Mechanisms of cytoplasmic hemoglobin and myoglobin function.

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Annu. Rev. Biophys. Biophys. Chern. 1990. 19:217-41 Copyright © 1990 by Annual Reviews Inc. All rights reserved

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CYTOPLASMIC HEMOGLOBIN AND MYOGLOBIN FUNCTION Jonathan B. Wittenberg and Beatrice A. Wittenberg Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, New York 10461 KEY WORDS:

leghemoglobin, oxygen, myoglobin-mediated oxidative phos phorylation, myoglobin-facilitated oxygen diffusion.

CONTENTS PERSPECTIVES AND OVERVIEW ................. . . . . . . . .. . . . . . ........................................ .. .. . . .. . . .. . . .. .

218

CYTOPLASMIC HEMOGLOBINS..........................................................................................

218

Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Occurrence . . ................................ .. . . . .................................. .. .... . .................. . ............ Plant Hemoglobins . ......................................... . .... . ...................................... .............

218 219 219

HEMOGLOBIN-DEPENDENT SySTEMS.................................................................................

22 1

Hemoglobins Function in States of Partial Oxygenation .................... ... ................... .

22 1

MYOGLOBIN-FACILiTATED OXYGEN DIFFUSION.............................. . .................. ................

224

MYOGLOBIN-MEDIATED OXIDATIVE PHOSPHORYLATION . . . . ".......................... . . . . . . . . . ...........

225

Isolated Cardiac Myocytes .... . . . . . .. . ....................... . .. .. . . . . . . . ....................... . . . . . . . . . . . .... . . Soybean Root Nodules..............................................................................................

225 226

......................... . . . . ................... . .... . .........

227

HEMOGLOBINS AS POTENTIAL TERMINAL OXIDASES...........................................................

228

Yeast Hemoglobin ................ . . . . . . . ..... .......... . ..................... .. . . . . . . ................................ Bacterial Hemoglobin ............ . . ................... . .................. . . . . . . . ............. . ...................... Horseradish Peroxidase......... . . ................... . ..................... . . . .... ................................. Myoglobin.............................. . ........................................................... ... . .................. Ferryl Hemoglobin in Tissue.....................................................................................

228 228 23 1 232 232

MYOGLOBIN AND LEGHEMOGLOBIN-ASSOCIATED IRON......................................................

233

A Proteinfrom Pigeon Breast Muscle ""'' ' ' ' ' ' ' ''''''''' ' ' ' ' ' ' ' ' ' ' ' ' ' ' '''''''''''''' ' ' ' ' '''''''''' ' ' ' ' ' ' ' Chelated Iron from Soybean Root Nodules .. ........................... . ................................. Chelated Iron/rom Symbiont-Harboring Mollusc Gills.............................................

233 233 234

HEMOGLOBIN-MEDIATED SULFIDE UTILIZATION

PERIPLASMIC HEMOGLOBIN.............................................................................................

234

OXYGEN SENSING .................................. .. . . . . .............. . .................. . ...................... .... .... ...

235

RECAPITULATION: MOLECULAR MECHANISM ............ . . .. . ............ . ................................ . . . ...

235

217 0883�9182/90/0610�0217$02.00

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WITTENBERG. & WITTENBERG

Annu. Rev. Biophys. Biophys. Chem. 1990.19:217-241. Downloaded from www.annualreviews.org Access provided by Emory University on 08/03/15. For personal use only.

PERSPECTIVES AND OVERVIEW Lankester (68) in 1872 marvelled at the hemoglobin in the nerve (actually in glial cells) of Aphrodite, an annelid, red as a drop of human blood. Since then, tissue hemoglobin has been observed in eubacteria, yeast, molds, protozoa, most phyla of higher invertebrates, protochordates (D. W. Kraus, J. E. Doeller, personal communication), vertebrates, root nodules, and growing root tips of diverse dicotyledonous flowering plants (7, lOa, 30, 79, 100, 101, 104, 112). This review addresses the molecular mechanisms by which cytoplasmic hemoglobins enhance oxidative phosphorylation and oxygen utilization. A central theme is the interaction of hemoglobin with intracellular target organs, mitochondria, and intracellular prokaryotic symbionts. Inter actions between cytoplasmic hemoglobin and intracellular organelles are abolished by disruption of the tissue and must be observed in living cells or tissue or in a reconstituted broken cell system. We shall examine in detail those five systems that have been studied in detail and construct an orderly statement of the facts, a sculptor's armature, around which to fashion detailed molecular mechanisms. We do not discuss myoglobin facilitated oxygen diffusion. Its role in the economy of the mammalian heart and muscle has been treated elsewhere (65, 121, 125). We also do not discuss oxygen storage. We use the expression tissue hemoglobin (Hb), modified by the organism or tissue of occurrence, to describe cytoplasmic protohemeproteins that in the ferrous state combine reversibly with oxygen. We retain the historical names myoglobin (muscle hemoglobin, Mb) and leghemoglobin (legume hemoglobin, Lb) for vertebrate muscle hemoglobin and legume root nodule hemoglobin, respectively. Nonlegume plant hemoglobin will be called plant hemoglobin.

CYTOPLASMIC HEMOGLOBINS Properties Except for those of yeast and nematodes, all cytoplasmic hemoglobins are similar globular molecules built on the myoglobin pattern, with molecular weights, Mn clustered around 18,000 and oxygen affinities in the range of 0.04-4.0 torr. Most are monomeric; some are isolated as dimers or tetra mers. Heme-heme interaction, reported occasionally in molluscan hemo globins (79), is the exception, not the rule. Oxygen affinity of myoglobin in situ (97, 119) and the partition between oxygen and carbon monoxide (120) are the same as those of the isolated protein. Usually only oxy- and deoxy-hemoglobin are observed in living plant and animal tissues. Ferric

CYTOPLASMIC HEMOGLOBIN FUNCTION

219


hemoglobin is never observed except in the bacteria-housing domain of certain clam gills (42, J. B. Wittenberg & c. A. Appleby, unpublished data). Ferric hemoglobin formed by the action of externally added reagents is soon restored to the ferrous state (61, 96, 99, 122), The higher oxidation state, ferryI myoglobin, Mb(IV), has been observed in living mollusc and annelid muscle and nerve (124, 131), suggesting that ferryl myoglobin may participate in respiratory electron flow. Occurrence

