Mb is a monomeric hemoprotein with a molecular weight of 17 kDa, carrying a single oxygen (O₂)-binding site per molecule. It is located near the contractile elements and cell membranes in the red skeletal and cardiac muscles of vertebrates 1. Previously, Millikan 23 proposed the following three possible physiological functions for Mb: (a) an O₂ store during temporary deficits in O₂ supply, (b) an intracellular O₂ transport agent and (c) an intracellular catalyst. Among them, the first function has traditionally been accepted. In the muscles of a beating heart and exercising skeletal muscles, Mb acts as a short-term O₂ store (i.e., an O₂ buffer), tiding the muscles over from one contraction to the next. The rich Mb content in skeletal muscles in aquatic mammals is considered to provide a long-term O₂ store during diving. However, this role of Mb, at least in human, is not significant because its oxygen storage capacity is so low that the total oxygen bound to Mb is exhausted within ca. 5.5 s after being cut off from the O₂ supply 4. The second function, called "facilitated O₂-diffusion by Mb", was based on findings in in vitro experiments 56. The conditions required for this facilitated diffusion to occur are 7: (a) existence of deoxygenated Mb in a certain fraction (or certain low intracellular partial pressure of O₂), (b) existence of a spatial gradient of oxygenated Mb concentration as a driving force for translational diffusion of Mb, and (c) sufficient mobility of the oxygenated Mb to permit diffusion. Although this mechanism has been widely accepted, several discrepancies remain unresolved 89101112. As for the third function, Doeller and Wittenberg 13 proposed the occurrence of Mb-mediated oxidative phosphorylation in heart myocytes under aerobic conditions. However, Mb concentration is not closely related to the oxidative capacity of muscles, that is, the concentration is higher in skeletal muscles (~0.5 mmole/kg wet wt.) than in heart muscles (~0.25 mmole/kg wet wt.) 7.

Thus, the physiological roles of Mb have not yet been established. Recently, alternative functions of (d) O₂ sensing and (e) nitric oxide scavenging were proposed 14. Another recent paper 15 seemed to have totally scrambled the past long-term disputes about the physiological significance of Mb. It was shown using gene-knockout technology that mice without Mb are fertile, exhibit normal exercise capacity, and have a normal ventilatory response to low O₂ levels, suggesting that Mb is not essential for apparently normal cardiovascular and musculoskeletal function in a terrestrial, homoiothermic mammal. However, it has been reported that the disruption of Mb results in the activation of multiple compensatory mechanisms such as increases in Hb concentration, hematocrit, coronary flow, coronary reserve, and capillary density 16. Further, a Mb-like hemoprotein, neuroglobin, has been found in the vertebrate brain 17 contrary to the long-held belief that Mb is restricted to vertebrate cardiomyocytes and oxidative skeletal myofibers. These studies imply that further investigations are required to reveal the physiological role of Mb in intact organs.

In contrast to Mb, which shows a rectangular hyperbolic ODC, the vertebrate Hb, a tetramer carrying four O₂ binding sites, shows a sigmoid ODC that is described in terms of a four-step cooperative O₂ binding. It is widely accepted that the sigmoid ODC enables Hb to transport O₂ with high efficiency: it is nearly fully saturated with O₂ in the lungs and it unloads O₂ sensitively depending on decreases in the partial pressure of oxygen (PO₂) in peripheral tissues. Here, no convincing explanation has been given for the question: does the hyperbolic ODC of Mb have any physiological adequacy or reasonability? The Bohr effect of Hb (pH dependence of O₂ affinity) has physiological significance, in that it enhances O₂ unloading from Hb in the capillaries where pH tends to decrease and in that it increases the solubility of CO₂ as bicarbonate in the venous blood through deoxygenation-induced uptake of protons by Hb. In contrast, Mb lacks the Bohr effect and it had long been believed that Mb was a totally non-allosteric protein, although recently lactate, a metabolic product, was found to cause a right-shift of the ODC for horse and sperm whale Mbs 18.

