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XI-BioEnergetics
The principal energy conserving
activities of the cell are found in the
mitochondrion (see left). The
mitochondrion is a fairly large
organelle (about 1-2 umeter long). It
is a major component of most cells;
e.g. about 10% of the protein of the
liver cell is due to the ca 10 4
mitochondria that are present. It is
probable that these mitochondria are
connected to one-another; I have heard
the term string-bag used to describe the
network of interconnected
mitochondria. The mitochondrion
consists of four domains as illustrated
below. The 4 domains are characterized
by the following principal activities:
Outer membrane (may be continuous with the endoplasmic reticulum): Fatty acid thiokinases (synthesis of acyl
CoA esters). The surface is smooth and full of holes (see next page); molecules of upto 6000 Daltons can pass
through this membrane.
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Inter-membrane space: -adenylate kinase (myokinase) and nucleoside diphosphate kinase (nudiki).
ADP + XTP ⇔ ATP + XDP
Inner membrane: Highly convoluted, a device for
increasing the surface area (some 15x). Contains the
enzymes for electron transport and oxidative
phosphorylation (OP), and transport systems for
various acids and cations. The inner membrane is
about 75% protein and about 25% lipid. However
about two-thirds of the protein protrudes out of the
membrane so that the proportion of protein: lipid
within the membrane proper is about 1:1. This
membrane is seen to have circular "knobs" attached to
its inner surface-these knobs are part of the OP
enzyme.
Matrix space: contains the enzymes for the
tricarboxylic acid cycle and for the β oxidation of fatty
acids.
There are several experimental materials:
1) Intact mitochondria.
2) Mitochondria with the outer membrane removed (mitoblasts).
3) Fragments of the inner mitochondrial membrane that have resealed into intact vesicles except
that the "knobs" are now on the outside.
4) Detergent extracts of mitochondria that have been purified to yield the individual enzymes.
The classical picture of the electron transport chain (ETC) was of a functionally and structurally linear
system in which the individual components were located in the membrane in a fixed spatial relationship to one
another; such a linear scheme was deduced from the response of the ETC to inhibitors.
Inhibitors
Just as we saw in glycolysis experimental advances in this field have relied strongly upon inhibitors. Several
inhibitors and their sites of action are (see next page):
Piericidin - (a Q analog)
Rotenone
⇒ Between Complex I and Q
Seconal, Urethane, amytal
Carboxamides
Thenoyltrifluoracetone
⇒ Between Complex II and Q
Antimycin
myxathiazol
⇒ After the b cytochrome
⇒ Blocks oxidation of the Fe/S center
CN, CO, N3
⇒ Binds to a3 and blocks its redox reaction
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The response of the individual redox components to these inhibitors was traditionally interpreted in terms of
a linear arrangement of electron carriers, with a 1:1 stoichiometry for each of the 4 relevant enzymes (Fig 20-8 V&V).
this picture arose because of the response to inhibitors. If an inhibitor blocks electron transfer between two sites (as
indicated above for antimycin) then in the presence of an excess of electron donor e.g. NADH the electron carriers to
the left of the inhibitory site become reduced while those to the right become oxidized. One can use the optical
absorption of the cytochromes (Ch X) to define the site of inhibitor action; in this example the b cytochromes
become reduced while the c and a cytochromes are oxidized.
In fact even early analytical data contradicted this representation and a consideration of the amounts of the
various redox-active prosthetic groups present in the inner mitochondrial membrane suggested that, for liver, the
"elemental composition" is 1:2:3:7 for Complexes I, II, III and IV, respectively. This is clearly inconsistent with a
simple, linear formulation and consistent with "floating enzyme complexes" along the lines of the fluid-mosaic model
(see next page). So the picture that you should get is of a system that is functionally linear in that electrons flow
from "left" to "right" but which is structurally more of a mosaic with the enzyme complexes randomly distributed in
the membrane with relative abundances given by the above stoichiometry. Rapid communication between the
complexes is provided by coenzyme Q (I & II with III) and by cytochrome c (III with IV).
