Myoglobin function reassessed
Department of Physiology and Biophysics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA
* Author for correspondence (e-mail: bwitten{at}aecom.yu.edu)
Accepted 13 January 2003
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Summary |
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Key words: myoglobin, oxygen, facilitated diffusion, dimensionality in diffusion, heart, red skeletal muscle, nitric oxide, mitochondria, cytochrome oxidase, Krogh cylinder
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Introduction |
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Within the sarcoplasm of the cardiac myocyte or of red skeletal muscle
fibers, translational diffusion of oxymyoglobin molecules, each carrying
pickaback a diatomic oxygen molecule (with an equal back flow of
deoxymyoglobin molecules), is believed to support a flux of oxygen from the
sarcolemma to the mitochondrial surface. The molecular mechanism of this
process, which has been called myoglobin-facilitated oxygen diffusion, has
been elucidated for solutions of hemeproteins that bind oxygen reversibly
(Wyman, 1966; Wittenberg,
1966
,
1970
; Murray,
1971
,
1977
;
Keener and Sneyd, 1998
). The
mechanisms of intracellular oxygen diffusion have not been described. We note
that oxygen is very insoluble in water, and that the ratio of myoglobin-bound
oxygen to free oxygen approximates 30:1 within working vertebrate heart or
muscle cells at 37°C. Accordingly, a large fraction of the oxygen flux
through the cytoplasm must be myoglobin supported. More impressively, in the
legume root nodule the cytoplasmic leghemoglobin concentration may exceed
millimolar; the dissolved oxygen concentration is vanishingly small
(10-8 mol l-1) and the ratio of leghemoglobin-bound
oxygen to free oxygen exceeds 105:1. Essentially all of the oxygen
flux must be leghemoglobin mediated.
Here, we describe myoglobin-augmented oxygen supply to heart and red muscle, taking into account their three-dimensional structures and the elevated concentration of myoglobin in the cytoplasmic domain to which it is restricted and recognizing the large area of mitochondrial surface available for oxygen diffusion. [A mathematical formulation of oxygen diffusion in the cardiac myocyte will be presented elsewhere.] Heart and muscle, having available an almost unlimited supply of oxygen, actually operate at controlled low oxygen pressure, at or near 0.33 kPa (2.5 torr), where myoglobin is about half-saturated with oxygen. Partial saturation of myoglobin enables oxymyoglobin to play a pivotal role; by converting endogenous nitric oxide to the innocuous nitrate, oxymyoglobin controls the level of nitric oxide (NO) within the cell. This, in turn, may control both the rate of capillary oxygen delivery to the cell and the rate of oxygen utilization by cytochrome oxidase.
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Formulations of oxygen diffusion in muscle |
---|
Krogh
(1919a,b
)
and later Hill (1928
)
considered that oxygen flowed from the capillary down a continuous gradient of
oxygen pressure towards a plane (or cylinder), about half-way between two
capillaries, where oxygen pressure would be minimal. Groebe
(1995
) has expanded this
model, and his treatment is used in the recent calculations of Gros and
collaborators (e.g. Jurgens et al.,
2000
). However, the Krogh cylinder model is not in accord with
present day concepts of oxygen gradients in muscle. Currently, gradients of
oxygen pressure around the capillary are thought to be discontinuous. A large
oxygen pressure drop across the capillary wall is followed by a very shallow
gradient across the sarcoplasm. Furthermore, the simple model of diffusion
between two concentric cylinders is not in accord with electron micrographs
showing a convoluted oxymyoglobin diffusion path and a very large area of
mitochondrial surface.
Wyman (1966), working with
Wittenberg's (1966
) data,
formulated a description of the, then relatively new, phenomenon
carrier-mediated oxygen transport. Wyman's equation was solved analytically by
Murray (1971
,
1977
; see
Keener and Sneyd, 1998
) and
used to construct profiles of oxygen concentration within muscle cells.