Hemoglobin in mammalian muscle is expressed in response to sustained work (74, 121, 125) and increases in the heart with the age of the animal. Hemoglobin is found in all legume root nodules that fix nitrogen (7, 20, 127) and in nodules formed on roots of the nonlegume Parasponia in sym biosis with Rhizobium (133). Nitrogen fixation in the legume-rhizobium symbioses occurs only when functioning hemoglobin i s present (7, 20). Hemoglobin is expressed in many actinorhizae [nitrogen-fixing nodules formed on the roots of diverse woody dicots in response to invasion by the actinomycete Frankia (7, 38, 44, 103, 104)], but has been reported absent from others (103, 104). Hemoglobin is easily missed in plant tissues; accordingly, we treat reports that hemoglobin is absent from members of an otherwise hemoglobin-dependent homologous series with caution. The volume-average concentrations of cytoplasmic hemoglobin in micro organisms and some plant and animal tissues are listed in Table 1 . Local concentrations in the cytosolic compartment to which the hemoglobin i s confined may b e much larger. Hemoglobin concentration may differ within popUlations of a single species. For instance, hemoglobin is more than millimolar in nerves of Aphrodite (an annelid) collected in the English Channel but is present at trifling concentration in nerves of the same species collected in Scotland. Within a homologous series of symbiont-harboring molluscan gills, hemo globin concentration varies IOOO-fold, from about micromolar to 1.6 mM (37, 128), and some members of the series apparently lack hemoglobin (36). In the growing plant root tip, strong evidence supports a functional role for hemoglobin at micromolar concentration (lOa). We posit multiple functions for cytoplasmic hemoglobins. Plant Hemoglobins

Hemoglobin occurs at high concentration in root nodules. Only very recently, hemoglobin has been discovered at 2- to an estimated 30-micromolar concentration in growing root tips, where it is confined to rapidly respiring meristem cells near the root cap or in the zone of elon gation (lOa, S. Craig, personal communication). The single hemoglobin OCCURRENCE

220

WITTENBERG & WITTENBERG

Table 1

Representative cytoplasmic hemoglobins Abundance"


Organism and tissue

(11 M )

M ammalian skeletal muscle

100-350

Mammalian heart

200-300

Oxygen affinityb (nM)

(torr)

3200

2.3

References 93 93, 121

Pigeon breast muscle

285

93

Pigeon heart

210

93

Pigeon gizzard' Tuna muscled

530

93

Marlin heater organd Symbiont-harboring mollusc gill Soybean root nodule

Parasponia root nodule Casuarina actinorhiza Myrica actinorhiza Yeast Vitreoscilla (bacterium) 'In

600-2100 400 2.5-1500

62,128

565'

0.31'

700

48

0.026

47

70

90

0.049

12, 133

80

135

0.074

103

38,44 103

10

20

30

6000

0.01

83,84

3.3

40,69

!,mol/kg fresh weight.

bValues al20"C, excepl mammalian hearl al3TC. < Avian gizzard is a smooth muscle.

dB. A. Block & J. B. Wittenberg, unpublished data.

, Lllrina peetinala, oxygen-reactive hemoglobin.

gene of Parasponia is expressed in root tips of aseptically cultured Para sponia (29, 67) as well as in Rhizobium-harboring nodules (66). The Para sponia hemoglobin gene introduced into the nonnodulating tobacco is expressed in the root tips (67). The roots of the nonnodulating Trema (29) and the cultured roots of the nodulating Casuarina also contain hemoglobin. Casuarina root and nodule hemoglobins differ in molecular size, suggesting that they are coded by different genes (c. A. Appleby, unpublished). Appleby et al (10) suggest that a hemoglobin gene may be a component of the genome of all plants, with widespread expression in the growing root tip. Parasponia hemoglobin and Trema root hemoglobin, which have similar amino acid sequences (29), may react similarly with oxygen. Hemoglobin concentration in roots may be too small to facilitate significant oxygen diffusion. Oxygen sensing, discussed later, has been suggested as a plausible function of root hemoglobin. The plant hemoglobins share the three-dimensional structure of myoglobin (14, 81) and retain the conserved amino acid residues of the heme pocket required for reversible oxygen binding (7). Two of the three intervening sequences (iutrons) of the genes coding plant hemoglobins interrupt the amino acid coding regions (exous) at exactly the same posi tions as those of animal globin genes. Modern vertebrate globin genes lack EVOLUTION


221


the third and central intron of plant globin genes. The third intron of plant genes falls where one had been predicted from analysis of vertebrate protein structure domains (51a). These findings support the hypothcsis that all plant and animal hemoglobins have evolved by vertical descent from hemoglobin present in a common ancestor, and that genes for wide spread root hemoglobins have been the source of the sporadic appearance of symbiotic hemoglobins (8-1 0a). PROPERTIES Gibson et al (47) studied fourteen plant hemoglobins, includ ing those from legume-Rhizobium symbioses; nonlegume, Parasponia-Rhi zohium, symbiosis; and an actinorhizal, Frankia-Casuarina, symbiosis. The symbionts of these associations vary widely in their patterns of terminal oxidases (7). Nonetheless, the hemoglobins have similar kinetics in their reactions with oxygen. Although geminate reactions vary widely, all achieve extraordinary oxygen affinity by rapid combination with oxygen together with moderate rates of dissociation. The rates of oxygen binding and the oxygen affinities of the plant hemoglobins may be near the maximum possible [or hemeproteins based on the myoglobin pattern. We suggest that selection pressure for function in a common environment may have forced the plant hemoglobins into a common mold.

HEMOGLOBIN-DEPENDENT SYSTEMS

Hemoglobins Function in States of Partial Oxygenation MAMMALIAN MUSCLE AND HEART Myoglobin in vertebrate heart and muscle is presumed to be in free solution in the sarcoplasm. Since myo globin is presumably excluded from the mitochondrial and nuclear volumes and the volume occupied by the contractile elements, the con centration in the volume to which myoglobin is confined must exceed several-fold the volume-average concentration given in Table 1. The mito chondrial outer mcmbrane and mitochondrial intermembrane space sep arate sarcoplasmic myoglobin from cytochrome c oxidase and the proteins of electron transport and oxidative phosphorylation, which are located in the inner mitochondrial membrane. Separation of cytoplasmic hemoglobin from terminal oxidase systems is general to all systems describcd. Millikan (73), using his oximeter, first reported that myoglobin in blood perfused mammalian skeletal muscle in situ functions in states of partial oxygenation. Fabel & Lubbers (43), using reflectance spectrometry, con firm this finding with the saline-perfused beating heart. In a series of papers (e.g. 45), Gayeski & Honig have examined myoglobin oxygenation in skeletal muscle and heart that was quick frozen while contracting or beating in situ. They find myoglobin always partially oxygenated. The


