Lecture notes on Electron Transport

THE COMPLEXES OF THE ELECTRON TRANSPORT CHAIN

Complex I is the NADH Dehydrogenase complex.  The mammalian complex is composed 
of at least 42 subunits.  The complex molecular weight is about one million Daltons.  Seven 
of its subunits are coded in the mitochondrial genome.  This is highly significant, since 
only 13 protein coding genes are found in the mitochondrial genome and more than half of 
them are for complex I.  The complex is composed of two domains, the membrane arm and 
the peripheral arm.  These seem to be assembled separately.  The peripheral arm contains 
most of the redox active centers.  The membrane arm contains all of the mitochondrially 
encoded subunits.  For a schematic diagram see Fig 21.3, p. 679 2nd ed. Fig 20.6, p.636 1st ed. 
and your handout.

The whole May 6, 1998 issue of BBA Bioenergetics is about complex I.  In this issue, I 
learned to my surprise that yeast do not have complex I.  Instead, they have a 57kDa protein 
that is an NADH-ubiquinone oxidoreductase like complex I, only smaller.  I have received 
an email about this.  Apparently not all yeast are missing complex I, but Bakers yeast is.

The E. coli equivalent of this complex has only 14 subunits.  Of these 14, seven are 
homologous to the 7 subunits coded in the mitochondrion.  If this bacterial complex is 
cleaved by proteolysis, three fragments are produced:  a membrane fragment with all seven 
subunits equivalent to the mitochondrially encoded subunits of complex I;  a soluble 
fragment with 3 subunits that contains 5 iron-sulfur centers and one FMN; a connecting 
fragment of 4 subunits with one iron-sulfur center.  A soluble piece with 5 subunits has 
been over expressed and crystallized, but the solution of the structure has not been 
completed.  When it is done, we will have a good idea of how the iron-sulfur centers are 
laid out and how the FMN is situated in the protein.  

The inhibitor rotenone binds to complex I and competes at one of the ubiquinone binding 
sites.  There appear to be two ubiquinone sites in complex I.  One is tightly bound and does 
not seem to come off in purified preparations.  One is loose and presumably is the one that 
can transfer electrons from complex I to complex III.  When rotenone binds, electron 
transfer from complex I is blocked.

The stoichiometry of protons pumped to electrons transferred is 4 protons/2 electrons.
Two electrons are passed for each NADH oxidized.

We have introduced three new and important players in the function of electron transport, 
and they each need to be discussed.  

1. FMN
2. iron-sulfur centers
3. ubiquinone

FMN is a cofactor tightly bound to the peripheral arm of complex I.  FMN stands for flavin 
mononucleotide.  See pages 590-592 2nd ed. Pages 474-477 1st ed. for details on flavin structure 
and reduction by NADH.  The fact that FMN is bound to Complex I makes this a flavoprotein.  
Please note in Fig 21.4 p. 680 2nd ed., (Fig. 20.4 on p. 634 1st ed.) that there are four 
flavoproteins that feed electrons to ubiquinone.  

Flavoproteins that reduce ubiquinone in the mammalian electron transport pathway

1. Complex I
2. Complex II
3. Electron transfer flavoprotein dehydrogenase (ETF-QO)
4. sn-glycerophosphate dehydrogenase

Complex II is a tetramer of non-identical subunits.  It contains FAD and three iron sulfur 
clusters.  Succinate dehydrogenase is part of the TCA cycle.  It is the only TCA cycle 
enzyme to be an integral membrane protein.  None of its subunits are 
coded in the mitochondrial genome, and it cannot pump protons across the inner 
membrane, so it does not contribute to the proton gradient like complexes I, III and IV do.
This is being challenged now by examining the crystal structure and the location
of critical amino acids (FEBS Lett. 504, 133 2001).  
The crystal structure of the bacterial succinate dehydrogenase is being solved now.  
A homologous enzyme fumarate reductase from E. coli has been solved (Science 284 June 18, 1999
Pages 1941-1942 for commentary and pages 1961-1966 for the structure.  Fumarate reductase 
catlayzes the reverse reaction.  We talked about it last time as a step in running the TCA cycle 
backwards.  The structure of succinate dehydrogenase can be modeled on the fumarate 
reductase structure.  The protein is modular.  An FAD containing subunit is in contact with an 
iron sulfur containing subunit and this is in contact with two membrane bound subunits.  The 
Iron sulfur subunit has three different iron sulfur centers, a 2Fe-2S, a 3Fe-4S and a 4Fe-4S 
cluster. 

