Electron Transport Chain and OxPhos

The Electron Transport Chain and Oxidative Phosphorylation
The Electron Transport Chain: reductio ad energum- So far, we have not gotten much ATP out of all this
cycle turning and oxidizing and old-and-new acetating. Where is the payoff? Earlier, when we discussed the
complete oxidation of sugars and related molecules, we denoted that process by starting with the molecule to be
oxidized and O2, and ending up with nothing but CO2 and water. In contrast, you may have noticed in the above
description that the oxidation of the Krebs cycle molecules does not go “all the way” to CO2 and H20. For each
acetate added, 2 CO2 molecules are produced, but no O2 is converted into H2O. In fact, although the Krebs cycle
causes oxidation of its substrates as they go around the cycle, no oxygen is involved or required for the Krebs
cycle. (This is a popular “trick question” on tests. Be tricked no more!) Instead, the reducing equivalents that are
produced are held in the molecules NADH, and FADH2. These will then be used to reduce oxygen to water,
getting oxidized back to NAD+ and FAD in the process, restoring the electron carriers. So one way to look at it is
that the carriers are being reduced during the Krebs cycle, allowing the production of CO2, and then being
oxidized later, to produce water by reducing O2. This “separation” of the complete oxidation of acetate groups
into two phases (production of CO2 and reduced carriers, followed by production of H2O and oxidized carriers)
serves a very important purpose. The oxidation of the FADH2 and NADH carriers back to their “unloaded”
(oxidized) forms is part of a process that is responsible for most of the ATP energy that we derive from glucose
and other fuels we consume and break down. The set of reactions that carry the electrons from carriers like
NADH and finally to O2 is called the electron transport chain (ETC), or the respiratory chain. The function of
this process is to use the chemical energy released during the reoxidation of the carriers and the reduction of O2, to
make ATP from ADP and Pi. The production of ATP driven by these redox reactions is called oxidative
phosphorylation (sometimes OxPhos for short). The way this happens is a little cell biological miracle. So much
so that it took a paradigm-breaking change in thought to get to the correct answer. So first, let’s look at the wrong
answer. It is not a fruitless exercise, and the “coming around” of the community from the commonly-held
incorrect answer to the unexpected, and at one time unvoiced, correct answer is a beautiful example of the power
of consensus-driven hypothesis testing as the most reliable way to arrive at physical truth, even when that truth is
unexpected, surprising, or even revolutionary.
Energetic Expectations Dashed!- The history of the endeavor to figure out how ATP is produced in the late
oxidation steps of glucose metabolism (that is, the conversion of pyruvate into CO2 and H2O with the concomitant
generation of ATP) is worth talking about for a minute, because it shows an important, general and beautiful
aspect of experimental science as we practice it. The basic principle is that your hypothesis can be wrong, so long
as you do the right things to test it.
The reactions of glycolysis were worked out years before the oxidative production of ATP in mitochondria was
figured out. So the detailed and accurate knowledge gleaned from glycolysis reasonably led to mechanistic
expectations about how ATP would be generated after glycolysis. In simplest terms, glycolysis produces ATP
through what is called substrate-level phosphorylation. Although this sounds fancy, it is a pretty straightforward
idea. A phosphorylated compound with a greater energy of hydrolysis is used to run the reverse reaction of ATP
hydrolysis, that is, ATP synthesis from ADP and Pi. (You should convince yourself that the transfer of phosphate
from such a high energy compound to ADP can be represented as the chemically balanced combination of two
reactions: the phosphate donor being hydrolyzed, and the hydrolysis of ATP being run in reverse. ATP generated
in this manner is called substrate-level phosphorylation because the phosphorylated compound that drives the
reaction with its free energy of hydrolysis will be a substrate of the enzyme that allows transfer of the donor
phosphate onto ADP to produce the desire ATP. It is the intrinsic chemistry of the phosphate donating substrate
that allows the production of ATP from ADP to occur spontaneously. If you look at the reactions of glycolysis
through the “lens” of substrate level phosphorylation, you will see that the whole show is about the clever
generation of two such high energy phosphorylated compounds, 1,3bPG, and PEP, each of which is used to
directly phosphorylate ADP to make ATP. Voila, substrate level phosphorylation! See? below is the 1,3bPG
reaction, just for review.
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So when people were trying to figure out
how the oxidation of pyruvate to CO2
caused the production of ATP by running
the ATP hydrolysis reaction in reverse, it
was totally reasonable to expect that
nature would use the same trick as it had
in glycolysis, that is, by substrate level phosphorylation. Specifically, the field was reasonably and completely
dominated by the thought that somewhere in the chemical reactions that took acetate to CO2, and oxidized O2 to
H2O, the resulting free energy would be used to make a cranked up phosphorylated compound (or several of
them) that would, like 1,3,bPG in the example, react with ADP to make ATP in an energetically favorable
manner. The hypothesis went something like: “in the course of the oxidation-reduction reactions that convert
pyruvate into CO2 and O2 into H20, high energy phosphorylated molecules are produced that drive the production
of ATP from ADP by substrate level phoshporylation”. If you look at the old papers or even the textbooks on this
subject in the mid sixties, they all would draw graphical models that included an unknown intermediate X~P that
would serve as the high energy phosphate donor in the production of ATP caused during these late oxidative
steps. The term oxidative phosphorylation is a common shorthand to describe this coupling. And the simplest
model, which was understandably (mis)informed by the successful unraveling of glycolysis, included substrate
level phosphorylation as the final step in ATP production. And not surprisingly, both the Beatles and the Rolling
Stones (both first big in the sixties) also espoused the substrate level phosphorylation model in their early work.
