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Chapter 4. Oxygen
Oxygen and oxidative metabolism
Oxygen is drawn into the body down a concentration gradient maintained by
mitochondrial respiration, which continually converts molecular oxygen (O2) to water
(H2O). Each animal cell contains between hundreds and thousands of mitochondria (if
indeed they exist as distinct individual structures as opposed to a reticulum). The
gradient that is established between the atmosphere (~ 21% oxygen at sea level; equals
160 mmHg) and mitochondria also influences the steady-state concentration (partial
pressure) of oxygen around tissue cells. The organization of the process is encapsulated
in the figure below (from Gnaiger et al, J. Exp. Biol., 1998. Vol201, pp. 1129-1139).
There are a number of discrete steps in the pathway between atmosphere and
mitochondria. Because the lungs are a blind-ended sac, and air exchange with every
breath is <100% of total lung volume, the pO2 within the gas exchange structures
(alveoli) is lower than in the atmosphere, i.e. about 14% (~100 mmHg). Thus, cells that
comprise the lungs experience quite high pO2. However, cells in other body tissues do
not, in part because of the presence of haemoglobin.
In the alveoli, large quantities of O2 are bound by haemoglobin, present in red blood cells
that move through the alveolar capillaries. These RBCs then return to the heart (left
ventricle) to be distributed to the rest of the body by the pressure developed during
ventricular contraction.
Haemoglobin has two distinct functions: (1) increase the oxygen carrying capacity of
blood (amount of free oxygen in plasma is very low); (2) maintain pericellular pO2 at
relatively low values and stabilize pO2 between tissues.
The oxygen cascade (from Habler and Messmer, Transfusion Science, 1997. Vol 18, pp.
425-435):
There is close to no consumption of O2 as blood moves through the large conducting
vessels, such as arteries. However, once it enters local capillary beds where gas
exchange between blood and cells is actually occurring, a steep drop in blood pO2 occurs
as oxygen diffuses out of the blood and into cells. This diffusion is entirely passive,
driven by the ‘sink’ of O2 within cells. It is difficult to measure pO2 around tissue cells in
vivo (in the live animal), and even harder to measure pO2 within cells. However, the
former can and has been done for a variety of tissues in different physiological states.
Tissue pO2 is actually remarkably constant, at values between 20-40 mmHg (see above
figure from Habler and Messmer, 1997) across a wide range of tissues, and changing
little in many tissues (egs. brain, heart) regardless of the level of work being performed.
Within the cytosol of cells, average pO2 is even lower (as it must be since O2 is moving
down a gradient), at <20 mmHg. And, within the matrix of mitochondria where it is
finally consumed, pO2 is probably ~2 mmHg (equivalent to 3 μM O2; though very
difficult to measure, so this is a best estimate). Thus, cellular respiration operates at
relatively low pO2, which is nowhere near its level in earth’s atmosphere.
Mitochondria use oxygen as the terminal electron acceptor during oxidative
phosphorylation, the process whereby ADP is phosphorylated to resynthesize ATP.
The enzyme that consumes oxygen, by catalyzing the reduction of two molecules of
molecular oxygen to water, is cytochrome c oxidase (also known as complex IV).
Cytochrome c oxidase is a large, multisubunit enzyme complex (~200 kDa). The
measured Km of cytochrome c oxidase for O2 is about 0.07 μM. Functioning within the
context of intact mitochondria, the apparent Km of cytochrome c oxidase for O2 (often
given as p50) is measured as 0.1 to 1 μM, dependent on various aspects of the conditions
of measurement.
Thus, the ‘system’ is designed such that even though pericellular pO2 can be quite low,
mitochondrial oxygen consumption can proceed at a near maximal rate due to the
relatively high affinity for oxygen of cytochrome oxidase. The rate of mitochondrial
oxygen consumption probably begins to be limited by oxygen only when
intramitochondrial pO2 falls below about 0.1 μM.
The corollary of this is that the rate of mitochondrial respiration can not be increased
simply by increasing pO2, because usually O2 is already at levels that fully saturate its
binding site within cytochrome oxidase. One cannot increase respiration rate (in the short
term) simply by providing more oxygen.
There are some caveats:
Carbon monoxide can outcompete oxygen for its binding site and thus inhibit respiration
even above 0.1 μM O2.
Nitric oxide can also compete with oxygen for its binding site and thus increase the
apparent Km (O2).
This is a dynamical system. In most animal cells, there is no way to ‘store’ oxygen (but
see below), which necessitates its continual delivery. It must therefore be continually
supplied at exactly the same rate that it is used up, and a great deal of animal physiology
is aimed at regulating this balance. The rate at which O2 must be supplied to a cell is
dependent upon the rate at which that cell is cleaving ATP to ADP, the amount of O2
required to counter proton leak, and the amount that is consumed by non-mitochondrial
reactions. The rate of ATP turnover is quantitatively the most significant determinant of
respiration rate in most types of cells. It is also the most variable from moment to
moment in most cells. The rate of ATP usage is a reflection of the work the cell is
performing. ATP turnover is sustained by oxidative phosphorylation, i.e. the harnessing
of energy released during the oxidation of chemical compounds (from food) for
phosphorylation of ADP to ATP.