The proteins present in this membrane are not crowded. Even though the membrane is about 75% protein, for
the complexes shown in the cartoon on the next page only about 40% (III and IV) or 17% (V) is confined to the
membrane bilayer; the remainder sticks out into either the matrix space or intermembrane space, as shown (next
page). These three proteins account for 35% of the total membrane protein; note that all three are oriented with their
long axis perpendicular to the membrane plane and with 60-80% of their mass external to the membrane. Note also
that these complexes are free to move in the membrane plane. (Complexes I-IV all have diffusion constants of about
10-10 cm2sec-1 while diffusion constants for cytochrome c and UQ are at least a factor of 10 larger. The passage of 1
electron through the chain from NADH to oxygen takes about 10 msec; these complexes can move about 500 Å in
this time.)
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Replace this page with Mito Cartoon
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The important concept is that three of the protein complexes (I, III & IV) function to transport electrons
from their particular electron donor to their specific electron acceptor; this activity is obligatorily coupled to the
transport of protons from the matrix space of the mitochondrion to the inter-membrane space thus conserving, in
large part, the free energy associated with the reaction of “H”(NADH or QH2) with oxygen. Subsequently complex V
exploits this proton gradient to make ATP.
The remaining redox active complex (II) is not energy conserving but is left as part of the chain as it serves
to deliver electrons to III & IV that do conserve energy. We first briefly summarize the characteristics of complexes IIV.
Enzymatic Activities and Redox Composition of the Major Redox Complexes
NADH-Q Oxidoreductase (Complex I)
Catalytic activity:
NADH + UQ + 5H+ ⇔ NAD+ + UQH2 + 4H+ out
The isolated complex contains 41 polypeptides together with 1 FMN
(triple hexagon), 24 Fe and 24 S, and has a molecular mass of ca 900,000
Daltons. The number and type of iron-sulfur clusters (cubes and diamonds) in
Complex I is quite controversial. There at least 6 as deduced from features seen by
spectroscopy (EPR). If we assume that they are all 4Fe clusters we expect a
total of 6 while if they are all 2Fe clusters then there are 12; these must be the
lower and upper limits. The purified enzyme also contains about 3 coenzyme Q;
the significance of this is not known.
The potentials of most of the redox active components lie around –250
mV. The site of reaction with NADH is (almost) certainly the FMN; the site of
reaction with Q must be an iron-sulfur cluster but which one is not known. This
enzyme is the first site of energy conservation. The number of protons pumped is
shown in the balanced equation to be 4; this number is not universally accepted.
Succinate-Coenzyme Q Oxidoreductase (Complex II)
Catalytic activity:
succinate + UQ ⇔ fumarate + UQH2
This enzyme is composed of 4 subunits (A, B, C and D).
Subunits A and B contain the catalytic centers while C and D serve to
attach the enzyme to the membrane; they also appear to modify the
catalytic activity of A and B. The enzyme contains histidinyl FAD (triple
hexagon, see flavin lecture) plus 9 Fe and 9S =. The FAD is located in
subunit A. There are three iron centers, S1, S2 and S3. S1 (diamond) is a
2-iron cluster, S2 (cube) is a 4-Fe cluster while S3 (triangle) is a 3-Fe
cluster similar to that described for aconitase. S1, S2 and S3 are located
in subunit B.
The redox potential of the flavin and S1 are at about 0 mV, that
of S3 at 100 mV. The potential of S2 is much more negative (-200 mV)
than that of the succinate/fumarate redox couple (approx. 0 mV) which might suggest that it is not involved in
catalysis. However the x-ray structure of a related enzyme reveals an almost linear pathway FMN ⇒ S1 ⇒ S2 ⇒ S3
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⇒ UQ making it most likely that this center plays a role in electron transport. Again we believe that the flavin
reacts with the succinate and an iron-sulfur cluster with the Q. Note that there is no energy conservation with this
complex, which is really a component of the TCA cycle.
