The Journal of Experimental Biology 206, 2011-2020 2011 © 2003 The Company of Biologists Ltd doi:10.1242/jeb.00243
Review Myoglobin function reassessed Jonathan B. Wittenberg* and Beatrice A. Wittenberg Department of Physiology and Biophysics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA *Author for correspondence (e-mail: [email protected])
Accepted 13 January 2003
Summary The heart and those striated muscles that contract for oxygen pressure, buffered near 0.33·kPa (2.5·torr; long periods, having available almost limitless oxygen, equivalent to approximately 4·µmol·l–1 oxygen) by operate in sustained steady states of low sarcoplasmic equilibrium with myoglobin, falls close to the operational oxygen pressure that resist change in response to changing Km of cytochrome oxidase for oxygen, and any small muscle work or oxygen supply. Most of the oxygen increment in sarcoplasmic oxygen pressure will be pressure drop from the erythrocyte to the mitochondrion countered by increased oxygen utilization. The occurs across the capillary wall. Within the sarcoplasm, concentration of nitric oxide within the myocyte results myoglobin, a mobile carrier of oxygen, is developed in from a balance of endogenous synthesis and removal by response to mitochondrial demand and augments the flow oxymyoglobin-catalyzed dioxygenation to the innocuous of oxygen to the mitochondria. Myoglobin-facilitated nitrate. Oxymyoglobin, by controlling sarcoplasmic nitric oxygen diffusion, perhaps by virtue of reduction of oxide concentration, helps assure the steady state in which dimensionality of diffusion from three dimensions towards inflow of oxygen into the myocyte equals the rate of two dimensions in the narrow spaces available between oxygen consumption. mitochondria, is rapid relative to other parameters of cell respiration. Consequently, intracellular gradients of Key words: myoglobin, oxygen, facilitated diffusion, dimensionality oxygen pressure are shallow, and sarcoplasmic oxygen in diffusion, heart, red skeletal muscle, nitric oxide, mitochondria, pressure is nearly the same everywhere. Sarcoplasmic cytochrome oxidase, Krogh cylinder.
Introduction Myoglobin, a mobile carrier of oxygen, is developed in red molecules, each carrying pickaback a diatomic oxygen molecule muscle in response to mitochondrial demand for oxygen (with an equal back flow of deoxymyoglobin molecules), is (Millikan, 1939; Wittenberg, 1970; Williams and Neufer, believed to support a flux of oxygen from the sarcolemma to the 1996) and transports oxygen from the sarcolemma to the mitochondrial surface. The molecular mechanism of this mitochondria of vertebrate heart and red muscle cells process, which has been called myoglobin-facilitated oxygen (Wittenberg and Wittenberg, 1989; Takahashi and Doi, 1998). diffusion, has been elucidated for solutions of hemeproteins that Likewise, leghemoglobin, a protein similar to myoglobin but bind oxygen reversibly (Wyman, 1966; Wittenberg, 1966, 1970; with 10-fold greater oxygen affinity, transports oxygen from Murray, 1971, 1977; Keener and Sneyd, 1998). The mechanisms the cell membrane of the central cells of the legume root nodule of intracellular oxygen diffusion have not been described. We to the symbiosomes, which are membrane-bound intracellular note that oxygen is very insoluble in water, and that the ratio of organelles housing the bacteroids, the intracellular nitrogen- myoglobin-bound oxygen to free oxygen approximates 30:1 fixing form of the bacterium Rhizobium (Appleby, 1984). A within working vertebrate heart or muscle cells at 37°C. similar system is found in neural cells, including the Accordingly, a large fraction of the oxygen flux through the photoreceptors, of the vertebrate retina. Here, neuroglobin, a cytoplasm must be myoglobin supported. More impressively, cytoplasmic hemoglobin that has the classic globin fold but is in the legume root nodule the cytoplasmic leghemoglobin otherwise only distantly related to myoglobin (Pesce et al., concentration may exceed millimolar; the dissolved oxygen 2002), apparently mediates oxygen supply to the mitochondria concentration is vanishingly small (10–8·mol·l–1) and the ratio of (Schmidt et al., 2002). leghemoglobin-bound oxygen to free oxygen exceeds 105:1. Within the sarcoplasm of the cardiac myocyte or of red Essentially all of the oxygen flux must be leghemoglobin skeletal muscle fibers, translational diffusion of oxymyoglobin mediated. 2012 J. B. Wittenberg and B. A. Wittenberg