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striking result is that myoglobin oxygenation is narrowly distributed near half-saturation in all the heart muscle cells examined, implying active control of myoglobin oxygenation. A critical review has appeared (121). Wittenberg & Wittenberg (119) and Katz et al (53) find that gradients of oxygen pressure within the sarcoplasm of isolated mammalian cardiac myocytes are small, never exceeding 2-3 torr. Assuming mitochondrial oxygen pressure is approximately 1 torr and sarcoplasmic oxygen pressure is 2.3 torr, the largest part of oxygen pressure difference (20--25 torr) from erythrocyte to mitochondrion of the working heart must be extracellular. Myocyte oxygen consumption is large. Given the consequent large oxygen flux across the sarcolemma, even the small diffusion distance (0.2-0.5 j-tm) from the erythrocyte to the sarcolemma of the myocyte poses a sufficient barrier to generate the observed oxygen pressure difference (121). Hence, we formulate the general principle that myoglobin-dependent systems operate in steady states of myoglobin partial oxygenation maintained by rapid oxygen consumption operating in the face of a barrier to oxygen entry. Nitrogen-fixing nodules are formed on roots of legumes and of the nonlegume Parasponia in response to invasion by bacteria of the genus Rhizobium, which proliferate within the plant cells of the developing nodule. Nitrogenase, the nitrogen-fixing enzyme complex, occurs wholly within the bacteroids (bacteria modified for symbiotic life). Even traces of oxygen irreversibly destroy nitrogenase (91), but, para doxically, nitrogen fixation depends on a continuous flow of oxygen to support bacterial oxidative phosphorylation to meet the demand of nitro genase for ATP. The rate of bacteroid nitrogen fixation (measured as acetylene reduction or hydrogen evolution) provides a convenient real time measure of the rate of intrabacterial ATP genesis. The bacteroids are separated from the cytoplasm proper of the host plant cell by a membrane of plant origin modified by bacteroid genes, the peribacteroid membrane (39, 88, 89, 109, 116a). Although the volume enclosed by the peribactcroid sac may be large or small, Brownian motion brings the bacteroids into continuing contact with the membrane. Leghemoglobin is located solely in the plant cytoplasm proper (90, 110). D. J. Goodchild, using immunohistochemical staining, found no Lb in the peribacteroid space (personal communication). D. A. Day, D. J. Good child, and C. A. Appleby detected no Lb in isolated peribacteroid units (39; personal communication). Leghemoglobin, in the cytosolic space to which it is confined, may reach an estimated local concentration of 5 mM (22). In this system, as in others, leghemoglobin is separated from the terminal oxidases of the bacteroid plasma membrane. LEGUME ROOT NODULES



223

Leghemoglobin, observed spectrophotometricaIly, is 5�20% oxygenated in the intact soybean or sweet clover root nodule, implying 10 nM or less local oxygen concentration (5, 7, 61). Soybean nodules, exposed to abruptly decreased oxygen pressure, return shortly to their original rate of nitrogen fixation (35). Since nitrogen fixation rate responds to oxygen pressure, this implies return to the initial P02 within the nodule. Spectro photometric observation of nodules attached to the undisturbed living plant show dramatic control of fractional oxygenation of cytoplasmic leghemoglobin in the face of step changes in rhizosphere oxygen pressure (61). Effective bacteroid respiration (that coupled to ATP production) at low P02 and ineffective (not coupled) respiration at higher P02 is sufficient to maintain the large oxygen pressure difference across the cortex and the low oxygen pressure within the nodule (7, 20, 127). Whether the plant controls oxygenation by varying the rate of oxygen consumption or by changing permeability of the cortex is actively debated (7, 3Sa). In any event, a series of air passages transmits the subcortical oxygen pressure to every cell of the massive nodule (21, 51). Accordingly, leghemoglobin assures the flow of oxygen within each individual cell (reviewed in 7, 20, 127). Nitrogen-fixing nodules are formed on the roots of 22 genera (8 families) of woody dicotyledonous flowering plants in response to infection by the actinomycete Frankia (104). The endophyte is intracellular and, as in other symbioses, is separated from the plant cytoplasm by a layer produced by the host plant. Nitrogenase is within the endophyte eell and, in common with nitrogenase elsewhere, is irreversibly inactivated by oxygen (91). Nitrogenase activity in nodules and in the isolated symbiont is strictly oxygen dependent, presumably because oxidative phosphory lation is required to generate the large amount of ATP consumed. The endophyte of alder roots contains cytoehromes aa3, b, C, and 0 (32). In many actinorhizal nodules, gas passageways bring air close to the infected cells (104, 117), and local barriers limit oxygen entry. Presumably, hemo globin resides in the cytoplasm of symbiont-harboring plant cells. If so, it is separated from the terminal oxidases of the endophyte plasma membrane by endophyte hyphal or vesicle walls and by the plant-secreted enclosing wall. Each individual endophyte-harboring plant cell of the nodules formed on roots of the tree Casuarina by infection with Frankia is surrounded by a thick, lignified, presumptively oxygen-resistant wall (17, 19). Within the cell, the endophyte grows as a mass of intracellular hyphae, which lack the highly oxygen-resistant (77, 78) vesicles developed by the Casuarina endophyte cultured at atmospheric oxygen pressure (78). Vesicles are

ACTINORHIZAE

224


characteristic of endophytes of nodules developed on the roots of all other plant families (18, 105). Oxygen uptake and nitrogen fixation (measured as acetylene reduction) of cultured hypha! Casuarina endophyte become saturated at 8 ,uM oxygen (in the absence of hemoglobin) and become half-maximal at I {LM (78). This may be compared with the oxygen affinity of Casuarina hemoglobin, Ko 0.l 35 {LM (44). Therefore, the symbiosis may operate with partial saturation of Casuarina hemoglobin. Observation of slices of Myrica nodules confirms that another actinorhizal hemoglobin functions in a state of partial oxygenation (102).


=

Certain molluscs-clams, mussels, and a snail-which live in environments where hydrogen sulfide (or meth ane or other reductants) meets oxygen in disequilibrium, enter symbiotic associations with chemoautotropic bacteria (31). These environments include the hydrothermal vents of the Galapagos and other Pacific Ocean sites and, less dramatically, worldwide coastal and deep m arine sediments. The bacterial symbionts are intracellular and housed in specialized cells (bacteriocytes) of the enlarged and modified gill. They oxidize hydrogen sulfide (or other reductants) and fix carbon dioxide into hexoses that supply most of the carbon used by the host. Peribacterial membrane sacs surround the symbionts, effectively separating them from the host cytoplasm proper (31). Hemoglobin is present at high concentration in the gills of many of these clams (128). Gill hemoglobins are of two kinds: Oxygen-reactive hemoglobins, which react reversibly solely with oxygen (62) and sulfide-reactive hemoglobins (62�64). The latter react reversibly with oxygen but in the presence of hydrogen sulfide (and oxygen) arc converted reversibly in the living gill to ferric hemoglobin sulfide (42). Oxygen-reactive hemoglobins are found in the gill and many other tissues of the host; sulfide-reactive hemoglobin is probably restricted to the bacteriocyte domain of the gill (J. E. Doeller, D. W. Kraus & J. B. Wittenberg, unpublished observations). The oxygen affinities of both classes are about the same, Pso 0.1�0.2 torr, although the reactions of the sulfide-reactive hemoglobin with oxygen are lOOO-fold faster than those of oxygen-reactive hemoglobins (62). Hemoglobin in the intact gill of a sulfide-interface clam is half-saturated at ambient POl 6 torr, an oxygen pressure experienced intermittently in the natural environ ment of the animal (42). SYMBIONT-HARBORING MOLLUSC GILLS