Complex II has two hydrophobic membrane subunits that are fairly small.  In E.coli, There 
is a heme sandwiched between these subunits.  This heme is liganded by two histidine 
residues, one from each subunit.  Mutagenesis studies in Robert Gennis' lab have shown 
that the histidines can be changed to other side chains.  This causes the loss of heme, but 
the enzyme still assembles and works nearly as well as wild type, so the heme is not 
needed structurally or enzymatically it is probably an evolutionary relic.  The fumarate 
reductase has no hemes, but it does have two quinones that are tightly bound at opposite sides 
of the membrane.  Electrons flow from the quinone at the matrix side to the iron sulfur clusters 
and on to the FAD at the active site.  This is the direction in fumarate reductase.  In 
succinate dehydrogenase the electrons flow from succinate to FAD then to the iron sulfur 
clusters and on to the quinones.  

ETF-QO is a membrane protein with one FAD and one 4Fe4S center.  It has one subunit 
of 67 kDa with two predicted transmembrane segments, so it is not a large complex like 
complex I, but it is important.  At least nine mitochondrial matrix dehydrogenases feed 
electrons into ubiquinone by way of this intermediate electron carrier.

Dehydrogenases that funnel electrons to ubiquinone by ETF-QO

1-4.	4 different, chain length specific acyl-CoA dehydrogenases
5.	isovaleryl CoA dehydrogenase
6.	2-methylbutyryl CoA dehydrogenase
7.	glutaryl CoA dehydrogenase
8.	sarcosine dehydrogenase
9.	dimethylglycine dehydrogenase

You should not memorize this list. It is for your curiosity not for an EXAM.

Defects in the ETF-QO cause the substrates of these dehydrogenases to build up.  This 
can be fatal in infancy if the defect is severe.  The disease is called glutaric acidemia type 
II.

A paper in the Aug. 21 2001 Biochemistry 9758-9769 presents evidence that mitochondrial 
dehydrogenases in yeast are part of a supramolecular complex.  At least eight dehydrogenases and 
four TCA cycle enzymes plus other proteins (up to 38 different proteins) were identified in a 
single complex.  This supports the metabolon idea of Srere and also suggests that NADH may be 
channeled from several sources directly to the membrane bound dehydrogenases.

The mitochondrial sn-glycerophosphate dehydrogenase is part of the glycerophosphate 
shuttle for moving electrons from cytosolic NADH into the electron transfer pathway.  
Remember that NADH does not cross the mitochondrial inner membrane, so the electrons 
are fed in from the periplasmic space in the form of glycerol 3 phosphate.  This compound 
becomes oxidized to dihydroxyacetone phosphate by the mitochondrial 
sn-glycerophosphate dehydrogenase (see figure 21.33 p 703 2nd ed fig 20.32 p. 654, 1st ed.) 
located on the outside of the inner membrane.
(the figure shows it as a transmembrane protein, but that is not correct).  
The human cDNA was just sequenced in 1994 and the gene structure determined in 1996.  
There are 17 exons over 83 kb of DNA.  The protein has 727 amino acids and contains 
FAD.  There is a cytosolic glycerophosphate dehydrogenase, but it does not resemble the 
membrane bound form.  It actually performs the reverse reaction, converting 
dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate, but it is named for the 
forward reaction.  The soluble and the membrane bound glycerophosphate dehydrogenases 
together form the glycerophosphate shuttle shown in fig 21.33 2nd ed. (fig. 20.32. 1st ed.)  
Electrons from cytosolic NADH are transferred to DHAP to form glycerophosphate and then back 
to the membrane bound glycerophosphate dehydrogenase FAD coenzyme and then on to ubiquinone 
in the membrane.  Yeast have two additional genes NDE1 and NDE2 that are external faqcing NADH 
dehydrogenases.  These can oxidize cytosolic NADH directly, but they do not pump 
protons like complex I, so they return less energy than the glycerophosphate shuttle.  