Here, we describe myoglobin-augmented oxygen supply to formed adaptively in tissues in response to the demand for heart and red muscle, taking into account their three- oxygen and that myoglobin contributes to the oxygen supply dimensional structures and the elevated concentration of of these tissues. Subsequent work (reviewed in Wittenberg, myoglobin in the cytoplasmic domain to which it is restricted 1970; Wittenberg and Wittenberg, 1989) bolsters Millikan’s and recognizing the large area of mitochondrial surface conclusion that: ‘muscle hemoglobin is generally found in available for oxygen diffusion. [A mathematical formulation large quantities in those muscles requiring slow, repetitive of oxygen diffusion in the cardiac myocyte will be presented activity of considerable force…and whose action must be elsewhere.] Heart and muscle, having available an almost maintained over long periods’. Possibly, low tissue oxygen unlimited supply of oxygen, actually operate at controlled pressure may initiate myoglobin formation. Certainly, low oxygen pressure, at or near 0.33·kPa (2.5·torr), where myoglobin messenger RNA is elevated under conditions myoglobin is about half-saturated with oxygen. Partial eliciting myoglobin formation (Williams and Neufer, 1996). saturation of myoglobin enables oxymyoglobin to play a Myoglobin is necessary to support cardiac function in the fetus pivotal role; by converting endogenous nitric oxide to the (Meeson et al., 2001). innocuous nitrate, oxymyoglobin controls the level of nitric Blockade of myoglobin function in mammalian or avian oxide (NO) within the cell. This, in turn, may control both the skeletal muscle sharply decreases oxygen uptake and work rate of capillary oxygen delivery to the cell and the rate of output (reviewed in Wittenberg and Wittenberg, 1989). oxygen utilization by cytochrome oxidase. Blockade of myoglobin mimics hypoxia, monitored by the ratio of mitochondrial NADH/NAD in isolated cardiac myocytes stimulated electrically to contract (White and Formulations of oxygen diffusion in muscle Wittenberg, 1993). Blockade of leghemoglobin function Present descriptions of oxygen diffusion/transport in tissues sharply decreases bacteroidal oxidative phosphorylation within originate from the studies of Krogh, Hill and Jeffries Wyman, the soybean (Glycine max) root nodule (Bergersen et al., 1973). to whom this essay is dedicated. Shallow radial gradients of oxygen pressure, visualized as Krogh (1919a,b) and later Hill (1928) considered that myoglobin oxygenation or NAD(P)H reduction in the central oxygen flowed from the capillary down a continuous gradient region of isolated cardiac myocytes (Takahashi et al., 1998, of oxygen pressure towards a plane (or cylinder), about half- 2000; Takahashi and Asano, 2002), are abolished by blockade way between two capillaries, where oxygen pressure would be of myoglobin function. minimal. Groebe (1995) has expanded this model, and his treatment is used in the recent calculations of Gros and Mice without myoglobin collaborators (e.g. Jurgens et al., 2000). However, the Krogh Knockout of the myoglobin-encoding gene (Garry et al., cylinder model is not in accord with present day concepts of 1998; Godecke et al., 1999) induces multiple compensatory oxygen gradients in muscle. Currently, gradients of oxygen mechanisms that tend to steepen the oxygen pressure gradient pressure around the capillary are thought to be discontinuous. to the mitochondria (Godecke et al., 1999; Grange et al., 2001; A large oxygen pressure drop across the capillary wall is Meeson et al., 2001). These include a higher capillary density, followed by a very shallow gradient across the