Dr. Peter Mitchell received the Nobel Prize in Chemistry in 1978 for his chemiosmotic
theory, first presented in 1961. The theory postulated that the linkage between food
oxidation and ADP phosphorylation was the production of a chemiosmotic gradient
between the matrix of mitochondria and the inner membrane space.
We now know much more about how this process works, including the path the electrons
take, the sites at which H+ are translocated from matrix to inner membrane space and the
paths they take to re-enter the matrix (From Brookes, Free Radic. Biol. Med., 2005. Vol
38, pp. 12-23).
We also now know how the gradient produced by oxidation/electron transport is used to
drive an enzyme called the ATP synthase. In 1997, John Walker and Paul Boyer shared a
Nobel prize for their work in elucidating the structure of the ATP synthase:
More recently, Masasuke Yoshida has demonstrated that the ATP synthase is actually a
rotary motor. As H+ pass through the core of the enzyme, the membrane-bound Fo
portion rotates and this is linked to the phosphorylation of ADP. Yoshida and colleagues
created a movie showing their experiment directly showing the rotary behaviour of the
synthase. View a movie of their experiment in action at:
http://www.k2.phys.waseda.ac.jp/F1movies/F1Prop.htm
Reactive oxygen, hyperoxia and oxidative stress
Reactive oxygen species
Too little oxygen is eventually fatal because without enough, oxidative phosphorylation
will be unable to match the rate of ATP depletion and [ATP] in the cytosol will fall.
ATP-dependent processes (maintenance of ion gradients, muscular contraction,
transcription and translation) will cease (see below). However, too much oxygen is
actually a substantial problem as well. Just as metal oxidizes (rusts) in the presence of
oxygen and salt, so will the molecules and structures that compose living cells oxidize
and become dysfunctional (and possibly even dangerous) in the presence of high levels of
oxygen and the salts that are abundant within tissues. The problem here is ‘reactive
oxygen’ (often referred to as reactive oxygen species, or ROS), which occurs when
oxygen inside of cells participates in reactions that activate it into a radical form (i.e. such
that it possesses an extra, unpaired electron). By far the most common such reaction
occurs with individual mitochondrial respiratory complexes. The respiratory complexes
are all very large hetero-oligomers of protein subunits that are contained in the
mitochondrial cristae. Their function is to catalyze the continual reduction/oxidation
(red/ox) reactions that are linked to the pumping of protons out of the matrix. If we think
about there being thousands of each individual respiratory complex present in each
mitochondrion, we can then recognize that, at any point in time, some portion of them
will be reduced and some portion oxidized. The complexes can only donate electrons to
O2 when they are in a reduced state.
The chemical species produced when O2 accepts an extra electron is superoxide, O2∙-:
O2 + e- → O2∙The production of superoxide by animal cells is not limited to mitochondria. A number
of enzymes can produce O2∙- , including glucose oxidase, xanthine oxidase, monamine
oxidase, cytochrome P450 oxidases, and the NADPH oxidase of cells that function in
immunity (NADPH oxidase produces huge quantities of superoxide to bombard foreign
material with free radicals, thus destroying them). However, in most cells, superoxide
production from mitochondria during respiration is thought to be quantitatively the most
important source of ROS. While this assertion must be viewed with caution, as it is
difficult to determine experimentally because ROS are inherently unstable and therefore
short-lived, it is supported by inferential evidence (see below)
Superoxide is not itself responsible for the most toxic effects of reactive oxygen. Rather,
the entry of superoxide into other reactions appears to be problematic. For example,
dismutation of superoxide gives rise to hydrogen peroxide (the stuff of the ‘bottle
blonde’) via the following reaction mechanism:
2O2∙- + 2H+ → O2 + H2O2
Hydrogen peroxide is uncharged and able to diffuse across membranes. Thus, ROS
produced within mitochondria can have effects at other sites within cells.
Nonetheless, as the sites of most ROS production in many cells, mitochondria are also the
most proximal targets of ROS. These organelles contain many iron-sulfur containing
enzymes (respiratory complexes, aconitase, pyruvate dehydrogenase and others).
Hydrogen peroxide can undergo the following reaction with divalent metal cations:
Fe2+ + H2O2 → Fe3+ + HO· + OHThe hydroxyl radical (HO·) is an extremely toxic free radical that will abstract protons
from a variety of really important macromolecules (like DNA!) and thus chemically
modify them (see below). The formation of this radical species is likely responsible for
much of the really lethal toxicity of ROS, including their mutagenicity.
