Complex I: also called NADH dehydrogenase or NADHQ reductase

1
Complex I also called NADH dehydrogenase or
NADH-Q reductase (transfers a pair of electrons
from NADH to coenzyme Q) This is a large enzyme
880 (kD) of at least 34 polypeptide chains The
initial reaction is the binding of NADH on the
matrix side of the inner mitochondrial membrane
and the transfer of its two high-potential
electrons to the flaven mononucleotide (FMN)
prosthetic group of this complex to give the
reduced form (FMNH2) electrons are then
transferred from the reduced FMNH2 to a series
of iron-sulfer clusters (Fe2 S2 and Fe4
S4) iron atoms in these clusters cycle between
Fe2 (reduced) and Fe3 (oxidized) states
There are seven or eight Fe-S clusters in
Complex I
Complex I
the final step of the reaction involves transfer
of 2 electrons from iron-sulfur clusters of
complex I to coenzyme Q where they are used to
reduce coenzyme Q
complex I is a proton pump (protons pumped from
matrix to intermembrane space)
A0251201
141
2

• Complex II also called succinate dehydrogenase
  or succinate- Q reductase
• the only citric acid cycle enzyme that is an
  integral membrane protein in the inner
  mitochondrial membrane
• has a mass of _at_ 100 - 140 kD and is composed of
  4 subunits
• when succinate is converted to fumarate in the
  citric acid cycle concomitant reduction of bound
  FAD to FADH2 occurs in succinate dehydrogenase
• this FADH2 transfers its electrons immediately to
  Fe-S clusters which then pass them on to Coenzyme
  Q

Complex II
Complex II and other enzymes that transfer
electrons from FADH2 to Q do not transport protons
142
3
Two other enzymes Glycerol phosphate
dehydrogenase and fatty acyl CoA dehydrogenase
(encountered later in the course) also transfer
their high potential electrons from FADH2 to
ubiquinol (QH2)
Note that complex I and complex II independently
transfer electrons to Q (not sequentially)
143
4
Coenzyme Q (also called ubiquitone or CoQ)

• has a long isoprenoid tail (making it lipophilic
  and able to diffuse in the hydrophobic core of
  the inner mitochondrial membrane)
• has electrons transferred to it from complex I
  and complex II, thereby reducing it
• CoQ shuttles electrons from complex I and complex
  II to complex III
• other flavoprotein dehydrogenases involved in
  fatty acid oxidation also transport electrons to
  CoQ
• CoQ is subsequently oxidized by cytochromes so it
  can be seen as a collection point for gathering
  electrons from various flavoprotein
  dehydrogenases and passing them along to
  cytochromes for ultimate transport to O2
• since CoQ oxidoreduction proceeds one electron at
  a time through a semiquinone intermediate, CoQ
  provides an interface between two-electron
  carriers and the one-electron cytochromes

144
5
Complex III
Complex III also called CoQ -cytochrome c
reductase or cytochrome bc1 complex or cytochrome
c - Coenzyme Q oxidoreductase reduced coenzyme
Q passes on its electrons to cytochrome c
through the Q cycle involves three different
cytochromes and an Fe-S protein complex III
drives proton transport (as with complex I,
passage of electrons through the Q cycle is
accompanied by proton transport across the inner
mitochondrial membrane from matrix to
intermembrane space)
145
6
Cytochromes
cytochrome is an electron-transferring protein
that contains a heme prosthetic group (group of
red or brown heme proteins having a distinctive
visible-light spectra) cytochromes were first
named and classified b, c, or a on the basis of
their absorption spectra which depends on the
structure and environment of their heme groups
(figure 15.5 Mathews and van Holde) note within
each class there are smaller spectral differences
and therefore subclassifications of b, c, or
a note these subclassifications depend on how
their heme groups are complexed their iron atoms
alternate between a reduced ferrous (2) state
and an oxidized ferric (3) state during electron
transport Q What prevents cytochromes from
binding oxygen (like other heme containing
molecules such as hemoglobin or myoglobin)? A
The iron in the porphyrin ring of cytochromes
(using cytochrome c as an example) is coordinated
both to a histidine nitrogen and to the sulfur
atom of a methionine residue which prevents
binding of oxygen and other ligands
146
7
Structure of complex III
The crystal structure of complex III and IV were
solved 1995-98 Providing a structural framework
to integrate biochemical observation
Complex III is a dimer of identical monomers Each
with 11 different subunits
Note the Rieske center One of the iron ions is
coordinated by two histidines instead of two
cysteine residues
147
8
Q cycle the mechanism for coupling of electron
transfer form Q to cytochrome c
Q cycle also facilitates the switch from the
two-electron carrier ubiquinol (QH2) to the
one-electron carrier cytochrome c
The cycle begins as ubiquinol (QH2) binds in the
Qo site (on complex III) Ubiquinol transfers its
electrons, one at a time One electron flows first
to the Rieske Fe2-S2 cluster, then to cytochrome
c1 and finally to a molecule of oxidized
cytochrom c, converting it to a reduced form (the
reduced cytochrome c is free to diffuse away from
the enzyme) The second electron is transferred
first to cytochrom bL then to cytochrom bH and
finally to an oxidized quinone bound in the Qi
site The quinone (Q) is reduced to a semiquinone
anion As the QH2 in the Qo site is oxidized to Q
its protons are released to the inner membrane
At this point, a semiquinone anion (Q.-) is
residing in the Qi site A second molecule of QH2
binds to the Qo site (displacing Q) and reacts in
the same way as the first (one electron to the
Rieske cluster etc and when the second electron
binds to Q.- This semiquinone anion takes up two
protons from the matrix side to form QH2 (the
removal of these two protons from the matrix aids
the formation of the proton gradient)
148
9
At the end of the Q cycle
Two molecules of QH2 are oxidized to form two
molecules of Q One molecule of Q is reduced to
QH2 Two molecules of cytochrome c are
reduced Four protons are released to the inter
membrane space Two protons are removed from the
mitochondrial matrix
149
10
Cytochrome C
a small protein (13 kD) associated with the inner
mitochondrial membrane and easily
extracted note this is unique in that the other
cytochromes are integral membrane proteins and
difficult to extract electrons traversing
Complex III are passed through cytochrome c1 to
cytochrome c cytochrome c is a mobile electron
carrier which mirgrates along the membrane
surface in the reduced state, carrying electrons
to complex IV
1410
11
Complex IV