A cell biological solution to a biochemical problem- The actual answer to how the oxidation of acetate (or
pyruvate) causes ATP production was totally different from the substrate level phosphorylation model proposed
and tested for many years during work on OxPhos (which is how many people refer to oxidative
phosphorylation). The solution to the problem dragged researchers who were hard core biochemists into the
worlds of cell biology and biophysics. What do I mean by that? Biochemists, especially back in those days but
even now, work on biochemical reactions; how molecules are made, broken down, and interact with larger
systems in the cell to make life happen. They are often interested in the enzymes that make such things happen at
an acceptable rate, and because there are many, many sorts of enzymes, there are many, many scientific endeavors
where understanding the biochemical aspects or a process is important and fruitful. Cell biologists focus on how
cellular processes occur and many times their work involves or includes thinking about the structure and
dynamics of the membrane compartments that define the cell as we know it. This division is a bit arbitrary, since
all enzymes are contained in cells, and the dynamics, construction and function of membranes involves many
enzymatic processes. Nevertheless, there are definitely people who are more “biochemical” in their research, and
others who are more “cell biological” in their approaches. This is because successful science requires lots of focus
and lots of expertise in particular approaches. The degree to which this is the case can be seen in the gatherings
that scientists go to: Each year the American Society of Biochemisty and Molecular Biology (ASBMB) draws
thousands of people who are doing things by more biochemical approaches; in the same year, the annual
American Society of Cell Biology (ASCB) meeting attracts thousands of cell biologist. Not surprisingly, many
people go to both meetings. Rules of thumb, and not absolute rules, govern this view of science.
Anyway, it turns out that the way ATP is made from the oxidation of acetate into CO2 and H2O is a process that
intimately involves cell biology, a process that depends on the the properties and existence of membrane
compartments. It is through the use of membrane compartments that the energy for ATP production is collected
and harnessed. Instead of the free energy for ATP production being stored in some X~P high energy molecule, it
turns out that an electrochemical gradient, that is an imbalance of H+ ions and charge across a membrane is
created and used to drive the production of ATP. So there is no X~P, and there never was one involved in the the
redox reactions that produce H2O by reoxidizing the electron carriers (NADH, FADH2) generated by the Krebs
cycle. You can imagine that if a large group of people who are very good at what they do, who are, in fact, the
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world’s experts in the biochemistry of ATP production all strongly believed that some kind of substrate-level
intermediate X~P was being generated in oxidative phosphorylation, then it would have been very hard to change
that thinking without some serious counter evidence. It is important that you the student realize that when we say
“believe” here, we are talking about the most informed guess or hypothesis derived from experimentally derived
information. The fact that substrate level phosphorylation is the way that glycolysis converts ADP back into ATP
created the totally reasonable and very heartily embraced idea, or belief, that this same approach was happening in
OxPhos.
Don’t make a paradigm you can’t break- The entrenched, communal thinking that a phosphorylated
intermediate (X~P), that functions like 1,3bPG or PEP, was being produced to drive the mitochondrial synthesis
of ATP during oxidation of acetate and production of H2O drove a lot of excellent biochemistry. Gradually, the
results of these beautiful and still-valid studies caused the growing concern that the substrate level
phosphorlyation model was not describing how oxidative production of ATP was occurring. The previous
substrate-level phosphorylation model that came from understanding glycolysis, and was reasonably expected to
also be true in mitochondrial oxidative phosphorylation, was what some philosophers of science would call a
paradigm, that is, a view or model of a part of the world (in this case the mechanism of oxidative ATP production)
*. Before any more progress could be made on this key problem, someone had to propose a new paradigm, that is,
a new view or model to explain all the observations that didn’t sync with the old substrate-level phosphorylation
view. In other words, the old paradigm had to be broken, or shifted to the new view. These terms, paradigm shift
and paradigm break, are used in many cultural contexts, and there are whole books written about the important
role of paradigm shifts and paradigm breaking in scientific progress. The most commonly mentioned one is
Thomas Kuhn’s famous “The Structure of Scientific Revolutions”.
*A set of assumptions, concepts, values, and practices that constitutes a way of viewing reality for the community
that shares them, especially in an intellectual discipline.
Mitochondria: giving redox reactions space- Both the Krebs cycle and the ETC (and a lot of other processes)
occur deep inside the mitochondrion. Many of you have seen pictures of mitochondria, and they are usually draw
to look sort of like a kidney bean (or just a kidney come to think of it) or a fancy SoCal swiming pool; sort of an
ovoid shape. This is a useful way to introduce the mitochondrion here, and we will use the same graphical
convention. But in fact mitochondria are highly dynamic structures that fuse with each other, break into smaller
units, and interact with all kinds of cellular parts in the course of their biology. But for now, we will join the
legions of kidney shape depicters that have gone before us. The basic feature of the mitochondrion is that it is a
closed membranous compartment, with contents that must be imported and exported. The biochemistry going on
within the mitochondrion is separated from that going on in the cytoplasm, with lots of communication caused by
regulation of the import and export of molecules. This is our first introduction to cellular compartmentalization of
metabolism, but it will keep coming up again and again.
Mitochondria are membrane-bound organelles, with two separate membranes, an inner one and an outer one. In
this sense they are like the gram-negative bacteria from which mitochondria are thought to have evolved, after a
probably very rare entry event of one of these bacteria into the cytosol of some pre-mitochondrial eukaryotes.
Somehow the unwanted guest gradually became an essential part of
the eukaryotic cell, allowing so much of what we are today (). The
outer membrane is called… wait for it… the outer membrane (OM)
and the inner membrane is called the inner membrane (IM). The
space between the inner and outer membrane is called the
intermembrane space (IMS), and the interior region enclosed by the
outer membrane is called the matrix. The inner membrane is highly
folded to promote surface area, and the folds are called cristae. The
diagram depicts these sections in our kidney- or swimming poolshaped version of the organelle.
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Similar to the Gram negative bacteria from whence mitos came, the two membranes are very different in function
and composition. The outer membrane has fairly large protein pores, such that metabolites (like ATP, pyruvate,
ions, fatty acids, etc.) can get right through. Proteins are restricted from exit or entry, so the IMS has a defined
and controlled protein composition, but communicates with the cytoplasm in terms of metabolites and other small
molecules. The inner membrane (IM) is a membrane of a different color. This membrane is highly impermeable
to even small things like ions, H+, ATP, etc. All movement of things occurs via membrane bound transporters or
fancy molecular turnstiles that are embedded in the inner membrane. As you will see, it is this highly
impermeable nature that allows mitochondria to extract the majority of energy during the reduction of O2 to H2O.