=

=

MYOGLOBIN-F ACILIT ATED OXYGEN DIFFUSION

Translational diffusion of oxymyoglobin molecules, each carrying pick-a back a diatomic oxygen molecule, generates a flux of oxygen in a gradient



225

of oxygen pressure and myoglobin saturation. Although myoglobin diffuses at one twentieth the rate of free oxygen, myoglobin concentration in working heart muscle exceeds free oxygen concentration 30-fold, and fluxes of free and myoglobin-bound oxygen in sarcoplasm are expected to be of the same order (121, 134). Leghemoglobin concentration in the soybean root nodule exceeds free oxygen concentration 1O,OOO-fold (7, 1 27), and essentially the entire flux of oxygen to the bacteroids must be leghemoglobin facilitated. Facilitated diffusion occurs whenever oxygen is removed from combination with the macromolecular carrier at the low pressure boundary of the system, e.g.the sarcoplasm-mitochondrion inter face (134). In the event experiment shows that facilitated diffusion does bring the bulk phase oxygen pressure to the very boundaries of the sys tem-to the cell membrane and the outer mitochondrial or peribacterial membranes (34, 121). Facilitated diffusion is established as a physical entity (65, 125). Functional myoglobin augments translational transport of oxygen from sarcolemma to mitochondria of living muscle (122). The role of myoglobin-facilitated oxygen diffusion in the economy of muscle is discussed elsewhere (121). MYOGLOBIN-MEDIATED OXIDATIVE PHOSPHORYLATION

Isolated Cardiac Myocytes

Myoglobin-mediated oxygen delivery to mitochondria is best demon strated in suspensions of cardiac myocytes flooded with superabundant oxygen (1 20). In this circumstance, sarcoplasmic myoglobin is fully oxy genated; facilitated diffusion contributes no additional oxygen flux; the diffusive flow of dissolved oxygen through the sarcoplasm exceeds the mitochondrial oxygen demand at least l OO-fold, and mitochondrial cyto chrome oxidase experiences oxygen pressures that are 20- to 200-fold greater than that required to maintain the l argely oxidized state seen in resting myocytes. Carbon monoxide in this circumstance selectively blocks oxygenation of sarcoplasmic myoglobin without perturbing the optical spectrum of intracellular cytochrome oxidase. A carbon monoxide blockade of intracellular myoglobin function abol ishes about one third of the oxygen uptake of resting cardiac myocytes. The myoglobin-dependent component of the cellular oxygen uptake decreases linearly with increasing fractional saturation of sarcoplasmic myoglobin with carbon monoxide (that is, a decreasing fraction of oxymyoglobin) with a slope near unity. Half-inhibition was achieved at different Pea in experiments at different oxygen pressures but always when myoglobin was half-saturated with carbon monoxide. Carbon monoxide, therefore, exerts


226


its dominant effect on myoglobin alone and inhibition of respiration cannot be ascribed to interference with other cellular functions. We conclude that the myoglobin-dependent component of oxygen uptake is proportional to the fraction of sarcoplasmic myoglobin combined with oxygen. Myoglobin-dependent oxygen uptake is tightly coupled to phosphory lation of ADP and is blocked by oligomycin, a specific inhibitor of mito chondrial ATP synthase (B. A. Wittenberg, unpublished data). Oligomycin blocks ATP synthase in cardiac myocytes but does not change total oxygen consumption. Carbon monoxide inhibitable, myoglobin-mediated oxygen uptake disappears in the presence of oligomycin. The rate of ATP synthesis is not easily measured. Phosphocreatine, which acts as a buffer of intra cellular high-energy phosphates, changes with the balance between rates of A TP synthesis and utilization, resisting change in intracellular ATP concentration. Intracellular phosphocreatine concentration falls when myoglobin oxygenation is blocked by carbon monoxide (118). This decrease disappears in the presence of oligomycin, showing that mito chondrial ATP synthase implements myoglobin-dependent ATP synthesis (B. A. Wittenberg, unpublished data). Rotenone, which blocks electron flow from nicotinamide-adenine dinucleotide (NADH) to ubiquinone, also

stops myoglobin-mediated oxidative phosphorylation, which proves that myoglobin-mediated oxidative phosphorylation depends on electron flow in the mitochondrial electron transport chain. This ncwly found function of myoglobin is rightly called myoglobin-mediated oxidative phosphorylation. Soybean Root Nodules

Oxidative phosphorylation by Rhizobium bacteroids in the intact soybean root nodule depends on leghemoglobin (27). In a reconstituted system, oxidative phosphorylation by isolated Rhizobium bacteroids is small in the absence of leghemoglobin and increases monotonically with increasing leghemoglobin concentration (130). The effect is greatest near half-satura tion of Lb with oxygen, 0.026 torr, 48 nM dissolved oxygen (13, 23, 24, 26). Experiments in which other hemoglobins replaced leghemoglo bin suggcst that the oxygen pressure at the bacteroid surface cannot be greater than 0.002-0.02 torr, 4-40 nM (1 30), with a preferred value, deduced in part from experiments with cultured Rhizobium, of 5 nM (25, 26). A specific chemical interaction between leghemoglobin and bacteroids seems highly unlikely because ten different oxygen-binding proteins, including the nonheme iron protein hemerythin and the copper protein hemocyanin, can substitute for leghemoglobin in supporting oxidative phosphorylation by isolated bacteroids (130). Leghemoglobin-facilitated oxygen diffusion, which will bring the bulk-phase-solution oxygen pressure