QH 2 -Cytochrome c Oxidoreductase (Complex III)
Catalytic activity:
QH2 + 2 c+3 + 2H+ in ⇔ Q + 2 c+2 + 4 H + out
This is a very complicated enzyme with between 9-11 different subunits depending
on species. In this complex there are four metal-based catalytic centers. The most
unusual is the b cytochrome(s) which contains two b hemes (bL and bH, diamonds)
in a single polypeptide of about 42 kDa; each b heme is coordinated by a pair of
histidines. This sub-unit (cartoon below) plays a central role in the function of the
enzyme; note the two hemes located between TM helices B and C. The c1
cytochrome (diamond) has his-met ligation similar to soluble cytochrome c (Ch. X)
and the heme is also attached to the polypeptide via thioether bonds. The iron-sulfur
center is unusual in that it is the only [2Fe2S] center with a very positive redox
potential. This cluster has two histidines (rather than two cys) attached to the iron
atom that accepts the electron.
There appears to be a single Q specifically bound to a special polypeptide called the Q-binding protein.
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Redox potentials.
Species
E o'
bH (a.k.a., bK, b 562, b N)
bL (a.k.a., bT, b566 , b P )
120 mV
-20 mV
Q/QH
QH/QH2
FeS
c1
200 mV
100 mV
270 mV
270 mV
This enzyme complex is particularly important because there are several diverse areas of catabolism that can
"feed" electrons into the electron transfer system (via the Q pool) and it is only because complex II is present in the
same membrane as the electron transport chain that it gets special attention. The various sources for Q reduction are
summarized in the next figure:
NADH
Thus from Dr. Rudolph you will learn
that the first step in each cycle of the
oxidative degradation of fatty acids consists
Fatty acid
of the dehydrogenation of adjacent
methylenes by the acyl-CoA
Complex III dehydrogenase. This flavoprotein transfers
Succinate
Q-pool
its electrons to an electron carrier called
ETF (electron transferring flavoprotein)
Branched amino
which passes them onto ETF
acids
dehydrogenase (a flavin containing ironsulfur-protein). This enzyme passes its
electrons directly to Q. This is the only
1-carbon
metabolic sequence that has 3
metabolism
flavoproteins acting consecutively (see Fig
23-10 Voet & Voet). Furthermore the oxidation of certain amino acids (those with branched chains) also pass
electrons to ETF dehydrogenase while the oxidation of compounds such as sarcosine and dimethylglycine is catalyzed
by enzymes somewhat similar to succinic dehydrogenase.
The presumed mechanism of this enzyme will be in the section on oxidative phosphorylation later in this
chapter.
Cytochrome c.
The only water soluble protein in the chain; MW =12 kDa, Eo' = 270 mV. See the ribbon drawing in
Ch. X, p4. It is not catalytically active in the usual sense; rather it functions as an electron acceptor from complex
III and an electron donor to complex IV. This protein wanders over the outer surface of the inner membrane and also
through the space between the two membranes (i.e. it is not confined to the surface of the membrane). The attachment
of the heme to the protein is similar to that of cytochrome c1.
Cytochrome c-O 2 Oxidoreductase (Complex IV, cytochrome oxidase)
Catalytic Activity:
4 c+2 + O2 + 8H+ in
⇔
4 c +3 + 2H2O + 4H + out
There are 13 subunits; only the three largest (I, II and II) appear to have any catalytic activity.
XI-7
There are two heme a and three copper
ions. These seem to be organized
functionally as heme-copper pairs (see
cartoon). Cytochrome a and CuA are not
CuA
structurally linked to one another but are
referred to as a pair because they operate
together (i.e. they are functionally linked);
Mg
Cu A is the site where electrons enter the
enzyme from cytochrome c while
cytochrome a carries electrons from CuA to
the second pair-where catalysis occurs.
CuB
Cu A (Ch X) is present in subunit II. The
cyt. a
remaining redox centers are present in
subunit I; recall from Ch. IX that this
cyt. a3
subunit has 12 transmembrane helices.