We will also see later that the highly impermeable nature of the mitochondrial inner membrane requires some
pretty fancy molecular tricks to get molecules into and out of the matrix where a lot of biochemistry occurs.
Whole lotta oxidation goin’ on- The inner membrane of the mitochondrion is the site of the enzymes that
catalyse oxidation metabolism and ATP production in oxidative phosphorylation. Our primary focus for now will
be the movement of electrons captured during the Krebs cycle (in the form of FADH2 and NADH) along the
electron transport chain to their final resting spot in H2O. But this set of enzymes is employed to extract energy
from a variety of oxidation reactions, funneling the resulting electrons into a common and useful fate. So learning
the details of the electron transport chain will open many metabolic doors, and allow you to understand many of
the varied biochemical functions of this now-essential part of the eukaryotic cell.
Dr. Mitchell, we presume…To get the field of bioenergetics out of the substrate level phosphorylation rut, it
took someone to come forward and break the old paradigm, someone to look at the evidence and put together a
radical new idea for how ATP production was being driven. The person most directly responsible for that change
was named Peter Mitchell. In truth, these things often involve lots of people all talking, combating, interacting,
drinking coffee in the morning, drinking beer after hours and at scientific meetings, writing
letters (emails and tweets now (#substratelevelbs; #oxphosisboss) and generally trying to
figure out what the heck is going on (there are exceptions, like some of the things Albert
Einstein cooked up). And Dr. Mitchell definitely considered a large number of studies to
formulate his new model. But it is safe to say he went way out on a limb with his new idea,
in the context of the dominant thinking, the dominant paradigm, of that time. He published a
seminal paper in 1961 suggesting an entirely new, and it turns out correct, model of
oxidative phosphorylation that proposed that during the course of production of H2O from
the electrons carried by NADH and FADH2, an electrochemical gradient was produced
across the inner membrane of the mitochondrion, and THIS was the way that energy was
collected from these reactions to be used for regeneration of ATP from ADP and Pi. Radical! No, wait, Ion! Not
surprisingly, at first there were numerous people who were quite skeptical and critical. This is reasonable, because
science if full of great yet wrong ideas. But the Mitchell model made many predictions that were all testable, and
soon tested, and it re-energized and correctly oriented the field. For this Peter Mitchell was eventually awarded
the 1978 Nobel prize. So here is another principle that we can add to our one above. So our new, expanded
science adage is:
Your hypothesis can be wrong, so long as you do the right things to test…but if your new and radical hypothesis
is right, you can get very famous, and it is a lot more fun.
Don’t worry, there are still enzymes involved- Although the quotes above have a silly aspect, there is real truth
to the idea that correct testing of the wrong hypothesis can and will produce much good science. Correctly done
science frees us from the dangers of our unbridled imaginations or our false hopes leading us astray. But it can
take a while. The study of the electron transport chain (ETC) is a good example of this “hope-proofing” in action.
What I mean is that although the many excellent scientists working on the enzymes of the ETC never found the
desired X~P high energy substrate, their unsuccessful search still revealed a huge amount of critical and key
information about how oxidative phosphorylation worked. In fact, one could argue that not believing a
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membrane-oriented, cell biological process was operating helped the field. I say this because the incorrect thought
that highly purified enzymes would make it easier to observe the production of the hypothesized X~P
intermediate led to a deep and very important understanding of the enzymes that actually do catalyze electron
transport and do cause the production of the unanticipated and key transmembrane electrochemical gradient that
supplies the energy for ATP production. So lets explore these enzymatic reactions:
The enzymes of the ETC- The basic reactions of oxidative phosphorylation start with… ready.. oxidation. At the
end of the Krebs cycle, we have NADH, and FADH2, holding the electrons that have been pulled off of Krebs
cycle molecules during their oxidation. The net chemical reaction for the Krebs cycle (pay most attention to the
reducing equivalents NADH and FADH2 that are produced) is:
+
3NAD + FAD + GDP + Pi + AcSCoA + 2H2O
+
3NADH + FADH2 + GTP + CoSH + 3H + 2CO2
And as you would expect, during the ETC, the NAD and FAD are regenerated and head back to the Krebs cycle
for more action. In addition, O2 is reduced to H2O. But this is done in gradual steps, little by little. In this way,
small units of chemical energy are used to create the H+ gradient we talked about above. This strategy allows the
maximum “bang for the thermodynamic buck” and is the reason that oxidative metabolism is much more efficient
in terms of captured and usable energy than an internal combustion engine. This gradual movement of electrons
from carriers such as NADH to oxygen is accomplished by the action of 4 separate protein complexes that
catalyze individual redox reactions, in which intermediate molecules sequentially pick up and lose electrons. The
complexes were purified in the heyday of studying the enzyme biochemistry of oxidative phosphorylation, and
are named Complex I, II, III, and IV. The first two complexes have one biochemical job: to take electrons from
the reduced Krebs cycle carriers and place those electrons on a single membrane-bound electron carrier called
ubiquinone, or simple Q in its oxidized form. Complex I oxidizes NADH and reduces Q to its two electron
reduced form QH2. Complex II converts FADH2 into FAD and reduces Q to QH2. So complex I and complex II
are sort of like an “electron funnel” that channels NADH and FADH2 electrons onto the Q carrier to produce
QH2. This is a wonderfully simple electron carrier. It has a “business end” that has a structure called a quinone.
This structure can be converted with one or two electrons to form a single or doubly reduced molecule. The three
forms are shown below.
This simple benzoquinone business end is linked to a long lipid molecule (shown in the first form and then called
R in the other two in the picture) that keeps it anchored to the surface of the inner mitrochondrial membrane,
where all the action happens. This lipid molecule is called “coenzyme Q” (and it is the self-same material that is
sold as the supplement/vitamin CoQ10).