227

to the very surface of the bacteroid, may offer a sufficient explanation of the results of experiments in the simplified reconstituted system. Other effects may intervene in the intact nodule, where the peribacteroid membrane sac and peribacteroid space are interposed between leghemo globin and bacteroids. Experimcnts at high oxygen pressure are not pos sible in this system. Nonetheless, the absolute dependence of the nodule on leghemoglobin and the preferential use of leghemoglobin-bound oxygen by isolated bacteroids imply that leghemoglobin-mediated oxidative phos phorylation occurs in the plant system. The terminal oxidase of Rhizobium bacteroids and cultured Rhizobia, which accepts oxygen from oxyleghemoglobin with KM,02 5 nM (26), is not yct identified. Thc carly candidates, cytochrome P-450 (6, 13) and the soluble catalase-peroxidase hemeprotein b-590 (formerly called cyto chrome al) (6) may be ruled out because cytochrome P-450 is absent from some strains of Rhizobia that have the high-affinity oxidase pathway, and hemeprotein b-590 reacts with hydrogen peroxide in preference to oxygen (c. A. Appleby & R. K. Poole, personal communication). A copper flavo protein (6) remains a viable candidate. The carbon monoxide-reactive cytochromes c-552 and c-554 (7) are probably on the high-affinity oxidase pathway, but may not be the actual oxygen-reacting terminal oxidases (c. A. Appleby, personal communication). =

HEMOGLOBIN-MEDIATED SULFIDE UTILIZATION

About half of the hemoglobin of the symbiont-harboring gill of sulfide interface clams is rapidly and reversibly converted to ferric hemoglobin sulfide when the gill is exposed to hydrogen sulfide at low P02 (42; D. W. Kraus, 1. E. Doeller & 1. B. Wittenberg, unpublished data). Ferric hemoglobin sulfide is a stable compound in which hydrogen sulfide (pos sibly HS ) is ligated to the ferric heme iron atom in the distal ligand position (63, 64). Hemoglobin in these clam gills is normally a mixture of oxy- and ferric hemoglobin (perhaps 10-15% fcrric Hb). Hydrogen sulfide introduced into the sea water shifts the equilibrium by trapping ferric hemoglobin as ferric hemoglobin sulfide, which results in the conversion of essentially all the sulfide-reactive hemoglobin to ferric hemoglobin sul fide. The reaction is accelerated (enzymatically?) in the living gill. The oxygen-reactive hemoglobin of the gill remains oxygenated. Rapid and reversible ferric hemoglobin formation in vivo is unique to the symbiont harboring clam gill and has never been detected in any other tissue (42). Sulfide-reactive hemoglobin isolated from the gill of the clam Lucina pectinata binds sulfide reversibly with extraordinary affinity, KD 3. 4 nM, 4000-fold greater than that of other hemoglobins (63). This fact, together -

=

228


with observations of the living gill, emphatically suggests that cytoplasmic, sulfide-reactive hemoglobin augments thc flow of hydrogen sulfide to the symbiotic bacteria. This hypothesis cannot be complete as it stands: The rate of dissociation of hydrogen sulfide from ferric Lucina hemoglobin sulfide, k 2.2 X 10-4 s- \ is slow, indicating a turnover time of 5000 seconds (63). This rate probably is inadequate both for facilitated diffusion and for hydrogen sulfide unloading at the symbiont. However, purified sulfide-reactive hemoglobin accepts electrons avidly from reductants with out prior dissociation of the bound ligand. Subsequently, sulfide dissociates rapidly from the now-ferrous hemoglobin. Ligand delivery in this system may require prior chemical reaction of the hemoglobin. The hypothesis that ligand delivery involves chemical reaction offers a model for possible chemical events in oxygen delivery to intracellular organelles.


=

HEMOGLOBINS AS POTENTIAL TERMINAL OXIDASES

Yeast Hemoglobin

Only certain strains dr yeast produce hemoglobin, and those strains do so i only sporadically (55, 84). Hemoglobin is found in the cytoplasmic fraction of protoplasts at a concentration commensurate with or slightly greater than that of mitochondrial cytochrome aa3 (84). Oxygen affinity in the intact cell is the same as that of the purified protein. There are two prosthetic groups, one protoheme IX and one flavin adenine dinucleotide, attached to a single polypeptide chain of Mr 50,000 (83, 84). Extraordinary affinity of the hemoglobin for oxygen is achieved by nearly diffusion limited oxygen combination, together with a moderate rate of dissociation (83). Yeast Hb has some structural resemblance to flavocytochrome b2 (L lactate: ferricytochrcime c oxidoreductase) from baker's yeast. The latter is a tetramer of identical subunits, Mr 57,500, each bearing one protoheme IX and one flavin mononucleotide, both bound covalently (reviewed in 135). Destruction of yeast Hb in the living cell does not result in any deficiency of growth or oxygen uptake (84). The same is true of terminal oxidases of many bacteria where alternative pathways are available (3). Bacterial Hemoglobin

Bacterial hemoglobin binds oxygen as a hemoglobin and shares the chemi cal reactivity of terminal oxidases. OCCURRENCE

Bacterial hemoglobin has been noted in free-living Rhizobia



229

(4) and in Vitreoscilla (111, 113). Vitreoscilla is a gliding, filamentous, strictly aerobic bacterium in the Beggiatoa family, which often live in oxygen-poor environments. The hemoglobin content of Vitreoscilla increases almost 50-fold when the oxygen pressure of the medium becomes limiting. The hemoglobin gene has been cloned and expressed in Escher ichia coli (40, 56, 57), in which the hemoglobin content may reach 200 flmol/kg wet weight, 8-fold the concentration in Vitreoscilla grown under comparable conditions. Expression of the gene is regulated by oxygen at the level of transcription (41). Hemoglobin-containing Escherichia cells use oxygen at increased rates, especially at low Po" and grow faster and to greater cell densities than cells lacking hemoglobin. Vitreoscilla hemoglobin, over-expressed in its native Vitreoscilla or in Escherichia, is partitioned between the periplasmic space (35-45% of the total) and the cytoplasm (58). We shall consider a physical mechanism, two-dimensional facilitated diffusion, to model the function of periplasmic hemoglobin and a chemical mechanism, transfer of electrons to the ligated oxygen molecule, to model the function of cytoplasmic hemoglobin. Vitreoscilla Hb (reviewed in 113) is a dimer of identical subunits with two protoheme IX per dimeric molecule. The amino acid sequence shows extensive homology with those of eukaryote hemo globins. The histidine proximal to the heme iron is conserved, as is the invariant phenylalanine CD 1, but the residue distal to the heme probably is glutamine (86, I l l ). This residue could form a hydrogen bond to the bound oxygen just as the distal histidine does in myoglobin (87). The apparent oxygen affinity (Table I ) is relatively low, KM,o, 6 flM (69), comparable to the affinity for carbon monoxide, Ko 8 flM (106, 114), with partition between oxygen and carbon monoxide near unity. Heme-heme interactions are marked: carbon monoxide binding is strongly cooperative (106); recombination of photodissociated carbon monoxide is markedly biphasic (115); and reduction of the ferric protein is biphasic, with the sequentially reduced hemes showing very different oxidation-reduction potentials (107). Cooperative carbon monoxide bind ing indicates that reaction of one of two originally equivalent hemes strongly modifics the properties of the second heme. Inequivalence of the hemes is most striking in the mixed valance species blIb'lIl (107) and the oxygenated species WWll)02 ( l 08). Tn each, a ferrous heme fails to react with oxygen. In these and later formulations, "b" represents a protoheme IX residue, "d" represents the heme d (chiorin) prosthetic group of the cytochrome d terminal oxidase complex, and the superscript roman numeral indicates the oxidation state of the heme. Inequivalence of hemes and oxygenation of only one of two potentially oxygenatable hemes are