Other reactions in which superoxide participates also lead to extremely toxic endproducts
with the ability to permanently damage cellular material (and thus effectively ‘kill’ cells).
One of the most important amongst these is the reaction between superoxide and nitric
oxide (NO). NO is synthesized in a variety of cells types, and functions as a paracrine
signal. However, NO levels are significant within mitochondria, possibly due to the
presence of a mitochondrial nitric oxide synthase. The following reaction:
O2∙- + NO → ONOOproduces the powerful oxidant peroxynitrite (ONOO-), which is perhaps the second most
toxic form of ROS.
It should be noted here that the production of superoxide and nitric oxide is normal and
necessary to the physiology of animals. ROS play important roles in signalling, amongst
other things. Acute toxicity occurs when either ROS levels are too high, or the wrong
forms are produced. As you will see below, this occurs in a very wide variety of human
diseases.
Antioxidant enzymes
You might expect that endogenously produced toxins potentially would be a significant
enough problem that cells would possess the ability to render them harmless. And you
would be right. Some of the most important enzymes in animal cells are antioxidant
enzymes. We will consider these in the order in which they encounter ROS.
Superoxide is unstable and will enter an uncatalyzed dismutation reaction producing
hydrogen peroxide (above). However, the superoxide dismutases (SODs), enzymes
present in virtually all animal cells, increase the rate of this reaction several thousand-fold
to rapidly remove superoxide from cellular compartments. There are two major isoforms
of superoxide dismutases: manganese SOD (MnSOD), which is localized within the
mitochondrial matrix, and copper-zinc SOD (CuZnSOD), which is localized within the
cytosol. MnSOD is apparently a very important enzyme. Disruption of the MnSOD gene
in mice or flies (Drosophila melanogaster) is incompatible with life. In both species,
there is essentially no ability to exist in an oxygen environment without this enzyme.
Interestingly, in both instances, the pathology involves massive neurodegeneration and/or
a loss of locomotor control, which hints toward the particular importance of MnSOD in
nervous tissue.
CuZnSOD dismutates superoxide in the cytosol to hydrogen peroxide. Disruption of
CuZnSOD is less acutely lethal, though also associated with truncated lifespan and
disease. A significant proportion of patients with familial amyotrophic lateral sclerosis
(ALS; Lou Gehrig’s disease) have CuZnSOD gene mutations that underly the
neurodegeneration associated with the disease.
Hydrogen peroxide produced by MnSOD within the mitochondrial matrix can diffuse out
of mitochondria and into the cytosol. Here, it can be completely detoxified by the
enzyme Catalase. Catalase is present within peroxisomes, small membrane enclosed
organelles that participate in the oxidation of some dietary fats. Catalase catalyzes the
following reaction:
2H2O2 → O2 + 2H2O
This reaction thus completes the cycle that molecular oxygen follows beginning with
accepting an extra electron during respiration, by returning molecular oxygen (though
note the stoichiometry: two molecules of O2 gave rise to one O2 and two H2O).
Nonetheless, the combined actions of SOD and Catalase effectively removed the ROS
from the cell.
There are other antioxidant enzymes that also contribute are to ROS detoxification within
cells, including glutathione peroxidase (GPx) and glutathione reductase (GR), two
enzymes that transfer reactive oxygen from H2O2 to glutathione. Glutathione is an
endogenous antioxidant. GPx catalyzes the conversion of H2O2 to H2O, linking this
reaction to the oxidation of glutathione (which donates the electron). GR then catalyzes
the reduction of the oxidized glutathione, linking it to oxidation of NADPH.
There are numerous isoforms of GPx, some of which are mitochondrial and other
cytosolic. Similarly, there are both mitochondrial and cytosolic pools of glutathione, and
GR. Thus, an entire ROS detoxification circuit is present in the mitochondrial matrix
(MnSOD, GPx and GR) and also within the cytosol (CuZnSOD, Cat or (GPx + GR)).
Note though that GPx+GR do not regenerate the original O2, though they do rid the cell
of its potentially toxic byproducts.
Mitochondrial ROS production
Mitochondrial superoxide production can be considered as an alternative branch to
electron transfer reactions during respiration, and indeed it is sometimes given the term
‘electron leak’. At any time, there are in the inner membrane pools of respiratory
complexes and associated coenzyme Q and cytochrome C, all existing in different
proportions of reduced versus oxidized states. In the reduced state, any of these
complexes is potentially able to donate a single electron to oxygen, though normally this
represents only a tiny fraction of electrons passing through the red/ox chain. It is
nonetheless often stated in the literature that approximately 2-5% of total electrons being
passed down the ETC suffer this fate. If you use this number in this class, you will lose
marks.