sarcoplasm. smaller cell width, elevated hematocrit and increased coronary Furthermore, the simple model of diffusion between two flow and coronary flow reserve. Transitions of type I to type II concentric cylinders is not in accord with electron micrographs fiber types and increased expression of hypoxia-inducible showing a convoluted oxymyoglobin diffusion path and a very transcription factors (HIF)-1α and HIF-2 (endothelial PAS large area of mitochondrial surface. domain protein), heat shock protein 27 and endothelial growth Wyman (1966), working with Wittenberg’s (1966) data, factor were also observed, and these tend to increase energy formulated a description of the, then relatively new, supply when oxygen is limiting. Taken together, these phenomenon carrier-mediated oxygen transport. Wyman’s compensations demonstrate that myoglobin, when present, equation was solved analytically by Murray (1971, 1977; see assures the oxygen supply of normal heart and muscle. Keener and Sneyd, 1998) and used to construct profiles of Experiments in which myoglobin function is abolished oxygen concentration within muscle cells. Independently, acutely suffer the criticism that the inhibitor or blocking agent Kreuzer and Hoofd (1970) devised a nearly identical equation may have effects other than those intended. Hearts from and solved it with computer assistance (reviewed in Kreuzer, myoglobin-knockout mice served as the ideal control in a study 1970). It would be of great interest to adapt Wyman’s of acute carbon monoxide inhibition of myoglobin in the description to our current understanding of muscle and isolated mouse heart (Merx et al., 2001). The results provide cytoplasmic structure. conclusive direct evidence that myoglobin is required to assure oxygen flow from the vasculature to mitochondrial cytochrome oxidase. The requirement for myoglobin Any discussion of myoglobin function must begin with Fish without myoglobin Millikan’s (1939) notable review in which he assembles an Antarctic ice-fishes of the family Channichthyidae lack impressive body of knowledge to establish that myoglobin is blood hemoglobin and circulating red blood cells. Some Myoglobin function reassessed 2013 species lack cardiac myoglobin as well. The mechanical 0.5 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 0.4 ) Horse 1 able to maintain cardiac output in the face of the additional – insult of increased aortic arterial pressure (Acierno et al., 1997; Horse Ox 0.3 Sidell, 1998). Channichthyid fish without myoglobin served (L. dorsi) as a control of the effects of nitrite in an experiment Elephant Pig demonstrating decreased cardiac function following blockade 0.2 of cardiac myoglobin (Acierno et al., 1997). Sheep Myoglobin (mmol kg 0.1 Myoglobin supports oxidative phosphorylation Hare Essentially all of the oxygen consumed by skeletal muscle and heart is taken up by cytochrome oxidase. Myoglobin is 0 Rabbit 1000 2000 3000 developed in skeletal muscle more or less in proportion to the Cytochrome oxidase activity (Q ) cytochrome oxidase content of the muscle (Fig.·1; Lawrie, O2 1953). Fig.·1. Relation of myoglobin concentration in muscles of various Most of the oxygen used by the soybean root nodule is animals to their cytochrome oxidase activity. Modified from Lawrie consumed by the terminal oxidases of intracellular bacteroids, (1953). the plant mitochondria being relatively sparse. In turn, most of the ATP produced by bacteroidal terminal oxidases is put and should be regarded as optimized for the particular consumed by the intrabacterial enzyme nitrogenase; hence, muscle at a particular rate of work output. nitrogenase activity can be used as a measure of the rate of The rates of reaction of myoglobin/leghemoglobin with ATP formation. Carbon monoxide blockade of leghemoglobin oxygen are subject to natural selection. For instance, Gibson in the living, plant-attached nodule causes nitrogenase activity et al. (1989) have found that an array of disparate to collapse (Bergersen et al., 1973). In an in vitro system using leghemoglobins have strikingly similar, rather slow rates of isolated bacteroids, the rate of ATP production, measured as oxygen dissociation. These rates determine the length of the nitrogenase activity, was proportional to the concentration path explored during the random walk of an oxymyoglobin of leghemoglobin added (Wittenberg et al., 1974). These molecule, as discussed below. The rate constants for oxygen experiments show that ATP generation is leghemoglobin combination are close to the maximum achievable. Oxygen dependent. 