Independently, Kreuzer and Hoofd
(1970
) devised a nearly
identical equation and solved it with computer assistance (reviewed in
Kreuzer, 1970
). It would be of
great interest to adapt Wyman's description to our current understanding of
muscle and cytoplasmic structure.
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The requirement for myoglobin |
---|
Blockade of myoglobin function in mammalian or avian skeletal muscle
sharply decreases oxygen uptake and work output (reviewed in
Wittenberg and Wittenberg,
1989). Blockade of myoglobin mimics hypoxia, monitored by the
ratio of mitochondrial NADH/NAD in isolated cardiac myocytes stimulated
electrically to contract (White and
Wittenberg, 1993
). Blockade of leghemoglobin function sharply
decreases bacteroidal oxidative phosphorylation within the soybean
(Glycine max) root nodule
(Bergersen et al., 1973
).
Shallow radial gradients of oxygen pressure, visualized as myoglobin
oxygenation or NAD(P)H reduction in the central region of isolated cardiac
myocytes (Takahashi et al.,
1998
,
2000
;
Takahashi and Asano, 2002
),
are abolished by blockade of myoglobin function.
Mice without myoglobin
Knockout of the myoglobin-encoding gene
(Garry et al., 1998;
Godecke et al., 1999
) induces
multiple compensatory mechanisms that tend to steepen the oxygen pressure
gradient to the mitochondria (Godecke et
al., 1999
; Grange et al.,
2001
; Meeson et al.,
2001
). These include a higher capillary density, smaller cell
width, elevated hematocrit and increased coronary flow and coronary flow
reserve. Transitions of type I to type II fiber types and increased expression
of hypoxia-inducible transcription factors (HIF)-1
and HIF-2
(endothelial PAS domain protein), heat shock protein 27 and endothelial growth
factor were also observed, and these tend to increase energy supply when
oxygen is limiting. Taken together, these compensations demonstrate that
myoglobin, when present, assures the oxygen supply of normal heart and
muscle.
Experiments in which myoglobin function is abolished acutely suffer the
criticism that the inhibitor or blocking agent may have effects other than
those intended. Hearts from myoglobin-knockout mice served as the ideal
control in a study of acute carbon monoxide inhibition of myoglobin in the
isolated mouse heart (Merx et al.,
2001). The results provide conclusive direct evidence that
myoglobin is required to assure oxygen flow from the vasculature to
mitochondrial cytochrome oxidase.
Fish without myoglobin
Antarctic ice-fishes of the family Channichthyidae lack blood hemoglobin
and circulating red blood cells. Some species lack cardiac myoglobin as well.
The mechanical performance of isolated perfused hearts from two very similar,
congeneric channichthyids show little difference at normal work loads, but
that of the species with myoglobin is far more able to maintain cardiac output
in the face of the additional insult of increased aortic arterial pressure
(Acierno et al., 1997;
Sidell, 1998
). Channichthyid
fish without myoglobin served as a control of the effects of nitrite in an
experiment demonstrating decreased cardiac function following blockade of
cardiac myoglobin (Acierno et al.,
1997
).
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Myoglobin supports oxidative phosphorylation |
---|
|
Most of the oxygen used by the soybean root nodule is consumed by the
terminal oxidases of intracellular bacteroids, the plant mitochondria being
relatively sparse. In turn, most of the ATP produced by bacteroidal terminal
oxidases is consumed by the intrabacterial enzyme nitrogenase; hence,
nitrogenase activity can be used as a measure of the rate of ATP formation.
Carbon monoxide blockade of leghemoglobin in the living, plant-attached nodule
causes nitrogenase activity to collapse
(Bergersen et al., 1973). In an
in vitro system using isolated bacteroids, the rate of ATP
production, measured as nitrogenase activity, was proportional to the
concentration of leghemoglobin added
(Wittenberg et al., 1974
).
These experiments show that ATP generation is leghemoglobin dependent.