It is well established that the O₂ affinity of Mb is higher than that of Hb but lower than that of Cyt. aa₃, as known from the relative positions of the ODCs for Mb and Hb and the oxidation curve for Cyt. aa₃ (Fig. 1). This fact led one to the idea that Mb acts as an intracellular O₂ transfer agent from Hb (vascular space) to Cyt. aa3 (mitochondria). Here, one must not overlook an important fact. The three curves in Fig. 1 are drawn with the same PO₂ scale. Therefore, they give O₂ saturation (Y) or the degree of oxidation for the individual proteins when dissolved in the same solution and are in equilibrium with oxygen at the given PO₂ value. However, in vivo, they sense different PO₂ values due to the presence of a PO₂ gradient along the path from the inside of red cells to the mitochondria in myocytes. Thus, the relative positions of the three curves in Fig. 1 must be considered with this precaution, and direct in vivo observations of Y or the degree of oxidation for these three individual proteins are required to get insight into their ensemble functional roles. Recently, using ¹H nuclear magnetic resonance spectroscopy, Mole et al. 19 and Richardson et al. 20 directly observed Y for Mb in human skeletal muscles under exercise of different intensities or during breathing of air with different O₂ contents. In these studies, Mb was used as an indicator of intracellular PO₂, and no attention was paid to the relation between Mb saturation and capillary PO₂.

In the present study, we directly measured Y for Mb in isolated rat hindlimb muscles, perfused with a Hb-free medium, under vigorous changes in respiration conditions. We plotted the Y values as a function of buffer PO₂ and found that the apparent ODC thus plotted for skeletal muscle Mb was rectangular hyperbolic under a suppressed metabolic activity condition but it became sigmoid under enhanced metabolic activity conditions, realizing virtually cooperative O₂ binding by monomeric Mb.

In the present study, by using computer-controlled rapid scanning fiber-optic spectrophotometry, I directly measured Y for Mb in isolated rat hindlimb muscles under extensive changes in respiration rate caused by mitochondrial activity control or perfusate PO₂ control. It is assumed that capillary PO₂ may be approximated by effluent PO₂ in the present experiment, and I plotted the Y values as a function of effluent buffer PO₂. Thereby, I expected that this treatment enabled a meaningful comparison of the ODCs for Mb and Hb. I found that thus plotted apparent ODC for skeletal muscle Mb was hyperbolic under a suppressed metabolic activity condition whereas it became sigmoid under enhanced metabolic activity conditions, realizing virtually cooperative oxygenation of the monomeric Mb.

It is generally accepted that cooperative O₂ binding by Hb is advantageous for efficient O₂ transfer from the alveolar gas to red cells and from red cells to peripheral tissues. Based on the Hill equation, Graby and Meldon 28 showed that an n value (here, n is a constant) of 1.5 to 2.0 is more favourable for minimizing the change in blood flow under resting conditions than the normal n value of 2.5 to 3.0, whereas an n value as large as 3 is beneficial for a large amount of O₂ extraction under vigorous exercise. Kobayashi et al. 29 showed that, under resting conditions, O₂ release from Hb becomes most sensitive to PO₂ change at Y = 38% where cooperativity measured by n (here, n is a variable of PO₂) is not maximal, whereas it becomes less sensitive at the mixed venous blood PO₂ where Y is around 70% and cooperativity is nearly maximal. These reports indicate that, under resting conditions, the blood reserves an O₂ transport capacity to meet possible increases in O₂ demand, e.g. under exercise conditions, and the sigmoid character of ODC becomes more important under such conditions. This situation is realized by maintaining Y at a rather high level (70%) below which the Y value drops sharply upon PO₂ decrease within the very steep middle portion of ODC.

The present study has clearly shown that the apparent ODC for Mb in intact skeletal muscle is sigmoid, the n_app value being 1.46 under the control condition (Table 2) and 2.23 under the maximal respiratory condition (Fig. 4). These n_app values greater than unity imply that the muscle Mb binds O₂ in a virtually cooperative manner with variation of effluent buffer PO₂. This phenomenon implies that the sensitivity of Y for Mb to vessel PO₂ change becomes higher for increased O₂ demands than for normal O₂ demand. In addition to this effect, the rightward shift of the ODC upon increases in oxygen demand will undoubtedly enhance O₂ unloading from Mb. These effects will facilitate Mb-mediated O₂ transfer from Hb to Cyt. aa₃, especially for heavy O₂ demands. Based on the Hill equation, the O₂ transport efficiency of Mb in the perfused muscle is estimated to increase ca. 4-fold under the control condition and ca. 11-fold under maximally respiring condition (Fig. 4 inset).

The Mbs isolated from body wall or radular muscle of a limited number of annelidan and molluscan species are dimers and show some cooperativity in oxygen binding (1 < n < 2) but no Bohr effect 30. The physiological significance of these dimeric Mbs is unknown. As shown in the present study, the ODC of monomeric Mb can exhibit virtual cooperativity and O₂ demand-dependent shifts. The virtually cooperativity and O₂ demand-dependent shifts of Mb oxygenation in vivo are probably common features at least for vertebrate Mbs, and this may provide a basis for explaining why the vertebrate Mb has been preserved as a monomer during molecular evolution.