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 Myoglobin suitable environment for the highly oxygen-intolerant bacterial Myoglobin is a relatively small protein (Mr 17·600) that is nitrogenase system. little affected by environmental conditions [with the exception Oxygen affinity is also subject to genetic selection pressure. of temperature and high concentrations of lactate (Giardina et The oxygen affinities and oxygen dissociation rate constants al., 1996)]. The molecules are slippery in the sense that they of myoglobins from predacious, oceanic fish that maintain slide past one another with little frictional interaction (Riveros- muscle temperatures well above ambient are similar to those Moreno and Wittenberg, 1972; Veldkamp and Votano, 1976). of a related fish whose muscle operates at cool ambient Myoglobin in the heart is generally close to temperature, when compared at the operating temperature of 200–300·µmol·kg–1·wet·mass·tissue but may reach the muscle (Cashon et al., 1997; Marcinek et al., 2001). 400–500·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 Diffusivity of myoglobin volume), the concentration in the remaining volume of the Until recently, the self-diffusion coefficient of myoglobin, heart cell becomes 330·µmol·l–1. The extent to which in solutions whose concentration was comparable to that of myoglobin penetrates the myofibrillar volume (47% of the proteins in the cell, was used in calculations of oxymyoglobin cell volume) is not known, but, in view of the intimate diffusion in tissue. These values have now been supplanted by association of mitochondria with the contractile elements of the significantly smaller values measured by microinjection cardiac muscle (Fig.·2A), we shall assume that it must. and photobleaching experiments in living muscle (Baylor and Leghemoglobin, 380·µmol·kg–1·wet·mass in nodules, is at a Pape, 1988; Jurgens et al., 1994, 2000; Papadopoulos et al., concentration of approximately 700·µmol·l–1 in the space to 1995, 2000, 2001). The value found in photobleaching which it is confined (Wittenberg et al., 1996). Myoglobin experiments (1.2×10–7·cm2·s–1 at 22°C or 2.1×10–7·cm2·s–1 concentration increases with the work to which the muscle is at 37°C) is about one-tenth that in dilute solution 2014 J. B. Wittenberg and B. A. Wittenberg
of the diffusing path by molecules or structures in the cytoplasm, with consequent tortuosity of the diffusion path.
The diffusion path The diffusion path for oxymyoglobin is always to some degree tortuous, and we must abandon the idea of a simple, unimpeded linear path implied in the Krogh cylinder model. For instance, mitochondria, from which myoglobin is excluded, comprise approximately 35% of the cell volume of mammalian heart, and any transept of a heart cell will be interrupted many times by mitochondria (Fig.·2A). Mitochondrial volume is optimized and, for instance, increases in cold-acclimated fish (Egginton and Sidell, 1989). Leghemoglobin is excluded from the symbiosomes (Wittenberg et al., 1996); the leghemoglobin-accessible volume is less than half the volume of the root nodule cell, and the diffusion path is indeed tortuous (Studer et al., 1992). An extreme is reached in eye muscles of blue marlin, which are modified for heat production (Block and Franzini-Armstrong, 1988; Fig.·2B). Here, mitochondria comprise approximately 70% of the cell volume; sarcoplasmic reticulum and T-tubules appear to occupy most of the balance, and myoglobin must be constrained to the tiny remaining space, where it may reach a concentration of 4·mmol·l–1 in the cytoplasm proper, a truly impressive value (B. A. Block and J. B. Wittenberg, unpublished data). Any model for myoglobin-augmented oxygen transport in muscle must be compatible with this structure. 