• Complex IV also known as Cytochrome c Oxidase
• transmembrane protein complex consisting of up to
  13 subunits (204 kD)
• (in eukaryotes, the largest three subunits are
  encoded by mitochondrial DNA and synthesized
  within the mitochondria and inserted into the
  inner membrane from the matrix. The smaller
  subunits are coded by nuclear DNA and synthesized
  in the cytosol)
• accepts electrons from cytochrome c and directs
  them to the four-electron reduction of 02 to form
  H20
• (the ultimate step in electron transport the
  reduction of oxygen to water)
• 4 cyt c (Fe2) 4 H O2 4 cyt c (Fe3)
  2 H20
• (four consecutive one electron transfers)

1411
12
Structure of Complex IV Cytochrome Oxidase
contains two heme centers (cyt a and cyt a3) and
two copper atoms which participate in electron
transfer by cycling between the reduced cuprous
(Cu) and the oxidized (cupric) Cu2 state
the reduction of oxygen in complex IV is
accompanied by transport of protons across the
inner mitochondrial membrane from matrix to
intermembrane space
1412
13
1413
14
1414
15
Calculating the G0 for the complexes of the
electron transport chain from standard reduction
potentials
NADH CoQ NAD
CoQH2 (reduced) (oxidized)
(oxidized)
(reduced)
Break down the equation into 2 half
reactions Written in the direction of reduction
Now subtract the half reaction that is actually
being oxidized (or change the sign and add)
and DEo 0.36V
Finish the calculation and DGo -70kJ/mol
1415
16
calculate the G0 for the remaining complexes of
the electron transport chain from standard
reduction potentials
oxidation of FMNH2 by coenzyme Q (this
example is on the previous slide) oxidation of
cytochrome b by cytochrome c1 cytochrome oxidase
reaction
1416
17
The energy of electron transport is efficiently
conserved in a proton gradient
The transfer of two electrons from NADH through
the respiratory chain to molecular oxygen can be
written as
NADH H ½ O2 NAD
H2O
½ O2 2 H H2O 0.82
DEo 1.14V
Much of this energy is used to pump protons out
of the matrix
For each pair of electrons transferred to
O2 Complex I pumps 4 protons Complex III pumps 4
protons Complex IV pumps 2 protons
1417
18
The electrochemical energy inherent in this
difference in proton concentration and separation
of charge represents a temporary conservation of
much of the energy of electron transfer

• 1 chemical potential energy due to difference
  in concentration of a chemical species (H) in
  the two regions separated by the inner membrane
• pH of the matrix is about 1.4 pH units higher
  than intermembrane space
• 2 electrical potential energy that results from
  the separation of charge when a proton moves
  across the membrane without a counterion
• (which can be measured)
• Both of these make up the Proton Motive Force
  (electrochemical gradient)

When you do the calculations you an answer in
positive volts Which means what in terms of DGo
1418
19
Inhibitors of electron transport
inhibitors Rotenone a plant product used as an
insecticide that blocks electron flow from NADH
to coenzyme Q (in some parts of the world the
roots of certain trees are beat along riverbanks
to release rotenone into the water where it
paralyzes fish and makes them easy to
catch) Amytal and other barbituates as well as
demerol act at the same site as
rotenone Antimycin A a Streptomyces antibiotic
that blocks electron flow from cytochrome b to
cytochrome c1 Cyanide, azide, carbon monoxide
are all cytochrome oxidase inhibitors (cyanide
and azide bind tightly to the ferric form of
cytochrome a3 while carbon monoxide binds only to
the ferrous form (the inhibitory actions of
cyanide and azide are very potent at this site,
whereas the principle toxicity of CO arises from
its affinity for the iron of hemoglobin because
animals have lots of hemoglobin compared to
cytochrome a3 - it only takes a little cyanide
to kill you while it takes a lot of CO)
1419
20
The effect of inhibitors can be followed by
looking at an absorbance spectra
When antimycin is added to mitochonria The
electron flow from cytb to cyt c1 is
blocked This inhibition causes all the electron
carriers beyond cyt b to become fully
oxidized While NADH, fMN (flavin) and cyt b are
reduced
1420