The virtual cooperativity in Mb oxygenation observed in the present study is explained in terms of the PO₂ gradient along the O₂ diffusion path. If the tissue O₂ demand was null, then the PO₂ gradient would be absent and the apparent ODC for Mb would be identical with the real ODC for Mb in solution. At a steady state with a certain level of O₂ demand a PO₂ gradient develops across red cell membrane, blood plasma, capillary wall, sarcolemma and sarcoplasm, making the PO₂ sensed by Mb lower than the capillary PO₂. Then, the apparent ODC will be shifted toward the right because a capillary PO₂ value higher than the PO₂ value sensed by Mb is needed to maintain the same Y value as that which occurs in the absence of a PO₂ gradient. When the tissue O₂ demand is kept constant, the ratio of capillary PO₂ to sarcoplasm PO₂ will become larger at low capillary PO₂ than at high capillary PO₂. This will cause a more extensive rightward shift of the apparent ODC in the low saturation range than in the high saturation range, making the curve steeper than the real one. Increase in tissue oxygen demand will enhance this mechanism and make the curve more right-shifted and sigmoid. All the apparent ODCs observed in the present study are shifted toward the right compared to the real one measured for Mb in solution (Fig. 3).

At present, detailed explanations for this cooperative mechanism is difficult. However, it could be argued that heterogeneous oxygenation in tissue 31 and in single myocytes 32 might be responsible in part for the shift and the shape change of the Mb ODC, and might also enhance intercellular O₂ transfer, i.e., re-distribution of O₂ among adjacent myocytes, although we adopted high and constant flow rate perfusion conditions (i.e., about 50 times higher than normal blood flow) and, thus, the perfused vessels of muscle were always passively dilated.

Unfortunately, it is not practical to use a Hb solution or a red cell suspension as the perfusate in our experiments because the absorption spectra for Hb and Mb are too similar and independent observations of Mb oxygenation are not feasible, especially when the concentration of Hb is much higher than that of Mb. To overcome the problem that the O₂ solubility of the buffer is much smaller than that of a Hb solution or a red cell suspension two strategies were employed: one was to make the PO₂ of the influent buffer as high as that of water vapor-saturated O₂ (ca. 700 mmHg) and the other was to use a high flow rate for the buffer, which was about 50 times higher than that of normal blood flow. As a result, the inflow of O₂ was about 5 times larger than that of the tissue O₂ consumption at the control metabolic rate. The large O₂ diffusion barrier (see Fig. 5) and the high PO₂ of the influent buffer (and consequently, the high capillary PO₂) are an additional (and probably, major) cause for the rightward shift of the apparent ODC of Mb. The apparent ODCs of Mb in the control and enhanced respiration activity states (Fig. 3A) are right-shifted compared to the whole blood ODC (Fig. 1). One may suppose that Mb cannot work when the capillary PO₂ is in the physiological range (40 to100 mmHg) because its O₂ saturation is too low to function, as judged from Fig. 3A. However, the actual apparent ODCs for Mb in muscles with blood circulation will be shifted much more toward the left compared to those shown in Fig. 3A, and Mb can be saturated with O₂ to practical levels. The important point is that the difference in in vivo O₂ saturation between Hb and Mb is not so large as that expected from the ODCs in Fig. 1. In fact, Y for Mb in working muscles is less than around 50% 1920313233. Red blood cell (RBC) in perfusion buffer appears to exert considerable effects on intracellular oxygenation in the beating heart 34, probably due to the facilitated O₂ transfer by RBC motion within capillary lumen 35. Therefore, the virtually cooperative oxygenation of Mb might be only demonstrated in organs perfused with RBC-free medium. However, it is well known that the blood flow in the capillary bed is not constant and frequently only plasma flow is observed. In this case, the virtually cooperative oxygenation of Mb may play a significant role for O₂ transfer from capillary to mitochondria.

In summary, I found that the ODC for Mb in intact skeletal muscle is sigmoid and right-shifted. This virtually cooperative O₂ binding by Mb and the right-shift of ODC become more marked as tissue respiration activity is increased. Hence, increase in O₂ demand in tissues makes the O₂ saturation of Mb more sensitive to capillary PO₂ change and enhances Mb-mediated O₂ transfer from red cell to motochondria. The virtual cooperativity and O₂ demand-dependent shifts of ODC may give a basis for explaining why Mb has been preserved as a monomer during molecular evolution. Preservation of a monomeric structure may be required to retain multi-functional role of Mb in vivo.