The actual proportion of electrons that leak from the ETC is almost certainly much, much
lower than 2%. The original measurements from which the 2-5% estimate is derived
used conditions that are not physiological: (1) they were made in atmosphere-saturated
media (~160 mmHg, or almost two orders of magnitude greater pO2 than mitochondria
are typically exposed to within most animal cells); (2) the experimental measurements
were made in isolated mitochondria respiring in state 4 (non-phosphorylating) under
which conditions membrane potential is higher (~160 mV) than is normal in most animal
cells (~120 mV); (3) the estimates have typically been made with mitochondria oxidizing
a single substrate at saturating concentrations, rather than a suite of substrates all at lower
than saturating concentrations as is likely in vivo; (4) in many instances, specific
respiratory poisons were used to exacerbate the rate of electron leak by blocking normal
transfer reactions. The importance of each of these points will be considered below.
Recall that the O2 consuming reaction catalyzed by cytochrome oxidase takes place
within the mitochondrial matrix, where pO2 is extremely low, about 2 mmHg. The
reaction producing superoxide has two substrates: an electron and O2 and, the less O2 is
available, the slower will be the rate of the reaction simply because the substrates will
encounter one another less often. Thus, the low physiological pO2 is actually quite
effective at keeping this spurious ‘electron leak’ reaction relatively contained. The graph
below shows what happens when mitochondria are isolated and the measurement of
electron leak is made at different values of pO2 (Turrens et al, Arch. Biochem. Biophys.
1982 Vol 217, pp. 401-410). Note that it is currently impossible to quantitatively
measure intramitochondrial superoxide production, because it is possible to measure the
hydrogen peroxide produced from superoxide that leaks out. The pO2 of media in which
cells are studied is actually an important consideration. Though tissue pO2 is relatively
invariant in vivo, many cell physiology experiments have been done at atmospheric pO2.
Also, cells are cultured in vitro typically under near-atmospheric pO2, which represents a
substantial hyperoxia and may impose a significant ‘oxidative stress’ on the cells.
Recall that ROS are produced also by a number of other reactions within the cell,
including those catalyzed by xanthine oxidase, monoamine oxidase, NADPH oxidase,
and cytochrome P450 oxidases. These reaction rates are strongly influenced by pO2 in
the near-physiological range because of they have Km(O2) that place the reaction in a
range that is highly responsive to this value (From Leach and Treacher, 1998, British
Med. J. 1998. Vol 317, pp. 1370-1373):
This is another reason why maintaining cellular pO2 at relatively low levels is important.
The rates of these other ROS-producing reactions increases markedly at higher pO2.
Note also the very low Km(O2) of cytochrome aa3 (part of cytochrome c oxidase, complex
IV). Thus, the ‘system’ is designed for very low pO2 to drive respiration at maximal rates
while maintaining the rates of ROS-producing reactions low.
The second consideration in interpreting measured rates of mitochondrial superoxide
production noted above is that membrane potential strongly influences the rate of electron
leak. This is shown below for isolated mitochondria in which membrane potential is
experimentally varied, while the rate at which hydrogen peroxide is produced is
measured (Korshunov et al, FEBS Lett, 1997. Vol 416, pp. 15-18). Note that membrane
potential (ΔΨ) is shown as % maximal and not as absolute values.
It is obvious from the graph above that the rate of mitochondrial ROS production
becomes very high when membrane potential is allowed to approach its maximal values.
This would happen when both proton leak and/or the rate of ATP synthesis are inhibited.
This may explain why mitochondria become very permeable to protons, thus limiting
membrane potential, as the membrane potential becomes high – it is likely very important
in preventing the destruction of mitochondria under some conditions.
Important sites of superoxide production in mitochondria (Brand et al, Free Radical
Biology and Medicine, 2004. Vol 37, pp. 755-767):
Three important sites of superoxide are: (1) the matrix face of complex I; (2) the matrix
side of complex III, and (3) the intermembrane side of complex III. Thus, during
respiration superoxide is released both within the matrix and into the cytosol from the
intermembrane space.
Oxidation of proteins:
ROS enter a wide variety of chemical reactions with amino acids within proteins. Some
of these are shown in the figure below.
Depending on where these occur, the outcome may be: (1) inactivation of the active site
by preventing interactions with substrate; (2) altering the conformation or flexibility of a
protein, thus preventing it from converting bound substrate to product; (3) modifications
that result in unfolding and complete denaturing of a protein. Many human diseases,
including several of the major neurodegenerative diseases, are characterized by
accumulation of oxidized proteins within cells. These have the potential to interfere
catastrophically with protein function and cellular homeostasis, and may underlie the cell
death that occurs.