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Myoglobin |
---|
Myoglobin in the heart is generally close to 200300 µmol
kg-1 wet mass tissue but may reach 400500 µmol
kg-1 wet mass in skeletal muscles. Since myoglobin is excluded from
mitochondria (35% of cell volume) and the sarcoplasmic reticulum (4% of the
cell volume), the concentration in the remaining volume of the heart cell
becomes 330 µmol l-1. The extent to which myoglobin penetrates
the myofibrillar volume (47% of the cell volume) is not known, but, in view of
the intimate association of mitochondria with the contractile elements of
cardiac muscle (Fig. 2A), we
shall assume that it must. Leghemoglobin, 380 µmol kg-1 wet mass
in nodules, is at a concentration of approximately 700 µmol l-1
in the space to which it is confined
(Wittenberg et al., 1996).
Myoglobin concentration increases with the work to which the muscle is put and
should be regarded as optimized for the particular muscle at a particular rate
of work output.
|
The rates of reaction of myoglobin/leghemoglobin with oxygen are subject to
natural selection. For instance, Gibson et al.
(1989) have found that an
array of disparate leghemoglobins have strikingly similar, rather slow rates
of oxygen dissociation. These rates determine the length of the path explored
during the random walk of an oxymyoglobin molecule, as discussed below. The
rate constants for oxygen combination are close to the maximum achievable.
Oxygen affinities in the nanomolar range are achieved, and the oxygen pressure
in the functioning nodules, close to 0.03 kPa (0.02 torr; equivalent to
approximately 10 nmol l-1) provides a suitable environment for the
highly oxygen-intolerant bacterial nitrogenase system.
Oxygen affinity is also subject to genetic selection pressure. The oxygen
affinities and oxygen dissociation rate constants of myoglobins from
predacious, oceanic fish that maintain muscle temperatures well above ambient
are similar to those of a related fish whose muscle operates at cool ambient
temperature, when compared at the operating temperature of the muscle
(Cashon et al., 1997;
Marcinek et al., 2001
).
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Diffusivity of myoglobin |
---|
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The diffusion path |
---|
Because of its simpler cytoarchitecture, heart muscle is favored over
skeletal muscle for construction of a model of oxygen inflow. [A mathematical
formulation of oxygen diffusion in the cardiac myocyte will be presented
elsewhere.] Within the heart muscle cell, mitochondria are arrayed in long
columns parallel to the long axis of the cell and often about one sarcomere in
length. In cross-sections of heart muscle, the columns of `...mitochondria
are not randomly distributed but are so evenly spaced as to suggest that each
serves only a very limited area of the myofilament mass immediately
surrounding it.' (Fawcett and McNutt,
1969; Fig. 2A).
Simple diffusion may suffice to distribute ATP, newly generated in the
mitochondria, throughout this limited area
(Meyer et al., 1984
).
Morphometric analysis, assuming a hexagonal array of capillaries, detects
increased density of the mitochondrial columns in a band about 45 µm
removed from each capillary (Kayar et al.,
1986
).
The oxygen dissociation rate constant, together with the diffusion
coefficient, determine the distance traveled in the random walk of myoglobin
molecules during the time that an oxygen molecule is resident. A remarkable
feature of the random walk is that `...a diffusing particle that finds
itself in a given region of space is destined...to wander around that region
for a time, probing it rather thoroughly before wandering away for good.'
(Berg, 1983). The mean radii of
the region of cytoplasm explored by oxymyoglobin molecules during the time
that an oxygen molecule is resident are given in
Table 1. These radii are large
compared with the narrow spaces available between mitochondria in muscle or
symbiosomes in the root nodule. If diffusing oxymyoglobin molecules are for
the most part reflected off the surfaces of the confining mitochondria, the
volume explored will be flattened from something like a sphere to something
more like a disc, whose radius will be greater than that of an unconstrained
sphere. Oxymyoglobin molecules will be displaced further in the plane of the
confining mitochondrial surface during the residence time of an oxygen
molecule, and myoglobin-facilitated oxygen diffusion will be accelerated. An
elegant description of this effect (Adam
and Delbruck, 1968
) is that the dimensionality of oxymyoglobin
diffusion is reduced from three dimensions towards two dimensions.