Please note that progress in cloning and sequencing these electron transport components is 
current research not old history.  John Walker and Paul Boyer were just awarded the Nobel 
Prize October 15, 1997 for work done on the X-ray crystal structure and mechanism of ATP 
synthase.  We will talk about that in our next lecture.  John Walker has also been a major 
contributor to understanding all the mammalian complexes at the level of their genes.  
The last eight human genes were just sequenced at the end of 1998. (Biochem. Biophys. Res. 
Commun. 1998 Dec. 18;253(2):415-22).

Levels of the mitochondrial form of the glycerophosphate dehydrogenase are high in 
pancreatic islet cells.  In patients and in rats with non-insulin dependent diabetes 
mellitus(NIDDM), the levels are quite low, suggesting that this protein might be a candidate 
gene involved in this disease.  

We began this discussion talking about three important players in electron transfer.  The 
first was FMN (and as we have seen FAD) and their flavoproteins.  The second was iron-
sulfur clusters.  These are a second type of redox active center that can accept and then 
donate electrons in the electron transfer pathway.  They come in three varieties called the 
2Fe-2S cluster (Fig. 20.16, p 654 2nd ed., Fig 19.16, p. 611 1st ed.) or the 4Fe-4S cluster (Fig. 
20.8 p 650 2nd ed, Fig. 19.8, p. 607 1st ed.)  The 3Fe-4S cluster is not shown.  The first three 
flavoproteins we talked about all have these iron-sulfur clusters.  Complex I from E. coli 
has six (three of each type), the mammalian Complex I has 6-7.  Complex II has three, and 
we already saw that the electron transfer flavoprotein dehydrogenase had one.  The 
glycerophosphate dehydrogenase has non-heme iron, but it is not clear if it is an iron-sulfur 
cluster.  There is a disease in humans that is caused by a defect in iron sulfur cluster 
formation in proteins.  As you can expect, complex I, complex II and other iron-sulfur 
containing proteins are pretty severely affected.  These include aconitase in the TCA cycle. 
and to a lesser extent complex III.

Animals have a ferredoxin protein in their mitochondria.  This protein gives electrons to 
several cytochrome P450 enzymes that require electrons for their catalysis.  A related protein 
is seen in yeast, but yeast do not have any p450s in their mitochondria, so it was not 
clear what this protein did.  If the gene is knocked out in yeast it is fatal, so it is doing an 
essential function.  It was shown recently that this function is assembly of iron sulfur 
clusters.

One thing in common among these flavoproteins is this: the flavin gets the electrons first 
and then passes them on to the iron-sulfur centers.  This is absolutely required since 
NADH is an obligatory 2 electron donor(see pages 588-589 2nd ed, 468-469 1st ed about hydride 
transfer) and Fe3+ is an obligatory one electron acceptor.  NADH cannot donate its electrons 
directly to iron.  There has to be an intermediate electron acceptor that can accept two 
electrons and give them to iron one electron at a time.  That is why the flavin gets the 
electrons first.  It cannot happen any other way.

In our journey down the electron transfer pathway, so far all our electrons have been 
passed to ubiquinone.  Ubiquinone is a lipid soluble electron carrier (Fig. 21.5 p 682 2nd ed., 
Figure 20.5, p. 636 1st ed.)  It exists in the mitochondrial inner membrane and it carriers 
electrons between complexes in the electron transport pathway.  Up until now we have not talked 
about how the electrons get between the complexes.  They do this on carriers.  Ubiquinone is one 
of these carriers. The other is cytochrome c, but we need to talk about ubiquinone first.  

Ubiquinone is like a flavin in that it can accept one electron at a time.  When it has accepted 
only one electron it is called a semiquinone(Fig. 21.5 p 682 2nd ed., Figure 20.5, p. 636 1st ed.). 
When it has two electrons it is fully reduced and when it has none it is fully oxidized.  Fig. 
21.4 p. 680 2nd ed. (Figure 20.4, p 634, 1st ed) shows ubiquinone picking up electrons from our 
four different flavoproteins.  As it exists in the membrane, it is either fully reduced or fully 
oxidized.  The semiquinone is apparently stabilized by binding to protein sites, but does not 
float around freely in the membrane.  