Highly damaged and/or denatured proteins are themselves substrates for the proteasomal
degradation system. It is normal for protein oxidation to occur and, so long as there is
balance between this kind of damage and the removal/replacement of damaged proteins,
homeostasis is achieved and normal function is possible. The proteosome (shown below)
is a large, barrel-shaped multi-subunit structure with a hollow core that acts to
disassemble the damaged proteins to their component parts (amino acids) that can be
recycled. There are two layers each of 7 alpha-subunits and two layers each of 7 betasubunits, the organization of which is shown in the figure below. Oxidatively damaged
amino acids are either re-reduced, or simply destroyed.
Lipid peroxidation:
ROS such as the hydroxyl radical are strong oxidants that readily abstract hydrogen
atoms from the double bonds of phospholipid fatty acyl chains. This not only modifies
the structure of the effected fatty acid, but initiates a series of conformational changes
that eventually result in formation of a peroxyl radical. This species, also a powerful
oxidant, then perpetuates a chain reaction of hydrogen abstractions from neighbouring
fatty acids. Other molecules present in biological membranes are capable of quenching
the chain reaction and thus preventing the complete oxidation of the membrane. For
example, vitamin E (i.e. α-tocopherol) functions in this way. Oxidized membrane
phospholipids have altered properties that affect the functions of membrane-bound
proteins. They may: (1) directly oxidize membrane proteins via peroxyl radicals; (2) alter
the immediate environment of proteins that require specific fatty acyl chain compositions,
or (3) alter the fluidity and phase behaviour of the membrane.
Membrane phospholipids can be (and are) synthesized de novo on a continual basis to
replace oxidatively damaged molecules. This and prevention of damage limit the ROSmediated destruction of membranes.
Oxidation of DNA
Nucleotides are also vulnerable to oxidative damage from ROS generated during
respiration. This may either by oxidation of nucleotide pools which are subsequently
incorporated into DNA during replication, or RNA during transcription. Alternatively, it
may occur via chemical reactions that occur between ROS and nucleotides within DNA
strands. Examples of DNA oxidative damage include modifications of individual bases,
loss of a base, or strand breakage. All of these are potentially lethal to the cell; unlike
protein and lipids, DNA that is oxidized to the extent that it loses its coding fidelity
cannot be recovered from. Therefore, chemical base modifications are reversed and
repaired by a wide variety of enzymes that continually scan DNA to detect and remove
such damage. One of the most important such repair pathways is base excision repair.
By far the most studied individual form of DNA oxidative damage is 8-oxo-2deoxyguanine (8-oxodG; shown below), a product of guanine oxidation that is readily
measurable and can be quantified in various physiological states. DNA oxidation is
highly correlated with neurodegeneration, aging and apopototic cell death.
Oxidative stress: a dyshomeostasis of ROS metabolism
A large number of human pathologies have been linked to ‘oxidative stress’, i.e. an
imbalance between the rate of ROS production and its removal. For example,
Parkinsonism can be reproduced in mice simply by interfering with mitochondrial
respiration. Alzheimer’s disease causes massive cellular oxidative damage and
degeneration. Ischemia-reperfusion injuries such as stroke and heart attack are
characterized by oxidative damage, particularly during the reperfusion phase upon return
of blood flow. Thus, the study of oxidative stress in disease has grown rapidly in recent
years. Experimentally, oxidative stress is typically estimated by selecting one or several
biomolecular markers within the cell that are readily modified by ROS. These may
include protein carbonyls or DNA adducts such as 8-oxo-2-deoxyguanine.
Roles of ROS in intracellular signalling
It is interesting that, while knockout of the MnSOD gene is usually lethal, transgenic
overexpression of MnSOD or other antioxidant enzymes can also negatively affect
viability. One explanation for this may be that reactive oxygen species (and reactive
nitrogen species) also serve important functions as intracellular signals.
* This section under construction *
Hypoxia, oxygen sensing and HIF-1α
Early life on earth developed (and evolved) in the presence of much lower levels of
atmospheric oxygen than we presently have. Perhaps for this reason, in almost all
instances, animal cells work best at relatively low pO2. Despite the relatively low Km(O2)
of mitochondria, tissue hypoxia of sufficient magnitude to limit the rate of oxidative
phosphorylation can and does occur.
In people, this is typically associated with disease. In other animals, it occurs in the
course of daily life and these animals possess specific adaptations that allow them to
survive it. The central problem with hypoxia in any animal is, of course, the inability to
maintain cellular energy charge (i.e. [ATP]) via oxidative phosphorylation, and therefore
the inability to sustain energy-consuming processes, which are going. As we saw in
Rolfe and Brown (1997), a constant and substantial transduction of energy from food
constituents to ATP is required to energize processes like, for example, the maintenance
of critical ion gradients and protein turnover.
Many human cell types are extraordinarily sensitive to hypoxia. For example neurons of
the human brain can survive a substantially hypoxic environment for less than 3 minutes.
On the other hand muscle cells can survive hypoxia for periods of minutes to hours
(From Leach and Treacher, 1998, British Med. J. 1998. Vol 317, pp. 1370-1373).