Complexes I and II: electrons to Q- As mentioned above, both complexes I and II catalyze reactions that
convert Q into QH2. The formal name of complex I is NADH:ubiquinone oxidoreductase, and like this long but
accurate name suggests, the reaction involves transfer of electrons from NADH to ubiquinone, to make NAD+
(ready for more service in the name of energy metabolism) and the reduced from of coenzyme Q known as QH2.
The net chemical reaction looks like this.
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Complex II is actually an enzyme that we have already encountered, in the Krebs cycle. It is none other than
succinate dehydrogenase, (it is at about 7 o’clock on the Krebs cycle). The Krebs version of the succinate
dehydrogenase reaction, as you remember from earlier studies, looks like this:
succinate + FAD
fumarate + FADH2
While the Complex II version looks like this:
So you might reasonably ask, in the manner of scholarly discourse, “WFT! How can these be describing the same
enzyme when the two reactions are different!?”. And that is a reasonable question. It’s really due to the fact that
these enzyme complexes are… complex and usually catalyze a set of internal transfers that start with a small,
discrete metabolite that is the electron carrier (or donor) and end with a product that is similarly small, discrete
electron acceptor and now carrying the electrons. In both cases, the succinate is the donor of the electrons. But in
the version we learned in the Krebs cycle, the succinate dehydrogenase reaction, the acceptor of the succinate
electrons is FAD, which ends up being reduced to FADH2. In the version that is described in Complex II, the
acceptor or recipient is ubiquinone, or Q which is converted to QH2. What gives? The answer lies in the fact that
these complexes usually involve a number of internal transfers of electrons onto and off of various carrier, that we
never see. It is true that the first place the succinate electrons go in the succinate dehydrogenase/Complex II)
reaction is FAD to become FADH2, and so that aspect of the Krebs cycle is true because we are focused on the
flow of carbon metabolites. Also, in the days of discovering the Krebs cycle, spectrophotometric methods were
employed that focused on the FAD/FADH2 pair. But when the whole ETC is operating, the FADH2 that is
produced in this reaction is tightly bound to Complex II and those electrons are then passed to other electron
accepting sites within the complex and ultimately delivered to Q to reduce it to QH2. So when people are talking
about the Krebs cycle, they usually leave the electrons on FADH2, but they wend their way through the Complex
II carriers and end up on QH2. It, like a lot of metabolism, is a matter of what one is emphasizing and what one is
ignoring. Often when people are studying the ETC in isolated mitochondria they will add succinate to isolated
mitochondria to start the electron transport chain going. Keep that in mind for later when we talk about this
approach to studying mitochondrial respiration.
The electrons from NADH or FADH2 (really succinate) end up on the carrier QH2 after the reactions of complexes
I and II, but we know they finally end up on H2O (as a result of reducing O2) it is not hard to guess that the next
two complexes (III and IV) are involved in getting the electrons from QH2 to O2. Ready…?
Cytochrome c: mind your Qs and Qs- We have the electrons from the Krebs cycle now held in the reduced
mitochondrial carrier QH2, and the Krebs cycle acceptors NAD and FAD have been restored to continue their
good work in oxidation of acetate groups that enter the Krebs cycle. Now what? The electrons from reduced QH2
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are next placed onto a new carrier called cytochrome c, or cyt c.
Complex III (NADH: cytochrome c oxidoreductase) catalyzes this
transfer of electrons. cyt c has simple chemistry (and fancy
biochemistry). It a protein with a heme cofactor that holds a Fe ion, ,
just like the iron that we encountered in hemoglobin that uses iron ions
to reversibly bind O2. The iron ion in cyt c can either be in the Fe3+ state (oxidized) or the Fe2+ state (reduced).
That is it. Complex III has the job of taking the electrons from reduced QH2 and transferring them to cyt c to make
the reduced (Fe2+) form. This simple transfer has the net reaction as shown.
So the products of the reaction are reduced cyt c (with Fe2+) and re-oxidized Q. One thing to notice about this
chemistry is that the transition from QH2 to Q involves the removal of two electrons while the reduction of cyt c
involves the transfer of one electron to the Fe3+ in the oxidized cyt c to make Fe2+ in the reduced form. The cool
thing about Q as an electron carrier is that due to its quinone chemistry, it can exist in three states: fully reduced
(with 2 electrons) partially reduced (with one electron) or fully oxidized, with no electrons. The transfer of
electrons from QH2 to cyt c is a fairly complex affair, called the “Q cycle”. The actual chemistry involves
simultaneous oxidation of one QH2 to Q, with its two electrons going separate ways. One is actually used to
reduce cyt c, and the other is used to reduce a separate Q to the intermediate form QH (I know, it sounds
counterproductive). There are actually two distinct sites in Complex III, one for “donor” QH2 (that being
oxidized) and one for the distinct “acceptor” Q. This “parting of ways” type reaction is then repeated with a new
QH2 and the half-reduced QH, to produce a second reduced cyt c, a second Q and a QH2 from the acceptor QH.