The next electron acceptor in the path is complex III also called the bc1 complex.  This 
structure has been solved in 1996 by Chang-An Yu of Oklahoma State University at 
Stillwater and Hans Deisenhofer who got the Nobel prize for the first crystal structure of a 
membrane protein complex (the light reaction center) He is in Dallas now.  This structure is 
the largest protein structure ever solved, with 16 monomers of bc1 complex in the unit cell.  

The bc1 complex (complex III) has 11 subunits in mammals, (Figure 21.11 p. 686 2nd ed.)
but only three in bacteria.  These three each contain important redox centers.  The extra 
subunits in mammals have no known function and they are often called supernumerary subunits.  
One of these poses a curious problem of biogenesis that I will mention in a minute. 

The redox centers that you must know to understand complex III are two b type hemes that 
are contained in the same subunit and are directly above one another in the membrane.  
They are perpendicular to the membrane(see Fig 21.11, p. 686 2nd ed., Fig. 20.11, p. 639 1st 
ed).  At the cytosolic side of the membrane there is the Rieske iron-sulfur protein.  Also on 
the cytosolic side of the membrane is cytochrome c1.  (See Fig 21.10, p. 685 2nd ed, Fig 20.10, 
p. 639 1st ed. for the differences between the three types of heme found in cytochromes b, c and 
a.)  In mammals there are 13 transmembrane segments, with 8 coming from cytochrome b that 
contains the two b type hemes.  One of the transmembrane segments is actually the presequence of 
the rieske iron-sulfur protein.  This presequence is cleaved off by a matrix metallo proteinase 
located at the matrix surface.  The biogenesis problem I mentioned has to do with the 
orientation of this one transmembrane segment.  The cleavage site of this peptide is found on 
the cytosolic side of the membrane, not at the matrix side where it had to be cleaved.  This 
means that the transmembrane helix had to flip after it was cut.  This is the only example I 
know of where a leader sequence of a protein is used as a subunit in a complex.

Complex three operates in an unusual way.  The mechanism is called the protonmotive Q 
cycle, or just the Q cycle.  In this cycle, electrons from ubiquinone flow in two different 
pathways through the protein.  

Ulrich Brandt gives a detailed description of this process in a paper in Biochim. Biophys. 
Acta 1275, 41-46 (1996).  This special issue of BBA is in my lab if you want to read it.  It 
is a collection of papers from an Aug. 1996 conference I attended on bioenergetics, and it 
covers many items we will discuss in this class.

The Q cycle has a reputation for being difficult to understand and a bit abstract.  It helps a 
lot to pay attention to the structure of the complex and where the electrons are flowing to 
understand the Q cycle.  The electrons are donated to complex III from ubiquinone.  Up 
until now we have talked about electrons, though protons are also being transferred and 
these are not equivalent.  In the Q cycle it is important to keep in mind where the electrons 
and the protons are going, because they do not go to the same places.  

Note: your book is not completely up to date in its discussion of the Q cycle.  It suggests 
that electrons move before protons at the P center, which is probably not true, and it 
suggests that an electron flows all the way to cytochrome c before the other electron moves 
to BL.  See below for a more up to date account.  The Figure 21.12 on p. 687 2dn ed, fig 20.12 
on p. 640 1st ed. may be helpful in visualizing what is going on.

At the start, fully reduced ubiquinone diffuses to the cytosolic side of the membrane where 
it binds to a site known as center P (P for positive side of the membrane) or the oxidation 
center, because the ubiquinone will become oxidized here.  
Its two electrons will be split into two different paths, one going to 
the Rieske iron sulfur center (reduction potential +290 mV) and the other going to the first 
b heme called BL (for its low potential -20 mV).  As we discussed earlier, electrons will 
prefer to choose the pathway toward the most positive reduction potential so they would 
prefer to both go to the iron-sulfur center, one at a time rather than split up and go both 
ways.  This bifurcation of electron flow is unique to the bc1 complex.  It has a structure 
that forces this splitting to take place.  Remember that iron can only accept one electron at a 
time so both the Rieske protein and the BL cytochrome can only take on one electron.  The 
protein forces them to go different ways by requiring that both electrons are released from 
ubiquinone simultaneously.  This way they have to go to two different acceptors because 
each acceptor can take only one electron.  