Cytochrome a is a heme axially
coordinated by two histidine residues and
appears to be a typical cytochrome (apart
from containing heme a); cytochrome a
receives electrons from CuA. The second
heme and copper centers, cytochrome a3
Zn
and CuB are structurally linked and are
present as a binuclear metal center as
discussed in Ch.X; cytochrome a is also
structurally linked to cytochrome a3 via
helix X as shown on the next page.
Cytochrome a3 and CuB function as the
catalytic site for reaction with oxygen and are thus both structurally and functionally linked to one another. It is
currently believed that they are also the machine that pumps protons across the membrane in this enzyme.
The following 2 drawings shows the topological relationship between the metal ions with distances in Å;
the arrows show the path of electron transfer.
2.5
19
13
XI-8
5
Top view onto the membrane surface showing the arrangements of the two hemes (shaded rectangles), CuB (circle)
and several helices (in this presentation adjacent amino acids are approximately 1/4 turn displaced from one-another)).
The redox potentials of the four metal centers are all close to 300 mV.
The Mechanism of Action can be separated into two parts as shown in the figures on pages 11 and 12.
Conceptually the first events are the delivery of 4 electrons (e) to the enzyme. This must occur as a result of 4
consecutive collisions with molecules of reduced cytochrome c each collision resulting in the transfer of an electron
to the enzyme. Once this has been accomplished the 4-e reduced form of the enzyme is ready to react with oxygen.
Part 1: The enzyme (CcO) reacts consecutively with four molecules of cytochrome c+2 (above). In each
reaction an electron is transferred to CcO entering via CuA. Before CuA can receive a further electron it must transfer
the present electron to cytochrome a. The first two electrons accumulate on cytochrome a and CuA. When Cu A and
cytochrome a are reduced the pair of electrons are transferred to the binuclear center:
{a+2 Cu A+1 }{a3+3 Cu B+2 } ⇔ {a+3 Cu A+2 }{a3+2 Cu B+1 }
The next two electrons can then be added to {a+3 Cu A+2 } so that all four metal centers are reduced.
Part 2: At this point the enzyme is ready to react with O2 (R in p. XI-12). Oxygen first binds to CuB and
then migrates to the Fe of a3+2 (and binds as in oxyhemoglobin, A, p. XI-12).
XI-9
The oxygen then undergoes a 4-electron reduction which leads to the cleavage of the O-O bond (P, p XI-12). This is
accomplished by electrons being provided by CuB (1 electron), cytochrome a3 (2 electrons) and cytochrome a (1
+4
+2
electron). with a, a3 and CuB all oxidized {a+3, a 3 , Cu B }. Both two oxygen atoms are at the level of water (O=).
One oxygen atom is bound to CuB as the mono-protonated species (i.e. OH-). The other oxygen atom is bound to a3
as the fully deprotonated species; this oxy-iron compound is usually written as Fe(IV)=O and called the oxy-ferryl
species
Finally CuA+1 donates its last electron to restore a3 to the Fe(III) state and the oxide atom gains a proton to
become OH- (H, Fig XI-12) At which point the two water species are released is unclear though each must capture an
additional protons as they leave the enzyme as H2O (O, p XI-12).. These 4 protons are called the scalar protons
because they are required by the balanced chemical reaction. I prefer to call them the chemical protons.
SOMEWHERE and SOMEHOW during this process an additional 4 protons are translocated across the
membrane and produce a net acidification of the outer space. These 4 protons are called the vectorial protons; I
prefer to call these the pumped protons. Current opinion is that two protons are translocated upon formation of the
oxy-ferryl state (Fe=O) and the additional two protons are translocated when the oxyferryl species is converted to the
final re-oxidized enzyme.
XI-10
XI-11
e
E
FeIII
FeII
CuI
FeIII
R
O2
e
O
CuI
CuII
FeII-O2 CuI
A
2 H2O
e ,H+
2 H+
H FeIII-OH HO-CuII
e , H+
XI-12
XI-12
FeIV=O HO-CuII
P
OXIDATIVE PHOSPHORYLATION.