Two-dimensional diffusion is much more rapid than three-dimensional
diffusion.
|
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Myoglobin operates in states of partial oxygenation |
---|
Leghemoglobin is approximately 80% deoxygenated in the living root nodule
(Appleby, 1969), and the
fractional oxygenation of leghemoglobin in the living, plant-attached nodule
responds immediately to a step change in ambient oxygen pressure but reverts
in minutes to the original value. (Klucas
et al., 1985
).
Millikan (1937), using an
oximeter that he had devised for the purpose, reported extensive deoxygenation
of myoglobin in skeletal muscle in situ as it was brought into
maximal contraction. Subsequent studies of the beating heart, either in
situ or saline perfused (e.g. Fabel
and Lubbers, 1965
; Hassinen et
al., 1981
), fully confirm Millikan's finding.
Cryomicrospectrophotometry of rapidly frozen tissue permits quantification of
myoglobin saturation (Voter and Gayeski,
1995
). Myoglobin in the in situ beating heart is
maintained near half-saturation in the face of a 20-fold change in work
output, a 5-fold change in heart rate and a 2-fold change in arterial oxygen
content (Gayeski and Honig,
1991
). Likewise, myoglobin saturation in red skeletal muscle,
working near maximum sustainable oxygen consumption, is controlled near 50%
saturation (Gayeski and Honig,
1986
,
1988
).
A quantitative nuclear magnetic resonance (NMR) study showed that myoglobin
saturation (near 76%) in the blood-perfused isolated heart was held invariant
in the face of an 8-fold increase in heart rate
(Jelicks and Wittenberg,
1995). A non-invasive NMR study of hard-working human leg muscle
reports 50% deoxygenation of myoglobin, with sarcoplasmic oxygen partial
pressure (PO2) near 0.32 kPa (2.4 torr;
recalculated from Richardson et al.,
2001
, taking P50=0.32 kPa at 37°C;
Schenkman et al., 1997
). On
the other hand, the consensus of recent NMR studies of the in situ
beating heart is that cardiac myoglobin may be only about 10% deoxygenated
under basal conditions (Zhang et al.,
2001
).
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The sarcolemmal boundary |
---|
Very shallow gradients of oxygen pressure encountered within the sarcoplasm
of isolated cardiac myocytes (Katz et al.,
1984; Wittenberg and
Wittenberg, 1985
) suggest that the largest part of the oxygen
pressure drop from the erythrocyte to the mitochondria of cardiac and red
skeletal muscle, approaching 2.7 kPa (20 torr), must be ascribed to the
pressure drop across the capillary wall
(Landis and Pappenheimer,
1963
; Wittenberg and
Wittenberg, 1989
). Partially desaturated sarcoplasmic myoglobin,
observed only micrometers away from capillaries, once again indicates that the
pressure drop from erythrocyte to sarcoplasm is large (Gayeski and Honig,
1986
,
1988
; Honig et al.,
1984
,
1992
).
The large oxygen pressure difference across the capillary wall, say
2.73.3 kPa (2025 torr), will not be much affected by small
changes in sarcoplasmic oxygen pressure. Accordingly, control over rate of
oxygen entry into the myocytes will be vested almost entirely in the number of
capillaries open at any one time (Krogh,
1919a,b
).
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The mitochondrial boundary |
---|
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Tissue oxygen supply is not limiting |
---|
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Sarcoplasmic oxygen pressure gradients |
---|
Furthermore, the sarcoplasmic oxygen pressure that supports half-maximal
mitochondrial function in isolated cardiac myocytes
(Wittenberg and Wittenberg,
1985) does not differ from that required to support half maximal
respiration of isolated cardiac mitochondria (J.B.W. and B.A.W., unpublished).