There is evidence that two ubiquinone molecules bind at the P center.  One is ten times 
more tightly bound than the other and acts as a prosthetic group, never leaving the enzyme.  
This tightly bound ubiquinone is oxidized at the start of the cycle.
In this paired arrangement, the major energy barrier to reaction is deprotonation of the loose 
ubiquinone.  This occurs first before the electrons move.  Then in a more speculative part 
of this model, one electron moves from the deprotonated ubiquinone to the tightly bound 
form and they both become semiquinones.  It is these two semiquinones that 
simultaneously give up their electrons to the two different pathways.  Only the loosely 
bound form is released as a fully oxidized ubiquinone

This splitting creates a problem.  One electron goes from the iron-sulfur protein to 
cytochrome c1 and is finally passed on to cytochrome c.  This electron eventually is used to 
reduce oxygen in complex IV, cytochrome oxidase.  What happens to the other electron?
This non-productive electron passes on to the second b heme BH (for high reduction 
potential +50 mV).  Finally this electron is given to a fully oxidized ubiquinone bound near 
the matrix side of the membrane at a site called the N center (N for negative) or the reduction 
center, because ubiquinone becomes reduced here.  This is a different site than the P center and 
the ubiquinones are different.  This electron transfer forms a semiquinone that stays tightly 
bound to this site.  In a second reaction,  another ubiquinone binds at center P and 
undergoes the same reactions as before.  The low potential electron moves down through 
the two b hemes to the semiquinone and fully reduces it.  This ubiquinone must pick up 
two protons from the matrix side of the membrane, then it is released.

Note that two protons were released on the cytosolic side at the P center each time 
ubiquinone was oxidized.  Two protons were picked up at the matrix side for each two 
electrons that were passed down the b heme branch of the pathway.  The net movement of 
protons is 4 out at the cytosol and two in from the matrix for every two ubiquinones that 
get oxidized.  Remember that the net movement of protons out of the mitochondrion has to 
be paid for by free energy release of the electrons flowing down their pathways.  The 
majority must come from transfer to the Rieske iron-sulfur protein since it has by far the 
biggest drop in reduction potential (see fig 21.3 p. 679 2nd ed, fig. 20.3, p. 633 1st ed.).

The movement of protons in membrane proteins is being revealed by studies of bacteriorhodopsin.
Crystal structures at high resolution have caught this proton pump in action (Science 286 8 Oct 
1999 p.252-253 for commentary and p255-260 for article.  The takehome message is that water 
molecules are very important, and only small side chain motions are required.  These shift water 
networks and cause the pK of side chains to shift.  Thus the proton flows as the pK of one group 
goes up and the pK of another goes down.  

Inhibitors that bind to the bc1 complex.

Antimycin binds very tightly to the N center at the matrix side of the membrane and 
prevents electrons from reaching ubiquinone from the b hemes.  Therefore these two hemes 
get completely reduced, backed up with electrons that now have nowhere to go.
Stigmatellin binds at the P center, at the interface between the iron-sulfur protein and 
cytochrome b.  It prevents entry of the loosely bound ubiquinone and stops electron flow 
through the complex.  The b hemes and the other redox centers become fully oxidized as 
their electrons leave and cannot be replaced.

COMPLEX IV CYTOCHROME c OXIDASE

Cytochrome c oxidase has made many careers in biochemistry.  In 1995 the structure of 
the bacterial form was reported (Nature 376, 660 (1995) and in May 1996 the 
mammalian form was published in Science 272, 1136-1144 (1996).  These were landmark 
events.  Cytochrome oxidase does the hard chemistry of reducing oxygen to water that is at 
the heart of all aerobic life on the planet.  It is a proton pump like complex I and complex 
III.  It also has numerous redox centers as we should expect for another member of the 
electron transport chain.  

