bo Chen.)

Figure 14-6Localization of mitochondria near sites of high ATP utilization in

cardiac muscle and a sperm tail

During the development of the flagellum of the sperm tail, microtubules wind helically
around the axoneme, where they are thought to help localize the mitochondria in the tail;
these microtubules then disappear, and the mitochondria fuse with one another to create the
structure shown.

Mitochondria are large enough to be seen in the light microscope, and they were first identified
during the nineteenth century. Real progress in understanding their function, however, depended on
procedures developed in 1948 for isolating intact mitochondria. For technical reasons, many of these
biochemical studies have been performed with mitochondria purified from liver; each liver cell
contains 1000–2000 mitochondria, which in total occupy about one-fifth of the cell volume.

Selama perkembangan flagel dari ekor sperma, mikrotubulus berputar secara

heliks di sekitar aksonem, di mana mereka dianggap membantu melokalisasi
mitokondria di ekor; mikrotubulus ini kemudian menghilang, dan mitokondria
bergabung satu sama lain untuk membuat struktur yang ditunjukkan.
Mitokondria cukup besar untuk dilihat dalam mikroskop cahaya, dan mereka
pertama kali diidentifikasi selama abad kesembilan belas. Kemajuan nyata
dalam memahami fungsi mereka, bagaimanapun, bergantung pada prosedur yang
dikembangkan pada tahun 1948 untuk mengisolasi mitokondria utuh. Untuk
alasan teknis, banyak dari studi biokimia ini telah dilakukan dengan
mitokondria yang dimurnikan dari hati; setiap sel hati mengandung 1000-2000
mitokondria, yang secara total menempati sekitar seperlima dari volume sel.

The Mitochondrion Contains an Outer Membrane, an Inner Membrane, and

Two Internal Compartments

Each mitochondrion is bounded by two highly specialized membranes, which have very
different functions. Together they create two separate mitochondrial compartments: the
internal matrix and a much narrower intermembrane space. If purified mitochondria are
gently disrupted and then fractionated into separate components (Figure 14-7), the
biochemical composition of each of the two membranes and of the spaces enclosed by them
can be determined. As described in Figure 14-8, each contains a unique collection of proteins.

Figure 14-7Biochemical fractionation of purified mitochondria into separate

components

These techniques have made it possible to study the different proteins in each mitochondrial
compartment. The method shown allows the processing of large numbers of mitochondria at
the same time. It takes advantage of the fact that, in a solution of low osmotic strength, water
flows into mitochondria and greatly expands the matrix space (yellow). While the cristae of
the inner membrane unfold to accommodate the expansion, the outer membrane—which has
no folds—breaks, releasing a structure composed of only the inner membrane and the matrix.

Mitokondria Mengandung Membran Luar, Membran Dalam, dan Dua Kompartemen

Internal
Setiap mitokondria dibatasi oleh dua membran khusus, yang memiliki fungsi
yang sangat berbeda. Bersama-sama mereka membuat dua kompartemen
mitokondria yang terpisah: matriks internal dan ruang antarmembran yang
jauh lebih sempit. Jika mitokondria murni dimusnahkan dengan lembut dan
kemudian difraksinasi menjadi komponen yang terpisah (Gambar 14-7),
komposisi biokimiawi dari masing-masing dua membran dan ruang yang tertutup
olehnya dapat ditentukan. Seperti dijelaskan pada Gambar 14-8, masing-
masing mengandung koleksi protein yang unik.
 
Gambar 14-7 Fraksinasi biokimia mitokondria murni menjadi komponen-komponen
terpisah
Teknik-teknik ini telah memungkinkan untuk mempelajari berbagai protein di
setiap kompartemen mitokondria. Metode yang ditampilkan memungkinkan
pemrosesan sejumlah besar mitokondria secara bersamaan. Ini mengambil
keuntungan dari fakta bahwa, dalam larutan kekuatan osmotik rendah, air
mengalir ke mitokondria dan sangat memperluas ruang matriks (kuning).
Sementara krista membran dalam terbuka untuk mengakomodasi ekspansi,
membran luar — yang tidak memiliki lipatan — pecah, melepaskan struktur
yang hanya terdiri dari membran dalam dan matriks.

Figure 14-8The general organization of a mitochondrion

In the liver, an estimated 67% of the total mitochondrial protein is located in the matrix, 21%
is located in the inner membrane, 6% in the outer membrane, and 6% in the intermembrane
space. As indicated below, each of these four regions contains a special set of proteins that
mediate distinct functions. (Courtesy of Daniel S. Friend.)

The outer membrane contains many copies of a transport protein called porin (discussed in
Chapter 11), which forms large aqueous channels through the lipid bilayer. This membrane
thus resembles a sieve that is permeable to all molecules of 5000 daltons or less, including
small proteins. Such molecules can enter the intermembrane space, but most of them cannot
pass the impermeable inner membrane. Thus, whereas the intermembrane space is chemically
equivalent to the cytosol with respect to the small molecules it contains, the matrix contains a
highly selected set of these molecules.