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 Fig.·2. (A) portion of a myocyte from the ventricular papillary columns of ‘…mitochondria are not randomly distributed but muscle of the cat. Reproduced with permission from Fawcett (1966). are so evenly spaced as to suggest that each serves only a very (B) A portion of a modified striated muscle cell from the heater limited area of the myofilament mass immediately surrounding organ of the eye of the blue marlin. Reproduced with permission it.’ (Fawcett and McNutt, 1969; Fig.·2A). Simple diffusion from Block and Franzini-Armstrong (1988). 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 (11.3×10–7·cm2 s–1 at 20°C) and about one-fifth that often capillaries, detects increased density of the mitochondrial used in earlier calculations of the magnitude of facilitated columns in a band about 4–5·µm removed from each capillary diffusion. The identical values found for the diffusion (Kayar et al., 1986). coefficients of myoglobin in the longitudinal and radial The oxygen dissociation rate constant, together with the directions in the muscle fiber (Papadopoulos et al., 2001), diffusion coefficient, determine the distance traveled in the together with nearly unimpeded rotational diffusion (Wang et random walk of myoglobin molecules during the time that an al., 1997), suggest that myoglobin may diffuse within an oxygen molecule is resident. A remarkable feature of the aqueous phase that is continuous for long distances in the random walk is that ‘…a diffusing particle that finds itself in cytoplasm. Furthermore, the measured value is not affected by a given region of space is destined…to wander around that contraction of the muscle between measurements and is the region for a time, probing it rather thoroughly before same in heart and red skeletal muscle. We cannot distinguish wandering away for good.’ (Berg, 1983). The mean radii of whether the impediment to myoglobin diffusion arises from the region of cytoplasm explored by oxymyoglobin molecules friction between molecules or whether it is due to obstruction during the time that an oxygen molecule is resident are given Myoglobin function reassessed 2015
Table 1. Estimated mean displacement of oxymyoglobin molecules in the sarcoplasm during the time that an oxygen molecule is resident a Temperature Oxygen dissociation t1/2 Mean displacement Protein (°C) rate (s–1)(ms)(µm) Sperm whale myoglobin 20 12 60 20 37 60 12 12 Blue fin tuna myoglobin 25 104 6.7 8 Blue marlin myoglobin 25 84 8.3 9 Soybean leghemoglobin 20 5.6 120 30
aCalculated from the relation
Myoglobin operates in states of partial oxygenation The sarcolemmal boundary The striking fact is that muscles and plant root nodules, The capillary endothelial surface offers by far the smallest having available an almost limitless supply of oxygen, actually cross-sectional area in the diffusion path for oxygen from operate at those low oxygen pressures where myoglobin or blood to cytochrome oxidase, and a large oxygen pressure leghemoglobin are partially desaturated with oxygen. These difference is expected at this point. Experiments by Cole et al. states of partial oxygenation resist change in the face of (1982), showing that partially desaturated myoglobin hastens changing workload or oxygen availability. the entry of oxygen from a gas phase into a myoglobin- Leghemoglobin is approximately 80% deoxygenated in the containing watery solution, serve as a model for the living root nodule (Appleby, 1969), and the fractional sarcolemmal boundary of the sarcoplasm. Entering oxygen oxygenation of leghemoglobin in the living, plant-attached combines with deoxymyoglobin adjacent to the sarcolemma, nodule responds immediately to a step change in ambient and the oxymyoglobin thus generated diffuses into the bulk of oxygen pressure but reverts in minutes to the original value. the sarcoplasm, dissipating the local concentration