Cytochrome c oxidase has 13 subunits in mammals and 28 transmembrane segments.  Its 
molecular weight is about 210,000 Da.  3 subunits are from the mitochondrial genome and 
these contain all the redox centers.  The 10 nuclear coded subunits seem to be 
embellishments, for regulatory purposes, or insulation.  The protein is purified as a dimer, 
but this is probably not relevant to the function, since the bacterial enzyme is monomeric, 
and so are several eukaryotic forms.  

One surprising feature is the channel for oxygen to reach the active site.  This channel 
appears to open in the lipid bilayer.  This makes sense since oxygen is more soluble in the 
lipid than in water, but it was not suspected and it was not seen by the authors on the first 
paper.

How do the electrons get to cytochrome oxidase?  They come on a carrier.  This time the 
carrier is cytochrome c.  Cytochromes can only deliver one electron at a time, and it takes 4 
electrons to reduce oxygen, so 4 cytochrome c proteins have to bind sequentially to one 
docking site on the periplasmic surface of the protein.  Adding a charge to a protein 
interior, such as placing an electron into cytochrome c oxidase is thermodynamically 
expensive.  One way to compensate for the cost is to take up a proton.  This is called 
charge compensation.  According to Peter Rich, cytochrome c oxidase is an extreme 
example of charge compensation.  Four electrons are taken in and four protons are taken 
up.  This is linked to the role of cytochrome oxidase as a proton pump.  

What are the redox centers?  There are 2 copper centers called copper A and copper B.  
Please refer to Fig 21.17, p. 690 2nd ed., Fig. 20.17 , p. 643 1st ed..  Copper A actually has 
two copper ions near one another.  There are two hemes of the a type.  These are called heme a 
and heme a3.  There is also a magnesium ion and a zinc ion, though the role these play is not 
understood.

The copper A site is the first place the electron goes upon leaving cytochrome c.  
Experiments have been done that modify this site so it binds only a single copper ion 
instead of two.  The enzyme activity is nearly completely lost, so both coppers are required 
for function (Biochemistry 36, 3232-3236 1997).  From the copper A site the electron 
migrates to heme a then to heme a3.  Heme a3 and copper B form what is called the 
binuclear center.  The heme a3 Fe is very close to the copper B ion.  This is critical in the 
chemistry of the enzyme.  There is no obvious reason to have two heme "a"s in the 
enzyme. Both hemes are about equal distance from the copper A site and theoretically, the 
electron from Copper A could go directly to heme a3, but it does not.  The structure 
suggests a network that would favor electrons moving to heme a first.  Peter Rich, who is 
an expert in the thermodymanics of this enzyme, proposes that the reason there are two 
hemes has to do with charge compensation.  One site that takes up a proton during the entry 
of the four electrons is the copper B.  When an electron moves from heme a to the heme a3, 
copper B binuclear center, the proton that was associated with copper B is forced to leave.  
This might be part of the driving force for proton pumping.

Since 4 electrons have to come in to reduce oxygen, what is the order of addition?  (see 
Figure 21.20 p. 691 2nd ed., figure 20.20 on p. 644 1st ed)  Experiments with various types of 
spectroscopy show that 2 electrons enter the heme a copper B site, one on each metal ion, then 
oxygen binds between them and both electrons are added to the dioxygen molecule at the same 
time.  This forms a peroxy bridge between the two metal ions Fe-O-O-Cu.  Now two protons come in 
and one more electron and the O-O bond is broken.  Another 2 protons and another electron finish 
the reduction to water.  The two waters leave through a water channel.  

Note that while this is going on 4 more protons are being pumped across the membrane 
through the protein.  There are some candidate proton conductance networks that are 
proposed and these are being tested by site directed mutagenesis.  This is difficult to do 
since the three large subunits with all the channels are coded in the mitochondrial genome 
and there are no plasmid based systems for making these mutants in eukaryotes, so this 
work is being done on the bacterial system.

It is also important to know that the protons that react with oxygen to form water move 
through the protein by different routes than the protons that get pumped.  These are called 
chemical protons and pumped protons and they have to be kept separate.  

For a nice web site on oxphos, bioenergetics and ATP synthase try this link
http://bmbpcu01.leeds.ac.uk/illingworth.oxphos/index.htm
click  here