section 14.2
Oxidative Phosphorylation
259
2. A hydrophobic complex of three or four proteins (also
called proteolipids), or Fo complex, located in the
inner membrane, which is thought to contain a
proton-translocating channel; and
3. A stalk consisting of protein components that connect
Fi with Fq.
FIGURE 14-13
Schematic representation of a transmembrane redox loop, in which 2H+
are ejected into the intermembrane space as the substrate is oxidized on the
matrix side.
(2 x 3) are ejected. Development of an electrochemical
potential for protons is the mechanism by which conser-
vation of free energy occurs during transport of reducing
equivalents in the respiratory chain.
The electrochemical potential is proportional to the
proton-motive force (pmf, designated as
A p
or A
fXH+),
given by the following equation:
A
jlH+
=
Ai/r
—
Z
ApFI
where A t/r is the membrane potential resulting from charge
separation, the matrix compartment being negative; ApH
is the pH difference across the membrane, due to proton
translocation; and Z is equal to —2.303 RT/F, the factor
used to convert pH units into millivolts, the unit of the
other two terms of the expression; Z equals —59 at 25°C.
The anisotropic arrangement of the respiratory chain
and the vectorial transport of protons are supported by
experimental observations. The distribution of the redox
carriers required by the chemiosmotic hypothesis is re-
markably similar to that derived from studies on enzyme
topology of the inner membrane. In respiring mitochon-
dria, the intramembrane space is more acidic than the ma-
trix space by about 1.4 pH units, and the transmembrane
potential is about 0.180-0.220 V. Thus, the basic premise
of the chemiosmotic hypothesis is
Transport of reducing equivalents —>
A jlH+
—> ATP synthesis
The proton-motive force drives ATP synthesis via the
reentry of protons. The ATP synthase (F„/Fi complex)
is driven by this vectorial transfer of protons from the in-
tramembrane space into the matrix. ATP synthesis occurs
in the inner membrane spheres (the Fi component of the
synthase).
ATP synthase consists of three parts (Figure 14-14):
1. The catalytic part, Fi, composed of five firmly
associated subunits with a subunit composition of
a 3p 3y8e;
When the ATP synthase forms a part of the intact mem-
brane system, it catalyzes proton translocation and ATP
synthesis. However, when Fi is dissociated from Fo, the
Fj no longer catalyzes ATP synthesis but rather ATP hy-
drolysis. Thus, Fi becomes an ATPase rather than an
ATP synthase. Submitochondrial vesicles devoid of Fi
can transport reducing equivalents, since they contain the
redox carriers, but are unable to support ATP synthesis.
Careful reconstitution of membrane vesicles by addition
of Fi allows the complex to regain its capacity for ATP
synthesis.
The antibiotics oligomycin and aurovertin and the
reagents N,N'-dicyclohcxylcarbodiimide (DCCD) and
4-chloro-7-nitrobenzofurazan (Nbf-Cl) inhibit ATP syn-
thesis by binding to different sites of ATP synthase.
Oligomycin and DCCD bind to proteolipid (Fo) compo-
nents and block translocation of protons, aurovertin binds
to a specific /3-subunit of Fi, and Nbf-Cl binds with a
specific tyrosine residue of the /
1
-subunit of F j.
The mechanism of ATP formation by the ATP synthase
driven by the passage of protons is attributed to confor-
mational changes that occur in the
aft
dimers of the Fi
complex (Figure 14-15). The Fi complex has three inter-
acting nucleotide-binding and conformationally distinct
af3
domains:
1. Open conformation (O): has very low affinities for
substrates and products and is catalytically inactive.
2. Loose conformation (L): binds loosely to nucleotides
and is catalytically inactive.
3. Tight conformation (T): binds nucleotides tightly and
is catalytically active.
In this model, originally proposed by Paul Boyer, the
conformational changes of a /3 assemblies are brought
about by the energy dependent rotation of-the
y
subunit.
The
y
subunit rotation occurs in discrete steps of 120° and
is fuelled by proton passage through Fo channels. In step
1 rotation (Figure 14-15), the conformation change at the
T site causes ATP release at the O site. In step 2 rotation,
ADP and P; bound at the L site, now bind at the T site
and undergo conversion to ATP. Thus, in the conforma-
tional cycle, ATP is synthesized only in the T site and is
released only in the O site. The energy dependent process
is the ATP release step which is accomplished by energy
dependent rotation of the
y
subunit.