As we explain in detail later, the major working part of the mitochondrion is the matrix and
the inner membrane that surrounds it. The inner membrane is highly specialized. Its lipid
bilayer contains a high proportion of the “double” phospholipid cardiolipin, which has four
fatty acids rather than two and may help to make the membrane especially impermeable to
ions. This membrane also contains a variety of transport proteins that make it selectively
permeable to those small molecules that are metabolized or required by the many
mitochondrial enzymes concentrated in the matrix. The matrix enzymes include those that
metabolize pyruvate and fatty acids to produce acetyl CoA and those that oxidize acetyl CoA
in the citric acid cycle. The principal end-products of this oxidation are CO2, which is
released from the cell as waste, and NADH, which is the main source of electrons for
transport along the respiratory chain—the name given to the electron-transport chain in
mitochondria. The enzymes of the respiratory chain are embedded in the inner mitochondrial
membrane, and they are essential to the process of oxidative phosphorylation, which
generates most of the animal cell's ATP.

The inner membrane is usually highly convoluted, forming a series of infoldings, known as
cristae, that project into the matrix. These convolutions greatly increase the area of the inner
membrane, so that in a liver cell, for example, it constitutes about one-third of the total cell
membrane. The number of cristae is three times greater in the mitochondrion of a cardiac
muscle cell than in the mitochondrion of a liver cell, presumably because of the greater
demand for ATP in heart cells. There are also substantial differences in the mitochondrial
enzymes of different cell types. In this chapter, we largely ignore these differences and focus
instead on the enzymes and properties that are common to all mitochondria.

High-Energy Electrons Are Generated via the Citric Acid Cycle

As previously mentioned, without mitochondria present-day eucaryotes would be dependent

on the relatively inefficient process of glycolysis (described in Chapter 2) for all of their ATP
production, and it seems unlikely that complex multicellular organisms could have been
supported in this way. When glucose is converted to pyruvate by glycolysis, less than 10% of
the total free energy potentially available from the glucose is released. In the mitochondria,
the metabolism of sugars is completed, and the energy released is harnessed so efficiently
that about 30 molecules of ATP are produced for each molecule of glucose oxidized. By
contrast, only 2 molecules of ATP are produced per glucose molecule by glycolysis alone.

Mitochondria can use both pyruvate and fatty acids as fuel. Pyruvate comes from glucose and
other sugars, whereas fatty acids come from fats. Both of these fuel molecules are transported
across the inner mitochondrial membrane and then converted to the crucial metabolic
intermediate acetyl CoA by enzymes located in the mitochondrial matrix. The acetyl groups
in acetyl CoA are then oxidized in the matrix via the citric acid cycle, described in Chapter 2.
The cycle converts the carbon atoms in acetyl CoA to CO2, which is released from the cell as
a waste product. Most importantly, the cycle generates high-energy electrons, carried by the
activated carrier molecules NADH and FADH2 (Figure 14-9). These high-energy electrons
are then transferred to the inner mitochondrial membrane, where they enter the electron-
transport chain; the loss of electrons from NADH and FADH2 also regenerates the NAD+ and
FAD that is needed for continued oxidative metabolism. The entire sequence of reactions is
outlined in Figure 14-10.

Gambar 14-8 Organisasi umum mitokondria

Di hati, diperkirakan 67% dari total protein mitokondria terletak di
matriks, 21% terletak di membran dalam, 6% di membran luar, dan 6% di ruang
antarmembran. Seperti ditunjukkan di bawah ini, masing-masing dari empat
daerah ini mengandung set protein khusus yang memediasi fungsi yang
berbeda. (Atas perkenan Daniel S. Friend.)
Membran luar mengandung banyak salinan protein transpor yang disebut porin
(dibahas dalam Bab 11), yang membentuk saluran berair besar melalui lipid
bilayer. Dengan demikian membran ini menyerupai saringan yang dapat
ditembus oleh semua molekul 5000 dalton atau kurang, termasuk protein
kecil. Molekul seperti itu dapat memasuki ruang intermembran, tetapi
kebanyakan dari mereka tidak dapat melewati membran bagian dalam yang tidak
tembus cahaya. Jadi, sedangkan ruang antar membran secara kimiawi setara
dengan sitosol sehubungan dengan molekul-molekul kecil yang dikandungnya,
matriks tersebut mengandung sekumpulan molekul-molekul yang sangat dipilih
ini.
Seperti yang akan kami jelaskan secara rinci nanti, bagian kerja utama
mitokondria adalah matriks dan membran bagian dalam yang mengelilinginya.
Membran bagian dalam sangat khusus. Bilayer lipidnya mengandung proporsi
tinggi dari "double" phospholipid cardiolipin, yang memiliki empat asam
lemak daripada dua dan dapat membantu membuat membran terutama kedap
terhadap ion. Membran ini juga mengandung berbagai protein transpor yang
membuatnya secara permeabel terhadap molekul-molekul kecil yang
dimetabolisme atau dibutuhkan oleh banyak enzim mitokondria yang
terkonsentrasi dalam matriks. Enzim matriks meliputi enzim yang
memetabolisme piruvat dan asam lemak untuk menghasilkan asetil KoA dan
enzim yang mengoksidasi asetil KoA dalam siklus asam sitrat. Produk akhir
utama dari oksidasi ini adalah CO2, yang dilepaskan dari sel sebagai
limbah, dan NADH, yang merupakan sumber utama elektron untuk transportasi
sepanjang rantai pernapasan — nama yang diberikan kepada rantai transpor
elektron di mitokondria. Enzim dari rantai pernapasan tertanam dalam
membran mitokondria bagian dalam, dan mereka penting untuk proses
fosforilasi oksidatif, yang menghasilkan sebagian besar ATP sel hewan.
Membran bagian dalam biasanya sangat berbelit-belit, membentuk serangkaian
lipatan, yang dikenal sebagai krista, yang diproyeksikan ke dalam matriks.
Figure 14-9How electrons are donated by NADH