So actually a case of “two steps forward and one step back”. There are a number of complex ways to draw this,
none of them are very succinct or revealing. Here is mine. The two donor QH2 that provide the electrons are each
a separate color (green and magenta) to indicate they are two distinct molecule. The acceptor Q in its various
forms is indicated with the brown color Q (so Q, QH. and QH2) to indicate it is the same molecule. In each of the
two reactions, the two donated QH2 electrons go to two separate places. One goes to reduce a cytochrome c, and
the other goes to reduce the blue acceptor Q. So we are oxidizing two QH2 to produce two Q, reducing two cyt c
to make two reduced cyt c, and reducing one Q to make one
QH2. That is, as you can check above, the net reaction for
complex III written above, but it has a fancier and even
inefficient-looking underlying set of occurrences. Why do
things run in this crazy way? It turns out that the special
separation of the two Q sites has a lot to do with the part of
the biochemistry that pertains to proton transport, discussed
below. Cytochrome c is the final resting place of the electrons
after the Complex III reactions, and now we are finally ready
to pass them on to O2 to make water. That is the job of
Complex IV
Complex IV: the last stop on an electron’s long trip home… The final net reaction in the action of the four
complexes involves the regeneration of oxidized cytochrome c and the reduction of oxygen to H20. It is called
cytochrome oxidase, and the net reaction is just what you’d predict, four reduced cyt c (with Fe2+) are oxidized,
and so donate their electrons to one O2 molecule (because that is how nature packages oxygen for us) to produce
two H2Os. Now if you think about it, that is a pretty elaborate reaction, since each cyt c can only donate one
electron at a time (since each has one iron that goes from the +2 to the +3 state), and but the oxygen is provided in
its molecular form and thus needs a total of 4 electrons to be converted into two H2O. Accordingly, the
biochemistry of complex IV is indeed complex. The catalytic cycle involves four separate cyt c molecules giving
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up their electrons, and a copper center complexing the oxygen atoms during their reduction, and a tyrosine
forming a free radical during the festivities, to name a few events during reduction of one oxygen molecule to two
H20. First the Q cycle and then these molecular shenanigans. Why is this all so complex? Read on! But first, here
is the net reaction of the Complex IV, or cytochrome oxidase enzyme:
Redox… is that it?Complexes I, II, III and
IV are correctly called
complexes because although they have names that sound like a single enzyme, and were originally purified as
distinct enzyme activities that are described by those names, they are in fact composed of many proteins, and they
are firmly embedded in the membrane. They are, in fact, integral membrane protein complexes that actually span
the highly impermeable mitochondrial inner membrane (IM). And it is this transmembrane orientation that is the
key to how the electron transport chain leads to production of energy that can be used to convert ADP back into
ATP. The simple redox chemistry of the complexes is accurately depicted by the indicated reactions, all grouped
together here.
But along with this, complexes I, III and IV during their catalytic cycles, push a set number of H+ ions across the
membrane to the side that faces the intermembrane space. That is, there is a vectorial or directional aspect to the
reactions that involves moving protons, that is, H+ ions, across the membrane. So this process does not affect the
chemistry in terms of bond making and bond breaking, but it is an integral part of the function, action and
energetics of these reactions. It is important to realize that when the reactions are run in the soluble form that was
so brilliantly devised in the enzymology labs of the 1950 and 60s, the “H+ pushing” aspect of the intact complexes
would be missed due to the lack of a membrane: when a membrane-embedded complex is solubilized into
detergent micelles, there is no membrane defining “inside” and “outside” to allow the generation of a gradient by
pushing H+ ions to the outside. Once Peter Mitchell devised his model that explained a lot of hard-to-fit data by
including intact membranes and gradient production, experimentalists turned to testing and examining the notion
that an H+ gradient was being created as a product of the individual ETC reactions. So the complex I action really
requires the membrane with complex I correctly situated in it. The reaction would be depicted like this:
Those 4 H+ are being moved across the inner
membrane of the mitochondrion in an orientation
so that the inside (matrix side) is a bit more
negative and the outer side (facing the IMS) is
more positive. The energy for this uphill battle is
provided by the redox reactions that move the
electrons from NADH eventually to Q (magenta
arrows). The increased H+ gradient can actually
be thought of as a product of the reaction. Hold
that idea because it will be a very useful way to
think about the mechanism of coupling between
the H+ gradient and the synthesis of ATP.
In the ETC, three of the four complexes have this added ability to “push” protons across the IM, and in this way
they each contribute to the electrochemical potential across the inner membrane of the mitochondrion. Those are
complexes I, III and IV. The full electron transport chain, including complexes I-IV, oriented in the mitochondrial
inner membrane is shown to the right. The two sides of the membrane are sometimes referred to as “P” and “N”.
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This stands for “positive” and “negative”, like a battery! And it is! You can see that complex I, III and IV each
have a red arrow signifying protons being pumped out across the membrane as part of their action. Complex II is
the only complex that does not
pump protons, but it is providing
electrons to the QH2 pool. Also
notice that the process of reducing
Q to QH2 is abbreviated here just
by showing Q as a “waystation”
for electrons flowing. We know
better, that both complex I and II
reduce Q to QH2 as part of their
enzymatic activity. Same with Cyt
c. The arrow comes to it and out of
it because Q first picks up
electrons, and then dumps them
into complex III.
Mitochondria living up to their potential- The movement of protons across the inner membrane produces an
electrochemical potential that is a powerful source of energy. Why? Two reasons: 1) there is a true charge
imbalance, literally a voltage, across the membrane that that is positive outside and negative inside, and 2) there is
a concentration gradient of protons, higher on the outer side of the membrane than on the inner side. These two
separate components will both provide energy for protons to move back down the gradient. That is, both
components contribute to free energy of the protons on the “p” or IMS side of the membrane by the following
equation, which looks fancy but really says there are two independent features of the proton gradient that
contribute to free energy (ΔG), them being the voltage across
the membrane (Ψ) and the difference in concentration across
the membrane (C2/C1) or ΔpH, depending on how one
specifically write the equation.
THIS is the source of the free energy that is used to make ATP. The energy stored in the H+ gradient acroos the
mitochondrial inner membrane. Directly. No phosphorylated intermediate. No X~P. A voltage and concentration
gradient across the membrane, that provides an energy-yielding path back across the membrane. The combination
of these two ways of energizing the protons (charge, concentration) is usually referred to as the “proton motive
force” or sometime PH+. Importantly, the protons are not consumed, but their movement across the membrane
provides energy that can do work. Just like a ball rolling down a hill is giving off energy that can be used to do
work even though the ball is not used up or consumed. Or just like water moving over a waterfall is giving off
energy that can be used to do work even though the water is not being used up or consumed, protons moving
across the energy gradient similarly can do work, and in the case of mitochondria that work is running the
“uphill” or endergonic reaction of converting ADP and Pi into ATP. Let’s consider that waterfall analogy a little
more. Your yacht wrecks on an uninhabited island in the south pacific, and you need to get your generator going
to radio for help, light lights to keep the unknown carnivores away from your campsite, and charge up your iPods.