CHEMICAL REACTIVITY

=

=


230


hallmarks of terminal oxidase function [for examples drawn from Vitreo scilla and Escherichia oxidases, see (2, 3, 92)]. Vitreoscilla Hb forms two oxygenated derivatives. That called oxyhemo globin dominates steady states of oxygen consumption in the intact bac terium (114) or in a reconstituted system (69, 114) and is formed by flash photolysis of diferrous, dicarbon monoxide hemoglobin in the presence of oxygen ( S2). This compound, (bllb'Il)02' has two ferrous hemes but prob ably only a single bound diatomic oxygen ( S2, lOS). Far infrared spectra indicate a bent, end-on configuration of the bound diatomic oxygen as in oxymyoglobin (33). The C-O stretch band of the carbonyl derivative is near that found for cytochrome oxidase and different from that of myo globin (33). Optical spectra of the oxygenated derivatives do not match the predicted sum of oxy- and deoxy hemes (10 S; D. A. Webster, personal communication), indicating extensive electronic coupling between the hemes. A second oxygenated derivative, Compound D, is formed by addition of limited oxygen to the ferrous protein or by stoichiometric reduction of oxyhemoglobin; it is tetrameric with four ferrous hemes and a single bound diatomic oxygen, (b"b'''h02 (lOS). Although binding of oxygen to Compound D may be reversed by reaction with carbon mon oxide (lOS), binding of oxygen to oxyhemoglobin is not. Optical spectra suggest formation of a compound with one ferrous and one ferric heme and a single bound carbon monoxide, presumably with liberation of superoxide anion (lOS). The lack of reversibility suggests that steady state respiratory uptake follows a pathway that does not involve deoxygenation. A soluble flavoprotein, NADH- Vitreoscilla Hb reductase, reduces the dimeric ferric protein when supplied with additional cofactors (50). BACTERIAL HEMOGLOBIN-A SITE OF OXYGEN REDUCTION? Anraku & Gennis (3, see also 71) proposed a scheme for the functional organization of the respiratory chain of Escherichia and, by extension, Vitreoscilla (46). In this scheme, proton liberation that accompanies ubiquinol oxidation on the outer face of the plasma membrane is separated from proton uptake that accompanies reduction of bound oxygen to water on the inner, or cytoplasmic face. Transmembrane proteins catalyze these functions and vectoriaJly transmit electrons from the ubiquinol-oxidizing center [e.g. the protoheme cytochrome bSS8 moiety of the Escherichia cytochrome 0 (70) and cytochrome d (71, 136) complexes] to the oxygen-binding and -reduc ing center [e.g. the cytochrome d moiety of the cytochrome d complex (72)]. Tn every case, binding of diatomic oxygen is an obligate first step in terminal oxidase action. We suggest that cytoplasmic Vitreoscilla hemoglobin, in temporary association with a transmembrane protein, could bind oxygen and transfer electrons to ligated oxygen.


231


Horseradish Peroxidase

By the very act of binding oxygen, heme protcins breach the defenses of the diatomic oxygen molecule against chemical reaction (85, 116). Sources of further reducing equivalents may be bound substrate, as in tryptophan dioxygenase (52) or indoleamine 2,3-dioxygenase (98); substrate and ancil lary proteins, as in mixed function oxidases; or additional oxidation reduction centers in the terminal oxidases. Here we discuss the few known examples of simple oxygen-binding proteins that do not have bound sub strate or additional oxidation-reduction centers and that accept four elec trons and reduce oxygen to water. One such protein is oxygenated horseradish peroxidase (132), which does not dissociate bound oxygen at a measurable rate. Oxyperoxidase is reduced by dithionite to ferroperoxidase and water without prior dis sociation of bound oxygen (132). Ferroperoxidase may also serve as the electron donor (132). The exact stoichiometry of this latter reaction, i.e. three ferroperoxidase plus one oxyperoxidase yields four ferriperoxidase, implies transient formation of a complex within which electron transfer may occur. Complex formation is not a unique reaction path; reaction may occur through three sequential, one-electron reductions. We favor a reaction path involving internal electron transfer within a protein-protein complex because the 250,000-fold decreased carbon monoxide affinity of horseradish peroxidase in concentrated solution (J. B. Wittenberg, unpublished data) proves protein-protein complexes are formed by horse radish peroxidase. Tn the presence of quinols or other two-electron reductants, oxy peroxidase i� re duced to ferroperoxida�e, which react� with addition al oxygen and reforms oxyperoxidase without intervention of ferric per oxidase as an intermediate (59, 60, 95). Bound oxygen is reduced to water, which retains all reducing equivalents donated by substrate (59). No intermediates have been detected (R. W. Noble, B. A. Wittenberg & J. B. Wittenberg, unpublished data). A probable reaction course is two sequen tial two-electron steps: Oxyperoxidase(VI) + 2e + 2H+

�

ferrylperoxidase(IV) + 2e + 2H+

ferrylperoxidase(IV) + H20

--+

ferroperoxidase(II) + H20

and, to complete the cycle, ferroperoxidase(II) + O2 --+ oxyperoxidase(VI). The roman numerals in these expressions denote the oxidation state of each complex. Oxidation state is defined as the number of reducing equivalents required in a hypothetical reaction to reduce the complex of the iron


232


porphyrin and its associated ligands, among which sharing of electrons may occur, to metallic iron and simple compounds such as water (85). It is not presently possible to formulate an unequivocal reaction path for the above reductive reactions. The converse, reaction of ferrous peroxidase with hydrogen peroxide to form oxyperoxidase, however, unquestionably proceeds in a sequence of two single-step, two-electron reactions, with the ferryl derivative [horseradish peroxidase(IV), Compound II] serving as an intermediate (80). In an analogous reaction, ferrous Ascaris pcrienteric hemoglobin reacts with hydrogen peroxide to form oxyhemoglobin in a sequence of two single-step, two-electron oxidations, again with the ferryl derivative Hb(IV) as intermediate (123). In yet another example, ferrous soybean \eghemogJobin reacts with hydrogen peroxide to form ferryl Lb, Lb(IV), in a single-step two-electron process (IS). Myoglobin reacts simi larly, but its reaction goes on to form oxymyoglobin in a second step (126). The two-step course of the oxidative reactions gives credence to the suggested course of the converse reductions. Myoglobin

