2713 The Journal of Experimental Biology 213, 2713-2725 © 2010. Published by The Company of Biologists Ltd doi:10.1242/jeb.043075 Review Myoglobin’s old and new clothes: from molecular structure to function in living cells Gerolf Gros1,*, Beatrice A. Wittenberg2,* and Thomas Jue3,*,† With an introduction and perspectives by Jonathan Wittenberg 1Zentrum Physiologie, Medizinische Hochschule Hannover, 30625 Hannover, Germany, 2Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, NY 10461, USA and 3Biochemistry and Molecular Medicine University of California Davis, Davis, CA 95616-8635, USA *These authors contributed equally to this work †Author for correspondence ([email protected]) Accepted 4 April 2010 Summary Myoglobin, a mobile carrier of oxygen, is without a doubt an important player central to the physiological function of heart and skeletal muscle. Recently, researchers have surmounted technical challenges to measure Mb diffusion in the living cell. Their observations have stimulated a discussion about the relative contribution made by Mb-facilitated diffusion to the total oxygen flux. The calculation of the relative contribution, however, depends upon assumptions, the cell model and cell architecture, cell bioenergetics, oxygen supply and demand. The analysis suggests that important differences can be observed whether steady- state or transient conditions are considered. This article reviews the current evidence underlying the evaluation of the biophysical parameters of myoglobin-facilitated oxygen diffusion in cells, specifically the intracellular concentration of myoglobin, the intracellular diffusion coefficient of myoglobin and the intracellular myoglobin oxygen saturation. The review considers the role of myoglobin in oxygen transport in vertebrate heart and skeletal muscle, in the diving seal during apnea as well as the role of the analogous leghemoglobin of plants. The possible role of myoglobin in intracellular fatty acid transport is addressed. Finally, the recent measurements of myoglobin diffusion inside muscle cells are discussed in terms of their implications for cytoarchitecture and microviscosity in these cells and the identification of intracellular impediments to the diffusion of proteins inside cells. The recent experimental data then help to refine our understanding of Mb function and establish a basis for future investigation. Key words: diffusion, heart, skeletal muscle, cytoplasm, seal, fatty acid. A mobile carrier of oxygen vertebrate myoglobins with oxygen are subject to Darwinian Myoglobin function in oxygen transport natural selection (Gibson et al., 1989; Wittenberg, 2007). Their Myoglobin, a mobile carrier of oxygen, is developed in red muscle oxygen binding properties are thereby optimized for transport of and heart cells in response to increased demand for oxygen during intracellular oxygen. exercise and transports oxygen from the sarcolemma to the A similar system is found in plants. Here, leghemoglobin, a mitochondria of vertebrate heart and red muscle cells (Wittenberg protein similar to myoglobin but with a tenfold greater affinity and Wittenberg, 2003; Kanatous et al., 2009). Random for oxygen, transports oxygen from the cell membrane of the displacement of oxymyoglobin molecules within a gradient of central cells of the legume root nodule to the symbiosomes, which oxymyoglobin concentration (translational diffusion of are membrane-bound intracellular organelles housing the oxymyoglobin) provides a flux of oxygen additional to the simple bacteroids, the intracellular nitrogen-fixing form of the bacterium diffusive flux (Wyman, 1966). The flux of oxymyoglobin is, of Rhizobium. Symbiosomes, in the root nodule cell, can be course, accompanied by an equal and opposite flux of considered analogous to the mitochondria of muscle cells. deoxymyoglobin (Hemmingsen, 1965). A myoglobin molecule Leghemoglobin is required to maintain the oxygen flux demanded carrying oxygen does not transverse the entire distance from by bacteroidal nitrogen fixation. The concentration of oxygen sarcolemma to mitochondrion. Instead, there is a continuing dissolved in the cytoplasm of the soybean root nodule is reaction in which myoglobin combines with and dissociates vanishingly small, about 10 nanomolar, near the P50 of oxygen, achieving near equilibrium. Direct transfer of oxygen leghemoglobin, whereas the concentration of leghemoglobin- from one myoglobin molecule to another does not occur (Gibson, bound oxygen can exceed millimolar levels. The ratio of 1959). The rate of dissociation of ligands from myoglobin (‘off’ leghemoglobin-bound oxygen to free oxygen exceeds one constant) plays an important role. If this rate is very small, as is hundred thousand, and essentially all of the