This differential cellular ability to withstand hypoxia is related to the metabolic rates of
the cells, their ability to recruit anaerobic pathways of ADP phosphorylation, and their
ability to suppress ATP-consuming processes. These vary quite widely, not just between
cell types, but particularly between animal species. Humans are relatively hypoxia
intolerant, but there are many animals with substantial tolerance to hypoxia, including
diving mammals, and some reptiles, amphibians and fishes.
Hypoxia tolerant cells are capable of doing several things that other cells do not in
response to low pO2. They can rapidly mobilize pathways of anaerobic metabolism;
they may have ‘non-traditional’ pathways of anaerobic metabolism with endproducts
other than lactate; they must also be able to reduce energy expenditure (thus reducing
ATP and therefore O2 requirements). The mechanistic details of these will be discussed
below. First, we will look at an apparently universal mechanism for regulating the
cellular response to hypoxia in which one protein, hypoxia inducible factor (HIF-1) plays
a central role.
The story of the discovery of HIF-1α begins about 100 years ago, with physiologists
identifying a blood-borne factor they named erythropoietin (EPO). EPO is produced
primarily in the kidneys in response to hypoxia. This makes sense as the kidneys receive
a very substantial proportion of cardiac output (blood flow) and therefore can respond
rapidly to changes in blood oxygenation. EPO travels via the bloodstream to the sites of
red blood cell (RBC) production in bone marrow, where it stimulates erythrogeneis (the
synthesis of new blood cells). This is a fundamental component of the whole body
adaptation to hypoxia: increasing the O2 carrying capacity of blood. The vast majority of
blood O2 is held bound to haemoglobin, so the most effective way to increase the amount
of O2 that can be held in blood is to produce more of this protein and more RBCs to carry
it.
For many years, that was the end of the story. Various details of the conditions causing
EPO production and secretion were elucidated, as were aspects of erythrogenesis. About
15 years ago, after molecular biology tools had become available and their application to
physiological questions begun, the following question was asked and answered using
these tools: is EPO synthesis regulated at the level of gene transcription?
The EPO gene was cloned and, upstream of the protein coding sequence, regulatory
elements were identified. These are now called hypoxia response elements (HREs).
Deleting the HRE associated with the EPO gene prevents its hypoxic induction.

A byproduct of the identification and cloning of the EPO gene was the
development of a new way for high performance athletes to cheat. Recombinant
human EPO, produced by and purified from E. coli, has become a popular way
injection drug used to boost hematocrit, and therefore blood oxygen carrying
capacity, prior to endurance races. In effect, the body is ‘tricked’ into believing a
hypoxic condition exists and responds by initiating an adaptive response though
this is not actually necessary.
At this time the transcription factor that interacts with the HRE was not known. An
intermediary experiment was performed to answer the question: Do all cells have the
machinery required to induce genes with an HRE?
By splicing the EPO gene HRE onto a reporter gene and transfecting this into a variety of
cell types, it was shown that virtually all cells have the ability to respond to hypoxia by
inducing the expression of HRE-linked genes.
It turns out that more than 100 genes (in people) have HREs. The functions of these
genes vary fairly widely (partly because the HREs are actually involved in other
physiological responses that are not strictly hypoxia related), but includes genes whose
products catalyze anaerobic glycolysis and stimulate angiogenesis (formation of new
blood vessels).
The identity of the transcription factor that binds to the HRE remained unknown. To
identify it, electrophoretic mobility shift assays (EMSA; or gel shift) were performed on
an HRE-containing cDNA. The binding protein (which showed up in this experiment
only when a sample from cells exposed to hypoxia was used, but not when the sample
was taken from normoxic cells) was purified and identified as hypoxia inducible factor
(HIF).
Hypoxia inducible factor (now HIF-1; we will ignore other HIFs for now) is a functional
heterodimer, with an α- and β-subunit. It is only capable of binding to an HRE under
hypoxic conditions, as shown above (N = normoxia; H = hypoxia). Further study showed
that HIF-1β is constitutively expressed in cells, whereas HIF-1α is present only under
hypoxic conditions. The method for regulating HIF-1α levels is interesting. HIF-1α is
constitutively expressed but rapidly degraded under normoxic conditions. HIF-1α in a
normoxic cell has a half-life on the order of one minute! This is the shortest half-life that
has yet been observed for a protein. In a hypoxic cell, on the other hand, HIF-1α persists
much longer.
It turns out that a single post-translational modification of HIF-1α is responsible for
changing the half-life of the protein. This modification was identified by mass
spectrometry: hydroxylation of an amino acid, proline, at sequence sites 402 and 564.