In the presence of an electron donor (hydroxyethylhydrazine), oxymyo globin is reduced to ferrous myoglobin, which immediately combines with additional oxygen to regenerate oxymyoglobin in a cycle already described for horseradish peroxidase (B. A. Wittenberg & J. B. Witten berg, unpublished data). The cycle continues until all oxygen present is consumed, at which point ferric myoglobin appears. Introducing new oxygen does not reestablish the cycle, proving that ferric myoglobin is not an intermediate. Spectral perturbation shows that a form of myoglobin other than oxymyoglobin is present during the reaction. Perforce the final step in the reaction sequence is a two-electron reduction because ferric myoglobin does not participate. We can formulate the overall sequence, by analogy with the reactions of horseradish peroxidase, as two sequential two-electron reductions with ferryl myoglobin as an intermediate. Reaction of oxyhemoglobin with the one-electron reductant aquopenta cyanoferrate(II) takes an entirely different course. Oxygen is reduced only to hydrogen peroxide, and ferric hemoglobin is the product (54). The reaction of oxyhemoglobin with nitrite follows a complex but similar path (94). We conclude that cytoplasmic oxyhemoglobins may undergo successive two-electron reactions, with reduction of bound dioxygen oxygen to water. Ferryl Hemoglobin in Tissue

Hemoglobin in certain invertebrate tissues, the muscle (124) and nerves (131) of Ap/ysia (a mollusc), and the nerves of Aphrodite (an annelid) (131;


233


J. B. Wittenberg, unpublished data), is completely converted to a spectral entity identified as ferryl hemoglobin whcn the tissues cxhaust their oxygen. On reexposure to oxygen, the hemoglobin reverts to oxyhemoglobin, and the cycle may repeat indefinitely. These findings reinforce the suggestion that cytoplasmic oxyhemoglobins undergo two successive two-electron reductions, with ferryl hemoglobin as intermediate. MYOGLOBIN AND LEGHEMOGLOBIN ASSOCIATED IRON A Protein from Pigeon Breast Muscle

Every gram mole of myoglobin in pigeon breast muscle matches precisely (± 5%) one gram atom of additional iron. This iron may be quantitatively isolated as an iron protein (1. B. Wittenberg, unpublished data). The protein has only recently become available in pure form; its properties are now being defined. It is a homotetramer of 64,000 Mr subunits. The iron is electron paramagnetic resonance (EPR) silent (129), suggesting a mononuclear ferrous center. Spin echo electron paramagnetic resonance spectra establish histidine ring nitrogen as a ligand to the iron atom (J. F. Lu, J. B. Wittenberg & J. Peisach, unpublished data). In the absence of oxygen, nitric oxide forms a complex with the protein. The electron paramagnetic resonance spectrum of the nitric oxide complex is structured and is centered at 9 2.03. One may consider it a unique signature for the iron protein-nitric oxide complex, and it may be used to localize the protein in tissue fractions. As isolated, the iron is ferrous; in air it becomes ferric and eventually denatures. The physiologic role of pigeon breast muscle myoglobin-associated pro tein is unknown. The exact molar equivalence of iron in this protein with myoglobin, however, implies binding between them. We consider two possibilities: that complex formation transiently tethers myoglobin to the iron protein located near an oxidation-reduction center in the tissue, or that the iron protein serves as a reservoir of reducing equivalents for reduction of diatomic oxygen ligated to the heme iron of myoglobin. =

Chelated Iron from Soybean Root Nodules

Supernatants from high-speed centrifugation of carefully prepared soybean root nodule extracts contain nonleghemoglobin iron in molar equivalence with leghemoglobin. The iron is recovered in a low molecular weight fraction from gel exclusion columns and is not bound to the protein (C. A. Appleby. B. A. Wittenberg & J. B. Wittenberg, unpublished data). Such fractions contain a mixturc of potential iron cheJators (some of which are clearly bacteroid products). The iron is distributed among the chelators.


234


The nature of the substance to which iron was bound in the nodule remains unknown. Peribacteroid units, which are peribacteroid membrane sacs and the bacteroids and soluble contents inside them, may be isolated intact from soybean root nodules (39). The soluble fraction from this preparation contains all of the leghemoglobin-associated iron. The plant cytosolic fraction contains no detectable leghemoglobin-associated iron (D. A. Day, C. A. Appleby & 1. B. Wittenberg, unpublished data). The iron con centration in the isolated peribacterial soluble fraction was 1-3 mM in agreement with the iron concentration calculated from the fraction of nodule volume occupied by the peribacteroid space (39, 51), which is greater than 2 mM. Chelated Iron from Symbiont-Harboring Mollusc Gills

Iron, 0.4 mmol/kg wet weight tissue, is recovered as a single chelated species in the low molecular weight, soluble fraction of gills from the clam Lucina pectinata (D. W. Kraus & 1. B. Wittenberg, unpublished data). The concentration of this iron is comparable with that of one of three different hemoglobins found in the gill at concentrations of 0.50, 0.50, and 0.58 mM heme/kg wet weight tissues, respectively (62). Recall that symbiotic bacteria in the clam gill, as in the root nodule, reside in peri bacterial sacs. One may infer that the peribacterial space contains the iron. PERIPLASMIC HEMOGLOBIN

About one third to half of the hemoglobin of bacteria in which the hemo globin gene is over-expressed is in the periplasm, external to the plasma membrane and internal to the bacterial outer membrane (58). One may regard the periplasm, which is about the thickness of a single protein molecule, as a two-dimensional domain. Within it, hemoglobin occurs at 200 11M concentration. In oxygen-limited growth when hemoglobin is of most benefit to the bacterium, the concentration of hemoglobin-bound oxygen in the periplasm may exceed 500-fold the concentration of free oxygen. Adam & Delbruck (1), in a celebrated paper, develop the idea that organisms achieve efficient delivery of ligands to their targets by reducing the dimensionality in which diffusion takes place from three-dimensional space to two-dimensions. They consider the case of diffusion of a particle, e.g. an oxyhemoglobin molecule, to a target, e.g. a bacterial terminal oxidase, in the bacterial plasma membrane. Murray (76) extended their idea to include hemoglobin-facilitated oxygen diffusion, in which case the description includes the probability of dissociation of bound oxygen in the vicinity of the target. The path traversed by a diffusing particle before



235

encountering a target is much shorter in two dimensions than in three dimensions, and, particularly if the target area is small, two-dimensional diffusion to capture will be much faster than three-dimensional diffusion. The finding that lateral diffusion of some proteins in the periplasm is 1 0to 1 00-fold slower than diffusion of comparable proteins in the cytoplasm (30a, 72a), too slow to support significant facilitated diffusion, casts doubt upon the attractive idea that periplasmic hemoglobin could facilitate diffusion of oxygen to plasma membrane terminal oxidases. OXYGEN SENSING