oxygen flux must be oxygen dissociation from hemoglobin H or carbon monoxide leghemoglobin mediated (Appleby, 1984). In support of this dissociation from myoglobin, carrier-mediated ligand diffusion assertion, carbon monoxide blockade of leghemoglobin in the nearly vanishes (Mochizuki and Forster, 1962; Wittenberg, 1966). living nodule essentially abolishes bacteroidal oxidative Gibson and colleagues and Wittenberg present strong evidence phosphorylation (Bergersen et al., 1973). that both the equilibrium and reaction rate constants of Myoglobin and leghemoglobin are partially deoxygenated in leghemoglobin (and similar plant symbiotic hemoglobins) and of both the nodule cell and the working myocyte (Wittenberg and THE JOURNAL OF EXPERIMENTAL BIOLOGY 2714 G. Gros, B. A. Wittenberg and T. Jue Wittenberg, 2003), thereby fulfilling the requirement that oxidative heart muscle has a lower myoglobin concentration than myoglobin must be desaturated with oxygen somewhere in the skeletal muscle. system for facilitated diffusion to operate (Wyman, 1966). With altered physiological demands, Mb levels can increase. Desaturation has two important consequences: (1) partially Some studies have detected with training an increase in both Mb desaturated myoglobin/leghemoglobin is available to buffer and and the levels of the oxidative enzyme succinate dehydrogenase optimize intracellular oxygen pressure near the P50 of the proteins, (Harms and Hickson, 1983; Beyer and Fattore, 1984). Even though and (2) bacteroidal nitrogen fixation, inhibited by mere traces of type I fiber usually has a higher concentration of succinate oxygen, is protected from intracellular oxygen. dehydrogenase than type II fiber, training can increase the amount The usefulness of myoglobin to the cardiac or red muscle cell in type II fibers to reach the level in type I (Henriksson and is established beyond doubt (Wittenberg and Wittenberg, 2003). Reitman, 1975). Other studies, however, have not detected any What is contested is the fraction of the total oxygen flux carried increase in Mb with training (Svedenhag et al., 1983; Masuda et by diffusing oxymyoglobin, relative to the fraction carried by al., 2001). At high altitude, Mb expression also appears to increase simple diffusive flux of dissolved oxygen. In the root nodule, as (Gimenez et al., 1977; Terrados et al., 1990; Weber, 2007). More we have seen, virtually all the oxygen is carried by recently, increased myoglobin gene expression, controlled by leghemoglobin. By contrast, the fraction of the oxygen flux intracellular calcium pools, has been shown to depend on a carried by myoglobin in vertebrate heart and muscle is very combination of both hypoxia and increased work output, which difficult to determine experimentally. Calculations presented might explain the earlier discrepancies (Kanatous et al., 2009). below suggest that it might not be large in some situations but could be considerable in others and is very dependent on the Determining the myoglobin concentration in muscles effective myoglobin diffusion coefficient within the cell and on To understand the function of Mb in the cell requires an accurate the sarcoplasmic myoglobin concentration. The calculation of determination of its concentration. Generally, researchers have the relative contribution, however, depends upon the cell model, utilized either an optical or an immunohistochemical approach cell architecture and the physiological condition. In agreement (Schuder et al., 1979; Möller and Sylvén, 1981; Nemeth and with these considerations, calculation from experimental data Lowry, 1984; Kunishige et al., 1996). Because of its simplicity, shows that, in the myoglobin-rich muscles of the apneic juvenile most studies have leaned on the optical method, which relies on elephant seal, a large fraction of the total oxygen flux is spectral differences to distinguish the Mb from the Hb contribution supported by myoglobin (see below) (Ponganis et al., 2008). (de Duve, 1948; Reynafarje, 1963). Alternatively, quantitative Thus, in both the root nodule and in juvenile seal muscle, where separation of hemoglobin from myoglobin by affinity the leghemoglobin/myoglobin concentration is high relative to chromatography has also been used (Schuder et al., 1979). intracellular oxygen, under steady-state conditions, myoglobin- In the visible spectrum, Mb and Hb exhibit almost mediated oxygen flux dominates. indistinguishable spectral features. However, as noted by deDuvet and Reynafarje, HbCO shows an equal absorbance intensity in the Mb concentration in muscle b (538nm) and the a (568nm) bands (de Duve, 1948; Reynafarje, Tissue and species 1963). By contrast, the MbCO b peak has a 20% lower intensity Mb isoforms and concentration vary across species