This hydroxylation occurred only in normoxic cells, and it identifies the protein for
ubiquitin-tagging and proteosome-mediated degradation. In hypoxia, the hydroxylation
reaction does not occur, HIF-1α is not degraded. It accumulates and is thus able to bind
HIF-1β, and the HIF-1 heterodimer subsequently binds to HREs and initiate the genetic
program of hypoxia adaptation.
One final remaining question was: what causes the hydroxylation of prolines 402 and
564 in HIF-1α? The answer is prolyl hydroxylases (PHDs). These ubiquitous enzymes
directly catalyze the O2-dependent hydroxylation of prolines in HIF-1α.
PHDs are a family of proteins (enzymes), but all are notable for having relatively high
Km(O2), in the range of 230-250 µM. Significantly, this is approximately the
concentration of O2 in atmosphere-saturated ECF. As a result, PHD activity is extremely
sensitive to pO2 in the physiological range, in which a near-linear relationship between
pO2 and PHD activity exists. Thus, PHD is effectively the ‘oxygen sensor’ in this
system.
Now, the question is: do these PHDs work only with HIF-1α? Or, like the enzymes that
catalyze reversible phosphorylations, do they have a broad suite of protein substrates that
they can regulate via O2-dependent hydroxylation of proline residues? The entire field of
protein phosphorylation-dependent signal transduction opened with the identification of a
single reaction (for which Edmond Fischer and Edwin Krebs won the Nobel prize in
1992). Could it be that the characterization of HIF-1α has revealed a new mechanism of
intracellular regulation based on the catalyzed regulation of proline? There is some
suggestion amongst researchers in the field that this could indeed be the case.
Hypoxia tolerance in animals
The difference between hypoxia sensitive and hypoxia tolerant organisms does not
appear to be presence or absence of HIF-1α, but rather differences downstream of this
transcription factor, as well as fundamental differences in metabolic organization. To
mount a successful adaptive response to hypoxic conditions, cells must accomplish
primarily two goals: (1) mobilize sufficient anaerobic fermentative flux to augment the
deficiency in ATP synthesis as pO2 declines to rate-limiting levels; (2) decrease the rate
of ATP usage so that lower rates of ATP production can support all necessary metabolic
reactions (remember that anaerobic glycolysis is more than an order of magnitude less
efficient than oxidative phosphorylation). Cells that do these two things poorly, like the
human brain, have limited hypoxia tolerance and will rapidly die when oxygen supply
becomes limiting. Human cells that always have very low energy requirements (eg. cells
of cartilage) are better able to withstand hypoxia because they are already positioned to
achieve (2). Generally, however, human cells have limited scope for achieving these
goals. On the other hand, cells from many animal species readily withstand prolonged
periods of hypoxia, and it is informative to investigate the means by which they achieve
this.
Examples of animals that employ alternative anaerobic metabolic pathways and produce
anaerobic endproducts other than lactate are particularly common amongst invertebrates,
but also include fish.
Turtles, particularly the aquatic species Trachyma scripta elegans, have revealed much
about the basic strategies of hypoxia tolerance. Studies of this turtle and other hypoxia
tolerant species have taught us that, of the two goals outlined above, (2) is by far the most
important to long-term survival. Thus the ability to suppress cellular activities that
consume large amounts of ATP will allow cells (and therefore organisms) to withstand
hypoxic conditions for durations that would be lethal to human cells, which are unable to
make significant reductions of metabolic rate (at least not acutely). Recall from Rolfe
and Brown (1997) that some examples of high ATP consumers are: (1) the
Na+/K+ATPase; (2) protein turnover and (3) gluconeogenesis. The table below shows the
rate of ATP use by each of these cellular processes in turtle hepatocytes under normoxic
and completely anoxic conditions.
Anaerobic ATP synthesis in facultative anaerobes
** under construction **
Intracellular Oxygen Storage: Myoglobin and Other Globins
The imperative for any cell is to maintain [ATP] at a level that will support the
essential biochemical reactions that maintain the integrity of the cell. Failure to do so is
not compatible with life. When the continual supply of oxygen to the cell is interrupted
for any reason, the main supply of ATP on an ongoing basis, oxidative phosphorylation,
is no longer possible. Flux of carbon fuels through anaerobic glycolysis will increase to
provide the required ATP in the short term. In some animals, anaerobic pathways are so
well developed that this can be an effective strategy also in the longer term. However,
another strategy is adopted by some animals that routinely encounter interruptions in
oxygen supply: oxygen storage.