of oxygen (Klucas et al., 1985). that would otherwise result near the sarcolemma. Millikan (1937), using an oximeter that he had devised for Very shallow gradients of oxygen pressure encountered the purpose, reported extensive deoxygenation of myoglobin within the sarcoplasm of isolated cardiac myocytes (Katz et al., in skeletal muscle in situ as it was brought into maximal 1984; Wittenberg and Wittenberg, 1985) suggest that the contraction. Subsequent studies of the beating heart, either largest part of the oxygen pressure drop from the erythrocyte in situ or saline perfused (e.g. Fabel and Lubbers, 1965; to the mitochondria of cardiac and red skeletal muscle, Hassinen et al., 1981), fully confirm Millikan’s finding. approaching 2.7·kPa (20·torr), must be ascribed to the pressure Cryomicrospectrophotometry of rapidly frozen tissue permits drop across the capillary wall (Landis and Pappenheimer, quantification of myoglobin saturation (Voter and Gayeski, 1963; Wittenberg and Wittenberg, 1989). Partially desaturated 1995). Myoglobin in the in situ beating heart is maintained near sarcoplasmic myoglobin, observed only micrometers away half-saturation in the face of a 20-fold change in work output, from capillaries, once again indicates that the pressure drop a 5-fold change in heart rate and a 2-fold change in arterial from erythrocyte to sarcoplasm is large (Gayeski and Honig, oxygen content (Gayeski and Honig, 1991). Likewise, 1986, 1988; Honig et al., 1984, 1992). 2016 J. B. Wittenberg and B. A. Wittenberg
The large oxygen pressure difference across the capillary unpublished). [A different relationship between oxygen wall, say 2.7–3.3·kPa (20–25·torr), will not be much affected uptake and ambient PO∑ is reported for isolated cardiac by small changes in sarcoplasmic oxygen pressure. myocytes in transient states of changing PO∑ (Rumsey et al., Accordingly, control over rate of oxygen entry into the 1990).] Independently, Gayeski et al. (1987) conclude that the myocytes will be vested almost entirely in the number of PO∑ experienced by cytochrome oxidase is virtually identical capillaries open at any one time (Krogh, 1919a,b). 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 The mitochondrial boundary membrane bound and one in solution, that the oxygen pressure The surface area of the mitochondria, which is 30- to 150- in the mitochondrial membrane proper does not differ from fold greater than the area of capillary lumen serving each cell that in the immediately adjacent solution, completes the proof (Clark et al., 1987; Wittenberg and Wittenberg, 1981, 1989), that the oxygen pressure experienced by mitochondrial is by far the largest area in the diffusion path for oxygen. cytochrome oxidase is very nearly the same as that established Accordingly, the oxygen pressure difference across the in the equilibrium between sarcoplasmic myoglobin and mitochondrial surface need only be small, and the oxygen oxygen. pressure experienced by cytochrome oxidase will closely approach sarcoplasmic oxygen pressure. Myoglobin does not interact detectably with the mitochondrial surface. The only Cytochrome oxidase requirement to sustain respiration of isolated cardiac Since myoglobin cannot cross the mitochondrial outer mitochondria is maintenance of a sufficient, myoglobin- membrane, cytochrome oxidase, located in the inner maintained oxygen pressure at the mitochondrial surface mitochondrial membrane and cristae, must be supplied by (J.B.W. and B.A.W., unpublished). dissolved oxygen diffusing from the sarcoplasm. The exceedingly thin mitochondrial outer membrane will scarcely impede oxygen diffusion (Vanderkooi et al., 1990). Tissue oxygen supply is not limiting Cytochrome oxidase is only partially (approximately 10%) Models of oxygen flow in muscle often assume that oxygen reduced in resting cardiac myocytes (Wittenberg and supply to mitochondria is barely sufficient and that diffusion Wittenberg, 1985). However, cytochrome oxidase (as CuA) in of oxygen limits muscle work output. This is not the case red skeletal muscle contracting in situ becomes