[A different relationship between oxygen uptake and ambient
PO2 is reported for isolated cardiac myocytes
in transient states of changing PO2
(Rumsey et al., 1990
).]
Independently, Gayeski et al.
(1987
) conclude that the
PO2 experienced by cytochrome oxidase is
virtually identical to mean sarcoplasmic oxygen pressure of red muscles
working near maximal oxygen uptake. Finally, the demonstration by Vanderkooi
et al. (1990
), using two
luminescent probes, one membrane bound and one in solution, that the oxygen
pressure in the mitochondrial membrane proper does not differ from that in the
immediately adjacent solution, completes the proof that the oxygen pressure
experienced by mitochondrial cytochrome oxidase is very nearly the same as
that established in the equilibrium between sarcoplasmic myoglobin and
oxygen.
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Cytochrome oxidase |
---|
Cytochrome oxidase is only partially (approximately 10%) reduced in resting
cardiac myocytes (Wittenberg and
Wittenberg, 1985). However, cytochrome oxidase (as CuA)
in red skeletal muscle contracting in situ becomes reduced in
proportion to increasing workload to about 90% reduction at maximum oxygen
uptake (Duhaylongsod et al.,
1993
; Boushel and Piantadosi,
2000
). If this effect reflects recruitment of motor units, we may
consider that the oxidase is largely reduced in each contracting myocyte. The
effect is to accelerate the combination of oxygen with cytochrome oxidase as
respiratory demand increases.
Fully reduced cytochrome oxidase combines very rapidly with oxygen to form
an oxygenated intermediate (Chance et al.,
1975; Verkhovsky et al.,
1996
). Equilibrium binding of oxygen in this complex is weak and
reversible at room temperature, but operational irreversibility is achieved by
kinetic trapping, i.e. fast electron transfer to the oxygen-bound center
(Chance et al., 1975
;
Verkhovsky et al., 1996
). The
operational Km for oxygen (the concentration yielding
half-maximal steady-state turnover) is not a constant but rather is affected
by many parameters and is linearly related to the flux of electrons through
the system (Chance, 1965
). The
operational Km in state III pigeon (Columba
livia) heart mitochondria, supplied with oxygen from oxymyoglobin or
other oxygenated heme proteins, is close to 0.09 µmol l-1 at
25°C [equivalent to 0053 kPa (0.04 torr) oxygen pressure; J.B.W. and
B.A.W., unpublished]. Competition between nitric oxide and oxygen for binding
to the heme a3/CuB reaction center of cytochrome oxidase
will tend to increase the effective operational Km in the
heart from 0.09 µmol l-1 to a value within the range of
myoglobin-buffered oxygen pressures obtaining in the sarcoplasm
(Moncada and Erusalimsky,
2002
). In vivo, sarcoplasmic oxygen pressure may, in
part, control the rate of reaction of cytochrome oxidase with oxygen.
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Oxymyoglobin controls oxygen utilization and supply |
---|
The interaction of NO and oxymyoglobin to control cardiac oxygen
utilization is demonstrated dramatically in a study by Flogel et al.
(2001) of the
myoglobin-knockout mouse. Firstly, they demonstrated using NMR spectroscopy
that infused NO actually converted oxymyoglobin to ferric myoglobin in the
surviving heart. They next demonstrated that infusion of NO, or of bradykinin
to stimulate endogenous NO formation, brings about a dramatic fall of coronary
perfusion pressure in hearts lacking myoglobin; myoglobin-containing hearts
from wild-type mice were little affected. The clear explanation is that
oxymyoglobin in the wild-type heart scavenges NO, a powerful vasodilator that
increases blood flow and the number of open capillaries.