Perhaps the best examples of oxygen storage are diving marine mammals, such as
seals and whales. These animals store oxygen in two ways: (1) in red blood cells that are
sequestered until needed; (2) in myoglobin within tissue cells. With respect to the first
strategy, diving marine mammals have unusually large spleens, and it has been shown
that oxygenated red blood cells stored within the spleen are released during a dive, as
blood pO2 becomes depleted. With respect to the second strategy, diving marine
mammals (as well as hypoxia-adapted humans) have particularly high levels of
myoglobin, a protein that is ancestrally homologous to haemoglobin, and which can
effectively store oxygen. The figure below shows the correlation between myoglobin
content of skeletal muscle and the average duration of an individual dive in various
marine mammals. Note that there is a reasonably good relationship between these two
variables, suggesting the importance of myoglobin in extending dive duration. In fact,
many marine mammals can remain submerged for more than twenty minutes without
accumulating any anaerobic endproducts, such as lactate. This is because myoglobin is a
store of oxygen within their muscle tissue, which can be provided to mitochondria as
haemoglobin in the blood is gradually depleted of its oxygen and cellular pO2 begins to
decline.
The aerobic dive limit (ADL) is the length of time an animal can dive without recruiting
anaerobic metabolism (and therefore accumulating lactate). The figure above (from
Kooyman and Ponganis, Annu Rev. Physiol. 1998) shows data from Weddel seals, where
their blood was sampled immediately after a dive. For dives up to 20 minutes, there is
virtually no accumulation of lactate in the blood, indicating that the animals remain fully
aerobic. Thus 20 minutes is the ADL, and longer duration dives begin to recruit
anaerobic pathways that produce lactate.
In addition to this role in oxygen storage, myoglobin may also be important in facilitating
the transfer of oxygen from the cell membrane to the mitochondrial inner membrane
where it is utilized. In fact, an ongoing debate in the field relates to whether there is
effectively an intracellular oxygen circulation: are myoglobin molecules sufficiently
mobile to have a role in promoting oxygen transfer? There is some evidence that the rate
of oxygen diffusion is greatly increased by myoglobin, perhaps as oxy-myoglobin is
actively transferred from the surface of the cell to mitochondria. However, there is
currently insufficient data to conclusively answer this question.
Before continuing our discussion of myoglobin, it is important to have some
understanding of its molecular biology. While functional haemoglobin is produced from
four separate proteins (and is therefore a tetramer), myoglobin is a monomer. While
haemoglobin is found exclusively in red blood cells, myoglobin is found in striated
muscle (both cardiac and skeletal), but nowhere else. They have quite similar structures,
as shown in the figure below.
However, the two molecules have some very important functional differences as well.
For example, while haemoglobin has a sigmoidal oxygen binding curve, making it less
able to bind oxygen at lower pO2 values (so that oxygen can enter solution and be used
by tissue cells), myoglobin shows a standard Michaelis-Menten type kinetic with respect
to oxygen, meaning at low pO2 myoglobin will bind much more oxygen than
hemoglobin.
The functional implication of these different oxygen affinities is that oxygen will transfer
from haemoglobin, through the extracellular fluid, to myoglobin at low blood pO2. This
could, in effect, facilitate the transfer of oxygen from blood to mitochondria. It is also
important in myoglobin’s role in oxygen storage. At normal blood pO2, myoglobin will
sequester oxygen from the blood, thus storing it within the cell. Myoglobin does not
readily release this oxygen until cellular pO2 becomes quite low. However, this system
works satisfactorily because the enzyme that ultimately utilizes oxygen, respiratory
complex IV (cytochrome oxidase) has an even greater affinity for oxygen (see chapter ?).
Thus, if the oxygen supply to the cell is disrupted, oxygen stored within cells can meet
the needs of oxidative phosphorylation for a period of time.
The advent of molecular approaches to studying physiology has had a significant impact
on our understanding of myoglobin’s function. Firstly, the creation of mouse strains in
which the gene for myoglobin has been ‘knocked out’, thus rendering them incapable of
producing the protein, has revealed a potential role in the handling of other gases,
specifically nitric oxide (NO). NO is a potent vasodilator. However, it also has
numerous other functions, including the ability to inhibit respiratory complex IV. The
knockout studies implicate myoglobin’s ability to bind NO as being possibly important in
regulating complex IV activity via the removal of NO. Myoglobin may also have
important roles in scavenging reactive oxygen (free radicals) within cells.
Molecular approaches to studying myglobin have also led to another exciting
discovery: the identification of other related globins. With the arrival of numerous full
genome sequences as well as statistical approaches to analyzing similarities between
sequences (eg BLAST), several homologous proteins and their genes have been
discovered. These newly discovered proteins, neuroglobin and cytoglobin, have quite
different tissue distributions. Neuroglobin is found in nervous tissue, including in the
brain. Cytoglobin is found in various tissues. A sequence alignment of these proteins is
shown below, with conserved regions and some important residues and domains
indicated.
Based on sequence data, it is possible to reconstruct the evolutionary history of globin,
beginning from a hypothetical ancestral molecule. Neuroglobin is only distantly related
to the other globins. The functional role of neuroglobin may also be only casually related
to that of myoglobin. There is evidence that its importance lies in cellular antioxidant
defence, but the study of this new protein is ongoing and will surely lead to more
surprises.