Oxidative phosphorylation is the process whereby ATP synthesis is coupled to the functioning of the
electron transfer chain. It is responsible for the production of > 95% of the energy used in metabolism. The tight
relationship between electron transport (ET) and oxidative phosphorylation (OP) is nicely demonstrated by the
phenomenon called RESPIRATORY CONTROL (next figure).
When carefully prepared mitochondria are incubated with an oxidizable substrate (e.g. malate) no oxygen is
consumed unless both ADP and Pi are present in the incubation mixture. As Pi is usually present as the buffer the
process can be controlled by addition of ADP.
+ malate (Why not NADH)
+ ADP (e.g. 3 umoles)
Conc of O
2
in solution
0.5 umoles
O consumed.
2
uncoupled
TIME
We define a quantity called the respiratory control ratio (RCR) as
RCR =
Rate of Oxygen consumption in the presence of ADP
Rate of Oxygen consumption in the absence of ADP
This value can be as large as 15. With damaged mitochondria the full activity is obtained without addition
of ADP-see the broken line above where full electron transfer is observed before ADP addition.
Intact mitochondria are said to be tightly-coupled, damaged mitochondria are uncoupled. Tightly-coupled
mitochondria operating with and without ADP are said to be in State 3 and State 4 respectively, and the
consequences of adding ADP is called the state 4 ⇒ 3 transition.
The interpretation of respiratory control is that electron transport is obligatorily coupled to ATP synthesis
(like meshed gears). If this synthesis cannot proceed because of the absence of ADP then electron transport is
inhibited. Aging or chemical or physical abuse breaks the coupling between the two enzymatic systems so that ET
can proceed in the absence of OP.
We define a second ratio, the P/O ratio, that expresses how much ATP is made for every pair of electrons
passing along the electron transport chain; 1 pair of electrons reduces 1 atom of oxygen to water1.
1
The following relationships are useful at this point: Oxidation of an organic substrate is equivalent to oxidation of
NADH is equivalent to 2 electrons being delivered to the electron transport chain. Two electrons reduce 1 oxygen
XI-13
P = umoles of ADP added
⇒
O
uatoms of oxygen consumed
(assumed equal to umoles of ATP synthesized)
The P/O ratio can be obtained from the respiratory control graph where the addition of x umoles of ADP leads to the
consumption of y µmoles of dioxygen or 2y µatoms of oxygen. In this case the P/O ratio would be x/2y. With
NADH (or NADH generating substrates such as malate) the P/O ratio is 3; with succinate it is 2.
What do these numbers mean? The classical view is that there are 3 sites of phosphorylation in the chain
and the passage of a pair of electrons through a particular site leads to the synthesis of an ATP. Two simple ways of
identifying these sites are:
A. From the standard redox potentials of the components of the chain we find that there are 3 locations
where the free energy changes considerably. As ∆Go' for ATP hydrolysis is -7.6 kcal/mole it requires a voltage gap
of ∆Go'/F = 7.6/23 = 0.3 volts to provide enough energy to make ATP. The 3 locations are
[1] Between NADH and Q .. (but not succinate and Q).
[2] Between cytochromes b and c
[3] Between cytochrome a and oxygen ( oxygen -> water = 800 mV/electron)
These assignments suggest that ATP synthesis is associated with complexes I, III and IV.
B. Confirmation of these assignments can be obtained by clever use of substrates
SUBSTRATE
Malate
⇒
Succinate
⇒
Succinate
⇒
Cyt. c
⇒
P/O
3
2 (Therefore complex I, but not II)
1 (therefore III)
1 (therefore IV)
Oxygen
Oxygen
Cyt c (anaerobically)
Oxygen
The deductions from these data agree with the thermodynamic results.