At higher concentrations of infused NO, cardiac contractility and
high-energy phosphate reserves were severely affected by NO in hearts isolated
from mice lacking myoglobin and, less so, in hearts from wild-type animals
(Flogel et al., 2001). As
already noted, NO is a potent but reversible inhibitor of cytochrome oxidase
(Moncada and Erusalimsky,
2002
). The probable explanation of the results of Flogel et al.
(2001
), as pointed out by
Brunori (2001b
), is that
intracellular oxymyoglobin, when present, continuously removes NO, thus
relieving inhibition of cytochrome oxidase.
The magnitude of the protective effect of oxymyoglobin on cytochrome
oxidase activity was demonstrated in an experiment using isolated heart cells
held at high oxygen pressures that are sufficient to fully oxygenate
intracellular myoglobin. In this condition, oxygen availability does not limit
respiratory rate, and myoglobin-facilitated oxygen diffusion contributes no
additional oxygen flux. Progressive conversion of intracellular oxymyoglobin
to carbon monoxide myoglobin (MbCO) now abolishes about one-third of the
oxygen consumption (Fig. 3A).
The oxymyoglobin-dependent portion of the oxygen uptake (defined in the legend
to Fig. 3A) decreases linearly
with increasing mole fraction of intracellular MbCO
(Fig. 3B). The probable
explanation (Brunori, 2001a)
is that intracellular oxymyoglobin continuously removes NO, a reversible
inhibitor of cytochrome oxidase. In accordance with the results of Flogel et
al. (2001
), the effect is
large, and about one-third of the total oxygen flux is dependent on
oxymyoglobin-mediated dioxygenation of NO. The histological location and the
isoform identity of the nitric oxide synthase forming the NO that controls
cardiac respiration remain matters of vigorous controversy
(Loke et al., 1999
;
Kanai et al., 2001
;
Moncada and Erusalimsky,
2002
).
|
These effects link intracellular oxymyoglobin, oxygen uptake by cytochrome oxidase, capillary oxygen delivery and intracellular NO generation into an integral controlled system. Any transient decrement in oxymyoglobin concentration will be countered by increased sarcoplasmic NO, increased oxygen input from the capillaries and decreased cytochrome oxidase activity, each tending to restore the initial oxymyoglobin level.
Pearce et al. (2002)
challenge the concept that oxymyoglobin regulates the level of NO in cardiac
muscle. They present evidence that NO endogenously generated in heart tissues
is catabolized with formation of neither nitrate nor ferric myoglobin, the
expected products of the reaction of NO with oxymyoglobin. Instead, they find
that NO is converted essentially quantitatively to nitrite. They explain this
on the basis of a plausible three-electron reduction of NO, catalyzed by
cytochrome oxidase. On this basis, they propose that cytochrome oxidase is the
major route by which NO is removed from mitochondria-rich cells. It must be
emphasized that there is absolutely no conflict between the data sets of
Pearce et al. (2002
) and those
of Flogel et al. (2001
) when
the experiments were performed under the same conditions.
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Towards a model for oxygen flow in muscle |
---|
Muscle and heart, despite access to almost unlimited oxygen, operate in steady states close to the oxygen pressure (0.33 kPa=2.5 torr) required for half-saturation of sarcoplasmic myoglobin with oxygen. In this steady state, the rate of oxygen utilization by cytochrome oxidase, the flux of oxygen across the sarcoplasm and the rate of oxygen entry into the muscle cell must all be the same. Sarcoplasmic oxygen pressure, we suggest, is a controlled parameter and resists change in response to changing workload or oxygen supply. Sarcoplasmic oxygen pressure, together with other parameters, including the concentration of cytochrome oxidase, the fraction of cytochrome oxidase molecules fully reduced and the operational Km of cytochrome oxidase for oxygen, determine the rate of oxygen utilization. Oxymyoglobin, by destroying NO, limits the blood flow supplying oxygen to the myocyte and relieves NO inhibition of mitochondrial oxygen utilization.
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Acknowledgments |
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