You find a waterfall and hooray, there is plenty of energy in that natural feature for your needs. But to harvest that
energy, you need a coupling mechanism to convert the energy of falling water into mechanical energy that turns
your generator. So you build a water wheel, that turns the generator crank, and viola, energy from falling water to
turn the generator. In the same way, the energy rich proton gradient must be harnessed by a protein complex to
couple the energy of protons moving back down the gradient to the formation of ATP. That coupling is
accomplished by a miraculous enzyme that is strangely similar to a sort of nano-scale water wheel. It is called
ATP synthase. But first, let’s explore the transmembrane proton motive force PH+ a bit more…
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An experimental interlude: experimental test of proton gradient-driven ATP synthesis- Before we get into
the nitty gritty of the ATP synthase (nature’s water wheel), I want to describe some experiments that indicate that
the proton gradient across the intact mitochondrial IM is the driving force for ATP synthesis. The first
observations that led to this idea were frustratingly negative ones. That is, the inability of really excellent
biochemical laboratories to ever see the reactions of the purified ETC complexes cause conversion of ADP and Pi
back into ATP. These biochemical experiments always had the complexes removed from the mitochondrial
structure, purified in a variety of detergents that allow proteins that normally sit in lipid bilayers to maintain their
structure in solution, but without a membrane with distinct sides like we see in the actual mitochondrion. This
absence of “coupling” between electron transport reactions (such as NADH transfer of electrons to Q) and ATP
synthesis in the test tube is in stark contrast to other biochemical systems. The glycolytic pathway can be
observed to make ATP from ADP and Pi when the reactions are run with soluble highly purified enzymes. And
this is because the reactions responsible for ADP conversion into ATP are catalyzed by enzymes that directly use
ADP and a phosphate donor such as 1,3 bPG (see glycolysis) to bring about the conversion. In contrast, the
inability to see direct production of ATP from ADP and Pi when the purified complexes of the ETC were
functioning in solution gradually indicated that something was missing or different from the expected substrate
level phosphorylation. This “absence of effect” didn’t prove that the effect was absent. But when some of the best
laboratories in the world, doing some of the hardest biochemistry in the world were unable to see ATP production
when the complexes were operating in solution, it allowed the possibility that ATP production was connected or
coupled to the ETC in a fundamentally different way. New answers require asking questions in new ways…
Keeping it together to study mitochondria- Another way to study mitochondrial function is…to study
mitochondrial function. No, this is not a Zen koan. It simply means that a different approach to studying
mitochondrial biochemistry is to look at the processes in the intact, purified organelle. This may seem like a step
back from the elegant study of highly purified, functional, detergent-solubilized ETC enzyme complexes; it is
certainly a lot easier. To look at functioning mitochondria one has to purify them from some cellular or tissue
source, typically from abundant tissues or those that that have high mitochondrial densities (liver, heart, etc.).
Then one needs to measure the biochemical processes going on upon adding things like NADH or succinate. To
do this, mitochondriists (probably not a real word) or
mitochondriacs (actually used in Nick Lane’s great
book about mitos) place the purified mitochondria
(made by classic cell biological techniques; not super
difficult with the correct centrifuges) in a chamber
with an electrode that can sense oxygen. This set up
is sometimes called an oxygraph, because it records
current oxygen concentration in the mitochondrial
mix. Since the electron transport chain consumes
oxygen, the rate of this process is indicated by the
oxygen consumption curve. A typical oxygraph
experiment of isololated mitochondria is shown to the
right. If everything needed to do oxidative
phosphorylation (ATP synthesis) is present except an
electron source, the addition of a molecule that can be
oxidized by complex I or II will get the ball rolling.
Normally people add succinate to the preparation, because NADH can not permeate the intact mitochondrial IM
(and because succinate is both cheap and permeant). The graph shows time along the horizontal x-axis and the
things being consumed or made on the vertical y axes. What happens is that the addition of the electron donor
succinate (“add succinate”) causes a sudden increase in O2 consumption, due to production of H2O. This means
the ETC is running in the intact mitochondria when an electron source, (and oxygen) is available. The graph
green line shows this process, and represent values on the left axis. Now suppose you measure ATP production,
which can also be done by the correct instruments. It is pleasing to see that if ADP (and Pi) are present, when the
succinate is added, production of ATP also goes up. This is shown in the red line in the picture, on the right axis.
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This is a very beautiful system to study how ATP formation is driving by the redox reactions that carry electrons
from succinate (or NADH) to 02. But first, lets remember something. This does not work with purified ETC
complexes, that is, when complexes I, II, III and IV are combined in solubilized form in the test tube, no matter
how much ADP and Pi you add. Those complexes will allow the ETC redox reactions to occur, but ADP and Pi
never combine to form ATP in the soluble preparations. So there was something about having the mitochondria
intact, with the complexes embedded and correctly oriented in the IM, that allows the electron transport chain to
drive ATP synthesis, and we now know that is the proton gradient generated across the IM. But how does one test
this notion with an experiment?
ATP production and ETC: a nice couple- The observation of ATP production being connected to and requiring
the ETC is called “coupling”, for obvious reason. This sort of coupling can also be seen with the glycolytic
pathway, but there the consumption of glucose that is coupled to ATP production can be seen with the soluble
enzymes, or more typically, in a cytoplasmic
preparation of only soluble molecules. Same
idea. As I said above, the soluble ETC
complexes work fine in doing their individual
redox reactions, but together they do not
drive the production of ATP. So now we will
show you a classic experiment that strongly
implicated the proton gradient across the IM
as being that entity that couples electron
transport to ATP production. First lets do a
few control experiments. We have shown that
succinate (an electrons source) needs to be
added to cause ATP production, just like
glucose needs to be added to cause ATP
production in glycolysis. What happens if
ADP is not present? The answer is shown in
an “order of addition” experiment to the
right. We show the same kind of experiment,
with O2 consumption on the left axis (green), and ATP production on the right axis (red), but instead of adding
succinate to a preparation of mitochondria where everything else is present, we first add succinate without ADP
or Pi. The arrow indicates where along the time axis the succinate is added. Nothing happens, meaning the slopes
for both O2 consumption and ATP production are unchanged. While the lack of ATP production is only expected
(since there is no ADP added yet), we also fail to see O2 consumption, despite having succinate present (as
indicated by lack of). Furthermore, when ADP and Pi are added after the succinate, Voila!, the whole process
starts working: both ETC (left axis; O2 consumption) and ATP production (right axis) go way up. Coupling! Each
process requires the other! It is again useful to think about glycolysis, where we would see the same sort of thing.