Mitchell, in advocating the chemi-osmotic theory, proposed that the passage of electrons from the organic
substrate (e.g. malate) to oxygen that proceeds in the plane of the membrane results in the translocation of protons
from the matrix space to the intermembrane space. He further postulated that this was accomplished by a special
arrangement of the electron carriers: he called this special arrangement REDOX LOOPS. The motion of the electrons
from one side of the membrane to the other as they moved along the electron transport chain would drive protons
across the membrane and generate the proton-motive force (see Fig 20-24 V&V). We have earlier (Ch. IV and IX)
dealt with the several components of the chemiosmotic hypothesis.
To place this idea in a more tangible form we can consider the most highly developed scheme for proton
translocation. The Q-cycle was developed to explain proton translocation in complex III (the properties of complex III
was described earlier in this chapter: it is shown schematically in the following figure). This scheme is rather
unusual and was developed to rationalize a variety of kinetic data and also the startling result that the passage of a
single electron through complex III leads to the appearance of 2 protons outside the mitochondrion. This
stoichiometry of 2 protons/electron is unusual; we usually expect either 0 (from metal based electron transfer) or 1
proton (from organic chemistry) to accompany each electron.
Complex III contains three active centers. The first is cytochrome c1 which is the site at which electrons
leave the complex, by being transferred to cytochrome c. The second catalytic center is called the quinol oxidase site
or center "o" (more recently it has been called center P (for positive). In some systems protons are actually moved
into an organelle; thus positive and negative give a more consistent notation; the quinol oxidase can be “outside” as
in mammals or “inside” as in plant chloroplasts). Center "o" is comprised of the FeS center and cytochrome b L. The
atom to water. A µmole of dioxygen (the molecule) = 2 µatoms of oxygen (the atom) = 4 µequivalents of electrons.
(When working with electrons we talk about equivalents; an equivalent is the electrochemical equal to the mole).
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third center is a quinol reductase site or center "i" (for inside, more recently center N, for negative). It is comprised of
cytochrome bH and a special quinone (shown as Q6 in the scheme) which is not part of the Q pool.
The scenario is as follows :
a) One QH2 (QpH2) from the Q-pool is oxidized at center "o" with one electron delivered to the FeS cluster,
the second electron to cytochrome bL and both protons departing to the inter–membrane space. The electron
on b L is immediately moved to b H while that on the FeS is equilibrated with c1 and can leave the enzyme
by virtue of the reaction of c1 with c. The oxidized Q vacates center “o”.
b) A second QpH2 now enters center “o” and reacts in precisely the same way. Thus 2 electrons and 4
protons have moved to the inter membrane space. The two electrons moved back across the membrane
accumulate on Q6 to yield Q 6H2 with the two protons being captured from the matrix space.
c) A Q (Qp) from the Q-pool now reacts with Q 6H2 to form QpH2 and regenerate Q6.
As a result a net of two electrons are transferred from the Q-pool to cytochrome c, four protons appear in the
inter-membrane space and 2 protons disappear form the matrix space.
From inspection of the redox potentials of the redox centers you would reasonably expect that the second
electron in step a above should also exit via the Fe/S center and cytochrome c1. However x-ray data show that the
Fe/S center can occupy two locations. When oxidized it is close to bL; however upon reduction the segment of
polypeptide to which it is attached moves such that the reduced Fe/S center is brought close to cytochrome c1. It is
this structural rearrangement that is the origin of this anomalous behavior.
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The unusual kinetic result that this scheme explains is as follows (Examine the scheme)
1) In the presence of the inhibitor antimycin (which blocks reaction at center "i") cytochrome b can be
reduced by QpH2 (via center o).
2) When the iron-sulfur protein is inactivated cytochrome b can still be reduced by QpH2 (via center i). The
iron-sulfur protein is most cleanly inactivated by reduction (in the absence of c), but it can also be
selectively destroyed by chemical agents or by inhibitors such as myxothiazol.
3) When the iron-sulfur protein is inactivated AND antimycin is present cytochrome b CANNOT be reduced
by QpH2.