In this diagram, the high-energy electrons are shown as two red dots on a yellow hydrogen
atom. A hydride ion (H- a hydrogen atom and an extra electron) is removed from NADH and
is converted into a proton and two high-energy electrons: H- → H+ + 2e -. Only the ring that
carries the electrons in a high-energy linkage is shown; for the complete structure and the
conversion of NAD+ back to NADH, see the structure of the closely related NADPH in
Figure 2-60. Electrons are also carried in a similar way by FADH2, whose structure is shown
in Figure 2-80.

Figure 14-10A summary of energy-generating metabolism in mitochondria

Pyruvate and fatty acids enter the mitochondrion (bottom) and are broken down to acetyl
CoA. The acetyl CoA is then metabolized by the citric acid cycle, which reduces NAD+ to
NADH (and FAD to FADH2, not shown). In the process of oxidative phosphorylation, high-
energy electrons from NADH (and FADH2) are then passed along the electron-transport
chain in the inner membrane to oxygen (O2). This electron transport generates a proton
gradient across the inner membrane, which is used to drive the production of ATP by ATP
synthase (see Figure 14-1).

The NADH generated by glycolysis in the cytosol also passes electrons to the respiratory
chain (not shown). Since NADH cannot pass across the inner mitochondrial membrane, the
electron transfer from cytosolic NADH must be accomplished indirectly by means of one of
several “shuttle” systems that transport another reduced compound into the mitochondrion;
after being oxidized, this compound is returned to the cytosol, where it is reduced by NADH
again.

A Chemiosmotic Process Converts Oxidation Energy into ATP

Although the citric acid cycle is considered to be part of aerobic metabolism, it does not itself
use the oxygen. Only in the final catabolic reactions that take place on the inner
mitochondrial membrane is molecular oxygen (O2) directly consumed. Nearly all the energy
available from burning carbohydrates, fats, and other foodstuffs in the earlier stages of their
oxidation is initially saved in the form of high-energy electrons removed from substrates by
NAD+ and FAD. These electrons, carried by NADH and FADH2, are then combined with O2
by means of the respiratory chain embedded in the inner mitochondrial membrane. The large
amount of energy released is harnessed by the inner membrane to drive the conversion of
ADP + Pi to ATP. For this reason, the term oxidative phosphorylation is used to describe this
last series of reactions (Figure 14-11).

Figure 14-11The major net energy conversion catalyzed by the mitochondrion

In this process of oxidative phosphorylation, the inner mitochondrial membrane serves as a

device that changes one form of chemical bond energy to another, converting a major part of
the energy of NADH (and FADH2) oxidation into phosphate-bond energy in ATP.

Electrons Are Transferred from NADH to Oxygen Through Three Large

Respiratory Enzyme Complexes

Although the mechanism by which energy is harvested by the respiratory chain differs from
that in other catabolic reactions, the principle is the same. The energetically favorable
reaction H2 + ½O2 → H2O is made to occur in many small steps, so that most of the energy
released can be stored instead of being lost to the environment as heat. The hydrogen atoms
are first separated into protons and electrons. The electrons pass through a series of electron
carriers in the inner mitochondrial membrane. At several steps along the way, protons and
electrons are transiently recombined. But only when the electrons reach the end of the
electron-transport chain are the protons returned permanently, when they are used to
neutralize the negative charges created by the final addition of the electrons to the oxygen
molecule (Figure 14-12).

Figure 14-12A comparison of biological oxidations with combustion

(A) Most of the energy would be released as heat if hydrogen were simply burned. (B) In
biological oxidation by contrast, most of the released energy is stored in a form useful to the
cell by means of the electron-transport chain in the inner mitochondrial membrane (the
respiratory chain). The rest of the oxidation energy is released as heat by the mitochondrion.
In reality, the protons and electrons shown are removed from hydrogen atoms that are
covalently linked to NADH or FADH2 molecules.