If you had the glycolytic pathway working in a test tube and the system was deprived of ADP, the whole pathway
would stop working very quickly because the ADP-requiring reactions would stop, their substrates would build up
and all the reactions would quickly cease. What about inhibitors of the mitochondrial enzymes? An inhibitor of
complex II/SDH would be expected to stop both O2 consumption and ATP production. There is an inhibitor of the
enzyme that produces ATP from ADP and Pi called ATP synthase. This inhibitor is called oligomycin, and that
drug also stops both ATP production (not surprisingly) and O2 consumption. In the same vein, inhibitors of the
intermediate steps (complex III and IV) also effectively cease both processes. And this is exactly what you would
see if similarly studying glycolysis: inhibitors of any intermediate enzyme would stop both glucose consumption
and ATP production. So far, the mitochondria looks exactly like a classic ATP producing pathway. But not for
long. It is worth mentioning that this “symmetrical” need for both electron donor (succinate) and ADP for either
process to run was precisely why people thought that a high energy X~P type molecule was being generated and
then consumed in OxPhos.
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Mitochondria on acid- So with this method to observe coupling between electron transport and ATP production,
the nature of coupling could be explored. One of the key observations that indicated a totally different mechanism
from substrate level phosphorylation is based on an incredibly simple type of molecule, weak organic acids. Like
all acids, these molecules are proton-releasing compounds (acids) that are charged when they are deprotonated,
and neutral when they are protonated. However, because they are organic molecules, they are sufficiently
hydrophobic that when in the uncharged protonated form, they are soluble in organic solvents, like you may have
learned about in O-chem lab. This turns out to be the perfect molecule to examine the importance of a
transmembrane proton gradient. That is because the presence of a weak organic acid will create a route for
protons to move across an otherwise impermeable membrane. The weak organic acid will exist as both the
protonated and deprotonated forms in each aqueous space, with the neutral protonated form moving freely across
the normally impermeable inner membrane. This way, a weak organic acid will tend to ferry protons across a lipid
bilayer. Thus, if there is a higher concentration of protons on one side of the membrane, this new route of
movement will rapidly break down the gradient until it is equal on either side, by simple mass action. The picture
below (for some reason, ever darn picture description starts just at the bottom of a page, requiring “page lag”.. I
am sorry!) shows an illustration of this process, and a “real” weak organic acid used in mitochondrial experiments
called dinitrophenol, or DNP.
DNP
So what happens when we treat isolated
mitochondria in an oxygraph with a weak
organic acid like dinitrophenol (DNP). The
picture illustrates an experiment in which
we observed the usual “coupled” processes
of ETC (as indicated by green O2
consumption) and ATP production (in
red), and then the effect of addition of
DNP at the indicated time. BOOM! Notice
two things: 1) that the O2 consumption
continues, but the ATP production drops
drastically. That is, the flow of electrons
and ATP production have been uncoupled.
This is why people frequently refer to weak
organic acids as “mitochondrial
uncouplers”.
It turns out that many weak organic acids
do this, because the effect of these types of
molecules is due to the general solubility
properties of weak organic acids rather than some special feature of DNP. This and similar experiments strongly
indicated that the proton gradient across the IM was responsible for powering the production of ATP. Put in other
words, this experiment indicated that the proton gradient was the “product” of the ETC that is used to drive
synthesis of ATP. In glycolysis, the products are actual molecules, that is 1,3 bPG or PEP. These two molecules
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are made and used to create ATP from ADP. In mitochondrial respiration, complexes I, III, and IV, move protons
across the membrane against a gradient, and this mode of energy storage is used to make ATP. That is why you
need intact mitochondria to see ATP production driven by the ETC. It is also why you can not see the purified,
soluble complexes make ATP in the test tube… there is no way to build up a proton gradient. I alluded to the idea
that the proton gradient is a “product” of some of the enzymes of the ETC. So instead of a molecule that is
produced in a typical enzymatic reaction, let’s consider the proton gradient across the IM as a reaction product. In
a sense it is, and behaves in many ways like a more typical product. As it builds up, the spontaneity of the
upstream reactions diminishes. ATP production expends the gradient in a useful manner (as we shall see) and
blocking ATP production causes the gradient to build up, just like a product building up in a more traditional
pathway like glycolysis. In the picture shown, the gradient is indicated by “PH+”, for proton motive force. The
entire (and very complex) ETC is
indicated by the net processes of
reduced carriers (like succinate or
NADH) getting oxidized and oxygen
getting reduced to water. This all
produces the “product” PH+. This
quantity consists of both the
chemical gradient and the charge
gradient, each caused by the
pumping of protons across the IM
during the ERC. PH+ is a source of
free energy, because the protons will
spontaneously move down the
gradient. And PH+ is the used up, just
like a substrate, to drive ATP
synthesis by ATP synthase. Keep this
in mind, and we will get back to it a
little later.