Synthesis of ATP
The utilization of the proton gradient for the synthesis of ATP is accomplished by Complex V, the protontranslocating ATPase, more accurately the ATP synthetase, popularly known as FoF 1. This has the following
general structure: (V&V, Fig 20-30)
Fo (eff-oh) is an integral protein (subunit composition a1b2c9)
that
appears
to be a proton conductor (vesicles of phosphatidyl
12
choline with Fo incorporated into the vesicle membrane can conduct
protons). F1 is readily detached from the mitochondrial membrane and
the soluble F1 is an effective ATPase. F1 has the composition
(αβ)3γδε. Each αβ pair makes up a catalytic unit. Each of the 6
interfaces between α and β contain a nucleotide binding site; the sites
which are largely contributed by β is the catalytic site (C) while those
that are mainly α is a regulatory site (R). The γ subunit looks like a
stalk and is believed to connect F1 to Fo; it is also called OSCP
(Oligomycin sensitivity conferring protein; it confers sensitivity to the
antibiotic oligomycin upon Fo. This antibiotic blocks ATP synthesis).
Note that the cartoon shows protons moving out (down); this is the
direction for the case when this enzyme hydrolyzes ATP and ejects
protons. We are currently concerned with the reverse process.
The ATP synthesis activity is only found with F1 attached to Fo in the
membrane. ATP is always synthesized on the side that bears F 1; in
mitochondria this is the matrix space.
This striking structure gives considerable support to current ideas on the mechanism of the enzyme –Boyer's
alternating sites mechanism – that has also received strong support from some mechanistic studies with the soluble
F 1. Boyer and John Walker (who determined the x-ray structure of F1) received the Nobel prize in chemistry in 1997.
The salient features of this mechanism are:
1) At low [ATP] ATP is bound very tightly to FoF1. At higher [ATP] the binding is very much less tight.
It is thus proposed that membrane energization is required for ATP release.
2) The interconversion of ADP + Pi and ATP that occurs in the C active center of F1 has an equilibrium
constant of 1, i.e. ∆G is 0, ATP synthesis occurs spontaneously.
The Boyer scheme uses three active centers, the C sites in the above figure (e.g. V&V Fig 20-31), that can
exist in three mutually exclusive conformations, called L(oose), O(pen) and T(ight) The existence of these 3 sites is
verified by the x-ray structure of F1. L can bind ADP + Pi weakly, O has low affinity for ATP while T is a closed
conformation in which ADP + Pi are in equilibrium with ATP.
XI-16
The enzyme (see cartoon below) always has 1 catalytic subunit in each of the three conformational states.
We start with a newly synthesized ATP in T. First the enzyme binds ADP + P to L. Then the enzyme is switched
between its 3 conformational states by the passage of three protons through Fo; this causes a series of conformational
changes whereby T ⇒ O, L ⇒ T, and O ⇒ L. ATP dissociates from O and the ADP and Pi in T are condensed to
yield ATP (the equilibrium constant ~ 1). The T sites appear to have an arrangement of charges such that cationic
amino acids assist in the removal of the hydroxide and anionic amino acids in the removal of the proton-thus effecting
the dehydration. We are now ready for another cycle (reiterate this paragraph).
ADP + Pi
Energy
ATP + H2O
Note that the arrow at the top left is the same as that at bottom right i.e. you should view the above sequence as
representing a closed figure.
Thus the proton-motive force is used to convert the loose binding of ADP and P to tight binding, and the tight
binding of ATP to loose binding.
There is direct experimental evidence for both the formation of proton gradients as a result of electron transfer
and for the ability of proton gradients to cause ATP synthesis The classical experiment was performed with the
photosynthetic electron transport system by Jagendorf. He incubated fragments of chloroplasts at pH 4 to render the
inside of the chloroplast acid (chloroplasts have their inside relatively acid and are thus inverted relative to
mitochondria). The chloroplast were then rapidly transferred to a neutral, pH 7, buffer thus creating a proton
gradient with the outside alkaline relative to the inside. When ADP was also present in the outside buffer ATP was
synthesized.