Appleby et al ( 1 0) suggest that hemoglobin occurring at micromolar con centration in rapidly growing cells of the plant root tip may sense oxygen pressure. Desaturation of the root hemoglobin may signal an oxygen deficit and initiate synthesis of a new suite of enzymes required for the switch from aerobic to anaerobic metabolism observed in roots starved of oxygen. The kidney and liver synthesize erythropoietin, the hormone that stimu lates red blood cell production, in response to hypoxia. Goldberg et al (49) report a human hepatoma cell line that increases its level of erythropoietin mRNA and its production of erythropoietin protein 20- to 50-fold in response to hypoxia. Carbon monoxide blocks hypoxic stimulation (48). This finding, together with other evidence, suggests strongly that the oxy gen sensor is an oxygen-binding hemeprotein. Desaturation of this putative hemoglobin is seen as triggering expression of the erythropoietin gene. As already noted, transcription of the hemoglobin gene in the bacterium Vitreoscilla is turned on at low oxygen pressure (41 ). Possibly hemoglobin itself acts as the oxygen sensor. Fractional saturation of erythrocyte hemoglobin reportedly controls sodium proton exchange in the erythrocyte membrane (75). RECAPITULATION: MOLECULAR MECHANISM

Cells of the legume root nodule, less than half a millimeter removed from the atmosphere, operate near 0.005 torr P02. Cells in the mammalian heart, a micron removed from red cells in the capillary, operate near 2 torr P02; sarcoplasmic myoglobin is half oxygenated. Little free dissolved oxygen is available to diffuse, and myoglobin-facilitated diffusion of oxygen plays a dominant role in movement of oxygen through the cytoplasm. Its role is to make oxygen pressure very nearly the same everywhere within the myoglobin containing domain. Myoglobin molecules carry away oxygen, entering heart cells at the sarcolemma, thereby steepening the gradient across the capillary wall and speeding oxygen entry. Oxygen pressure


236


in the bulk of the sarcoplasm is transmitted to the very surface of the mitochondrion, and oxygen is made instantly available to cytochrome oxidase. The path followed by facilitated diffusion may be tortuous indeed; mitochondria preempt one third of the volume of cardiac muscle cells and two thirds of the volume of muscle cells specialized for heat production in the marlin heater organ (Table I ) (28). Myoglobin, doubtless excluded from the volume occupied by the contractile machinery must be confined to the remaining cytosol. In this system, diatomic oxygen crosses the mitochondrial membrane(s). In the presence of experimentally imposed superabundant oxygen, car bon monoxide blockade of myoglobin function abolishes at least one third of the oxygen consumption and a significant part of the ATP synthesis of isolated cardiac myocytes. The phenomenon is known as myoglobin mediated oxidative phosphorylation. It depends on electron flow in the mitochondrial electron transport chain and requires functioning mito chondrial ATP synthase. Blockade of ATP synthase, at unchanged oxygen uptake, blocks the myoglobin� mediated effect -strong evidence for chemi cal interaction between cytoplasmic and mitochondrial systems. In this system, something other than oxygen must cross the mitochondrial mem brane(s). This could be a substrate or a carrier of reducing or o xidizing equivalents (e.g. NADH or a metalloprotein). Myoglobin-mediated oxi dative phosphorylation is a dominant function of myoglobin. We do not know the molecular mechanism but suggest two, not necessarily mutually exclusive, explanations. One explanation is essentially geometric, an extension of the concept of facilitated diffusion. An oxymyoglobin molecule may disengage its bound diatomic oxygen immediately adjacent to the mitochondrial surface. Or, alternatively, the myoglobin-associated iron protein, argumendo, located in a membranous domain, may interact with and tether oxymyoglobin, increasing the probability that a bound oxygen molecule will be disengaged within a limited domain of molecular dimensions. Computer simulation of the random walk of an individual molecule shows that " . . . a diffusing particle that finds itself in a given region of space is destined, by that very circumstance, to wander around that region for a time, probing it rather thoroughly before wandering away for good" (16). Perhaps a myoglobin disgorged oxygen molecule, released in immediate proximity to cyto chrome oxidase, is made available for rapid capture. A second explanation is chemical. We have shown that simple oxygen binding protoheme proteins without additional cofactors or oxidation reduction centers accept four electrons and reduce ligated diatomic oxygen to water in a reaction probably involving two sequential two-electron transfers. In an analogous situation, ferric hemoglobin sulfide, in the clam-



237

bacterial symbiosis, may well accept electrons before disengaging its bound ligand.We consider that cytoplasmic hemoglobins may be a site of electron transfer to bound oxygen in living tissue. To be effective in ATP generation, reducing equivalents for reduction of bound oxygen must come from a transmembrane protein capable of vectorial separation of electron charges or protons. In prokaryotes, this requirement implies complex formation between cytoplasmic bac terial hemoglobin and a transmembrane protein located in the plasma membrane. Together, the hemoglobin and membrane-spanning protein would make up a terminal oxidase system. In eukaryotes, the mitochondrial intermembrane space and outer membrane separate the inner membrane (site of terminal oxidases and ATP synthase) from cytoplasmic myoglobin. In the eukaryote-bacteria symbioses, the bacterial periplasm, the bacterial cell wall, the peribacterial space, and the peribacterial membrane separate the terminal oxidases of the bacterial plasma membrane from host cytoplasmic hemoglobin. For simplicity, we discuss the mitochondrion only. Wc propose that electron transferring proteins located either in the intermembrane space or mito chondrial outer membrane may couple an inner membrane-spanning pro tein with cytosolic myoglobin, a putative site of oxygen reduction. A role for the pigeon breast muscle myoglobin-associated iron protein is implicated in this process. Myoglobin, although spatially separated from membrane-bound ter minal oxidases, augments oxygen uptake by facilitating diffusion of oxygen to the mitochondrial surface. Here, diatomic oxygen crosses the mito chondrial membrane and is accepted by cytochrome oxidase. Indepen dently, oxyhemoglobin enhances mitochondrial electron flow by pro cesses called myoglobin-dependent oxidative phosphorylation. Something other than diatomic oxygen, such as a substrate or a carrier of reducing or oxidizing equivalents, must cross the mitochondrial membranes in this instance.

A CKNOWLEDGMENTS

We thank Dr. Cyril A. Appleby for ongoing discussion during 20 years of collaborative study of hemoglobin function. Supported by Grants HL40998 and HLl 9299 (to B. A. W.) from the United States Public Health Service and by Research Grants DMB 8416016 and DMB 8703328 (to 1. B. W.) from the National Science Foundation. 1. B. W. is a Research Career Program Awardee I -K6-733 of the U.S. National Heart, Lung and Blood Institute.

238


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