Necessity and sufficiency: light driven ATP synthesis- The experiments with intact mitochondria clearly show
that the proton gradient is a necessary condition for ATP production. The proton gradient is where the free energy
of the various reactions of complex I-IV ends up, stored in a chemical and charge gradient, a lot like a battery. By
removing or lowering this gradient with things like uncouplers, and through a variety of other like-minded
experiments, it became clear that the proton gradient was a necessary condition for ATP synthesis by
mitochondria. But mitochondria have a lot of things, and parts and stuff, so a related by distinct question was, “is
a proton gradient all that you need?”, or put another way, is the proton gradient sufficient to drive ATP synthesis
by the enzyme ATP synthase? This question was addressed in a most lovely and elegant experiment. It is one of
those cases where it is easy to describe, but very hard to do, because it requires some super challenging
biochemistry. But the basic idea is sort of the “Frankenstein” approach, building a biological system to test an
idea. Normally, the mitochondrial proton gradient is made though the complex actions of the ETC. But there are a
number of much simpler ways that nature can make an identical proton gradient. One of the simplest known is the
protein known as bacteriorhodopsin, which is a monomeric protein that spans the purple bacteria membrane seven
times. When this protein is properly functioning, it absorbs light and uses that light energy to pump protons across
the membrane, making a proton gradient. If the proton gradient is all that is needed, then a gradient produced by
this completely distinct way should still do the job just fine, and allow a mammalian ATP synthase to make ATP,
even though the relatives of these two proteins (ATP synthase and bacteriorhodopsin) parted ways like about 2
billion years ago! The laboratory of Effram Racker (who was instrumental in many of the key studies of the
individual complexes and mitochondrial energetics) combined bacteriorhodopson from the purple sulfer bacteria
with ATP synthase purified from beef heart into artificial membranes called “phospholipid vesicles” which are
very simple closed membranes made from purified lipid molecules. They set it up so the vesicles would get
protons pumped into them when the bacteriorhodopsin was activated, and the ATP synthase would then use the
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gradient to form ATP, by allowing protons to escape in a productive way. The cool thing is that because the
bacteriorhodopsin is light-activated, the experiment literally worked by turning a bright light on the vesicle prep
and watching ATP production start. The off and on states of this brilliant experiment are depicted in cartoon form
with the yellow squiggles being the activating light. This first version of the experiment was done in 1974, and
really put the nail in the coffin of about the chemiosmotic theory. In subsequent versions of the experiment, super
pure components were used, to the point that the system works with highly pure bacteriorhodopsin, ATP synthase,
and phospholipids. If you can build it, you understand it…
light on!
light-powered ATP synthesis
ATP synthase: what goes around comes around- ATP is produced during OxPhos by the catalytic action of the
enzyme ATP synthase. Long after the proton gradient was clearly shown to be necessary and sufficient condition
for ATP synthesis, the way by which this protein converts the proton gradient into ATP production was not clear.
Gradually the structure of the protein was learned in great detail, and this along with classic enzymology has led
to a deep understanding of the action of ATP synthase. The one totally weird thing is why it is called “ATP
synthase” when every other ATP-using enzyme almost without exception is called a “synthetase”. I don’t have an
answer for this. Oh well, you can’t know everything. The biochemistry text are full of extremely detailed
descriptions of this beautiful enzyme, but the basic “forest” view will suffice to gain you admiration and
understanding. There are two parts of the enzyme, called Fo and F1. Fo is embedded in the membrane, and F1 is
soluble, and can even be purified as a soluble multi subunit enzyme. The soluble F1 subunit has 3 sites (each
made up of two proteins, alpha and beta, so ababab is the total count of individual proteins in F1). The sites
defined by each ab dimer can be empty, bind ADP + Pi, or bind ATP. Not surprisingly these binding sites are the
active sites where the enzyme action is occurring, running the endergonic reaction of converting ADP and Pi into
ATP. The Fo subunit resides in the membrane and spans the membrane multiple times; exactly what you would
need to harness a proton gradient across a membrane. It turns out that the ATP synthase works by allowing H+
ions to flow back into the matrix by moving through the Fo subunit, and this movement is converted into
mechanical energy that pushes the F1 subunit into different conformations that promote ATP synthesis and
expulsion from the enzyme. Better still, the motion is a rotary motion, such that there is a shaft that sits in the
middle of the Fo protein which actually spins, and this spinning motion drives the changes in the F1 subunits that
promote the uphill battle of ATP synthesis. So the analogy of protons in the gradient being like water flowing
down a waterfall and turning a water wheel is spookily accurate. There have even been incredible experiments
showing that the ATP synthase actually turns like a piston, and this turning motion can be seen with the right
microscope and the correct clever tricks!
Acceptance is the key- One of the features of the ATP synthase is that the flow of protons through the Fo channel
and the movement of the subunits as they cycle through their different conformations are highly interdependent,
and this can be shown in two ways: If an inhibitor of the ATP synthase is added to a mitochondrial preparation the
ATPase will stop functioning, and the entire ECT will grind to a halt. This is again best viewed by remembering
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that the proton motive force PH+ can be viewed as a product of the ETC and substrate of the ATP synthase. If the
enzyme is inhibited, the proton gradient remains high, and the reactions that produce it eventually (and rapidly)
stop. In a similar vein, if there is no ADP, the ATP synthase can not function, and again, the flow of protons
through the Fo channel stops, and the whole ETC rapidly slows down. It is important to realize that this
connectivity, or coupling between the action of the ATP synthase and the redox complexes of the ETC is due to
the fact that the protons have only one way to go down their gradient: by passage through the ATP synthase Fo
channel. This tight coupling between the flow of electrons and the production of ATP has a very important
functional aspect, and it is this: when ADP is available, the electron transport chain will continue to function.
When ADP runs out, the flow of electrons will stop, due to a “pile up” of the gradient, and a slowing of all the
upstream reactions that make the gradient. If ADP increases, flow resumes as the protons start flowing through
the Fo channel. This is called “acceptor control” and it refers to the fact that the availability of ADP determines
the rate of the ETC. The term “acceptor” is used because ADP is a phosphate acceptor. A little byzantine but we
have to accept it (ha ha). So this provides a very natural control system for “deciding” how much ETC activity,
and hence how much consumption of NADH and succinate, will be occurring. The more ADP, the more
consumption of these key input substrates and the more oxidation of the fuels that produce them.
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