reduced in in the normally functioning heart nor in skeletal muscles proportion to increasing workload to about 90% reduction at operating below maximal work output (Jobsis and Stainsby, maximum oxygen uptake (Duhaylongsod et al., 1993; Boushel 1968; Gayeski et al., 1987; Murakami et al., 1999; Richmond and Piantadosi, 2000). If this effect reflects recruitment of et al., 1999; Zhang et al., 1999). Furthermore, oxidative motor units, we may consider that the oxidase is largely phosphorylation in cardiac muscle is independent of oxygen reduced in each contracting myocyte. The effect is to accelerate pressure above a low ‘critical’ PO∑ (Jobsis and Stainsby, 1968; the combination of oxygen with cytochrome oxidase as Wittenberg, 1970), and phosphocreatine levels in the in situ respiratory demand increases. dog heart decrease only when sarcoplasmic oxygen pressure Fully reduced cytochrome oxidase combines very rapidly falls below the normal 0.67·kPa (5·torr; Zhang et al., 2001). 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 Sarcoplasmic oxygen pressure gradients temperature, but operational irreversibility is achieved by Gradients of oxygen pressure within the sarcoplasm of kinetic trapping, i.e. fast electron transfer to the oxygen-bound isolated cardiac myocytes are found to be very shallow, center (Chance et al., 1975; Verkhovsky et al., 1996). The and sarcoplasmic oxygen pressure, within the limits of operational Km for oxygen (the concentration yielding half- measurement, is the same everywhere. For instance, the maximal steady-state turnover) is not a constant but rather is volume-average sarcoplasmic oxygen pressure of resting affected by many parameters and is linearly related to the flux isolated cardiac myocytes, probed spectroscopically as of electrons through the system (Chance, 1965). The myoglobin oxygenation, differs only slightly from operational Km in state III pigeon (Columba livia) heart extracellular PO∑ (Wittenberg and Wittenberg, 1985) and, in mitochondria, supplied with oxygen from oxymyoglobin or turn, does not differ from outer mitochondrial membrane other oxygenated heme proteins, is close to 0.09·µmol·l–1 at oxygen pressure, as measured by the enzyme monoamine 25°C [equivalent to 0053·kPa (0.04·torr) oxygen pressure; oxidase (Katz et al., 1984). J.B.W. and B.A.W., unpublished]. Competition between nitric Furthermore, the sarcoplasmic oxygen pressure that oxide and oxygen for binding to the heme a3/CuB reaction supports half-maximal mitochondrial function in isolated center of cytochrome oxidase will tend to increase the effective –1 cardiac myocytes (Wittenberg and Wittenberg, 1985) does not operational Km in the heart from 0.09·µmol·l to a value within differ from that required to support half maximal respiration the range of myoglobin-buffered oxygen pressures obtaining of isolated cardiac mitochondria (J.B.W. and B.A.W., in the sarcoplasm (Moncada and Erusalimsky, 2002). In vivo, Myoglobin function reassessed 2017 sarcoplasmic oxygen pressure may, in part, control the rate of 1.0 reaction of cytochrome oxidase with oxygen. A
Oxymyoglobin controls oxygen utilization and supply By acting as a scavenger of the bioactive molecule NO, 0.5 oxymyoglobin regulates both oxygen supply and utilization. NO is generated continuously in the myocyte. Oxymyoglobin Oxygen uptake reacts with NO to form the innocuous product nitrate, with concomitant formation of ferric myoglobin, which is recycled 0 0 26.7 53.3 80.0 through the action of intracellular metmyoglobin reductase. P (kPa) Sarcoplasmic NO concentration is determined by the balance CO between the rates of these two processes. 