Proton translocation coupled to electron transport is also well established. It was originally demonstrated
with whole mitochondria and with mitoblasts (mitochondria from which the outer membrane has been removed by
gentle chemical treatment). More recently the translocation has also been shown directly with both complex III and
complex IV. In these experiments the purified complex (either III or IV, as appropriate) was mixed with a pure
phospholipid which was then introduced into an aqueous buffer. The lipid vesicles that form spontaneously contain
some of the enzyme, typically one enzyme molecule per vesicle. These vesicles are incubated with a measured amount
of electron donor (i.e. substrate) together with an excess of a suitable electron acceptor and the pH of the external
medium measured. From the decrease in pH the amount of protons produced can be calculated while the quantity of
substrate added controls the number of electrons that passed through the enzyme. Typical results are:
For Complex III: 1 µmole of quinol was used as substrate so that 2 µequivalents of electrons were added.
4 µatoms of H + were produced. Thus the H+ :e ratio is 2:1.
For Complex IV: 1 µmole of reduced cytochrome c was used as substrate, thus 1 µequivalent of electrons
passes through the enzyme. 1 µatoms of H + were produced in the medium. Thus the H+ :e ratio is 1:1. These are the
vectorial protons. Remember that with complex IV a total of 8 protons disappear from the matrix 4 being used in the
XI-17
formation of water. The matrix space gets more alkaline due to the loss of 8H+ while the extra-mitochondrial space
gets more acid because of the appearance of 4H+.
The 2 schemes on the following pages are simply summaries of the information covered in this chapter and
you should use them as devices for organizing your thoughts. The first scheme summarizes the proton count and
charge (q) translocation occurring within each enzyme complex; the second scheme quantifies the proton count
associated with the synthesis of an ATP.
Proton Inventory for NAD-linked substrates
IN
OUT
AH2
2e
via NAD+
A + 2H+
4 H+
6 H+
Complex I
H+/2e = 4; q/2e = 4
QH
2
4 H+
H+/2e = 4; q/2e = 2
Q
Complex III
(Q cycle)
2 H+
Complex IV
2 H+
2e
c
2e
2 H+
H+/2e = 2H+/2e = 4
2e
[O] + 2H+
H2O
Overall: H+/2e = 10; q/2e = 10
q = charge
XI-18
Complexes III and IV, taken together, expel 3H+ :e. This is usually quoted as 6H+ :O (protons per oxygen
atom-it takes two electrons to reduce the oxygen atom to water).
When these experiments are done using succinate and whole mitochondria similar values are obtained. With
NADH the ratio is higher, typically 10 H+ :O, implying that 4 H+ are expelled as electrons pass through complex I.
It is believed that 4 protons are consumed in the synthesis of ATP; 3 of these protons are utilized to drive
the chemical reaction as noted above; the fourth proton is utilized by the transport system that moves Pi back across
the inner membrane:
H+ (out) + H2PO4= (out) ⇒ H + (in) + H2PO4= (in) (symport).
The synthesized ATP is moved out to the cytoplasm by the ADP-ATP translocator (antiport):
In-Out:
ADPout + ATPin ⇔ ADPin + ATPout.
11+
The process is electrogenic because ADP is 3- and ATP is 4 -, which leads to an increase in negative charge out of
the matrix. The exchange is driven by ∆φ (positive out).
XI-19
Proton Inventory: ATP Synthesis.
IN
OUT
4 H+
Redox
Proton
Pumps
3 H+
ATP
Synthase
H+
H2PO4-
Phosphate
Carrier
H+/ATP=3; q/ATP=3
H+/ATP=1; q/ATP=0
:
Cytoplasmic
ATP use
ADP 3 ATP 4 -
Adenine
Nucleotide
Carrier
4 H+
3 H+
H+
H2PO4-
ATP 4 -
ADP 3 ATP 4 -
H+/ATP=0; q/ATP=1
Overall: H +/ATP=4; q/ATP=4
The mitochondrial inner membrane contains a rich assortment of transport systems; these were summarized at the end
of the section on transport (Ch 9). You should also be familiar with the systems required to move
citrate and malate (Ch. 8) and acyl carnitine (Dr. Rudolph's lectures).
XI-20