1.0 The interaction of NO and oxymyoglobin to control cardiac B 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 0.8 actually converted oxymyoglobin to ferric myoglobin in the ) m surviving heart. They next demonstrated that infusion of NO, u xygen uptake im or of bradykinin to stimulate endogenous NO formation, brings o 0.6 ax m ent about a dramatic fall of coronary perfusion pressure in hearts d lacking myoglobin; myoglobin-containing hearts from wild- of n epen io 0.4 type mice were little affected. The clear explanation is that t -d c n oxymyoglobin in the wild-type heart scavenges NO, a a fr ( powerful vasodilator that increases blood flow and the number lobi g o of open capillaries. y 0.2 At higher concentrations of infused NO, cardiac contractility M 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 0.2 0.4 0.6 0.8 1.0 already noted, NO is a potent but reversible inhibitor of [MbCO]/[MbCO]+[MbO2] cytochrome oxidase (Moncada and Erusalimsky, 2002). The Fig.·3. (A) Steady-state oxygen uptake of suspensions of cardiac probable explanation of the results of Flogel et al. (2001), myocytes as functions of carbon monoxide partial pressure. Oxygen as pointed out by Brunori (2001b), is that intracellular uptake is normalized, taking the uninhibited rate in each experiment oxymyoglobin, when present, continuously removes NO, thus as unity. Oxygen partial pressure (PO∑) is equal to 10.6–12.0·kPa relieving inhibition of cytochrome oxidase. (80–90·torr) or 13.3–16.0·kPa (100–120·torr). The oxymyoglobin- The magnitude of the protective effect of oxymyoglobin dependent portion of the oxygen uptake is taken as the difference on cytochrome oxidase activity was demonstrated in an between the uninhibited rate and the plateau value at high carbon experiment using isolated heart cells held at high oxygen monoxide partial pressure (PCO). Carbon monoxide inhibition of pressures that are sufficient to fully oxygenate intracellular cytochrome oxidase becomes evident above PCO=80·kPa (600·torr). Reproduced from Wittenberg and Wittenberg (1987). myoglobin. In this condition, oxygen availability does not limit (B) Oxymyoglobin-dependent oxygen uptake of suspensions of respiratory rate, and myoglobin-facilitated oxygen diffusion cardiac myocytes as functions of mole fraction carbon monoxide contributes no additional oxygen flux. Progressive conversion myoglobin (MbCO). Since myoglobin (Mb) is essentially fully of intracellular oxymyoglobin to carbon monoxide myoglobin occupied by ligands, mole fraction MbO2 = 1 – mole fraction MbCO. (MbCO) now abolishes about one-third of the oxygen In different experiments, PO∑ is equal to 5.3–8.0·kPa (40–60·torr), consumption (Fig.·3A). The oxymyoglobin-dependent portion 9.3–12.0·kPa (70–90·torr), 13.3–16.0·kPa (100–120·torr) or 45·kPa of the oxygen uptake (defined in the legend to Fig.·3A) (340·torr). Reproduced from Wittenberg and Wittenberg (1987). decreases linearly with increasing mole fraction of intracellular MbCO (Fig.·3B). The probable explanation (Brunori, 2001a) is that intracellular oxymyoglobin continuously removes NO, remain matters of vigorous controversy (Loke et al., 1999; a reversible inhibitor of cytochrome oxidase. In accordance Kanai et al., 2001; Moncada and Erusalimsky, 2002). with the results of Flogel et al. (2001), the effect is large, These effects link intracellular oxymyoglobin, oxygen and about one-third of the total oxygen flux is dependent uptake by cytochrome oxidase, capillary oxygen delivery and on oxymyoglobin-mediated dioxygenation of NO. The intracellular NO generation into an integral controlled system. histological location and the isoform identity of the nitric oxide Any transient decrement in oxymyoglobin concentration will synthase forming the NO that controls cardiac respiration be countered by increased sarcoplasmic NO, increased oxygen 2018 J. B. Wittenberg and B. A. Wittenberg input from the capillaries and decreased cytochrome oxidase References activity, each tending to restore the initial oxymyoglobin level. Acierno, R., Agnisola, C., Tota, B. and Sidell, B. D. (1997). Myoglobin Pearce et al. (2002) challenge the concept that enhances cardiac performance in Antarctic fish species that express the protein. Am. J. Physiol. 273, R100-R106. oxymyoglobin regulates the level of NO in cardiac muscle. Adam, G. and Delbruck, M. (1968). Reduction of dimensionality in They present evidence that NO endogenously generated in biological diffusion processes. 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