I. INTRODUCTION

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Hemoglobin (Hb) is encountered in all five kingdoms of organisms. In the animal kingdom, apart from vertebrates, Hb occurs widely but sporadically in the phyla Platyhelminthes, Nemertea, Nematoda, Annelida, Vestimentifera, Pogonophora, Echiura, Phoronida, Arthropoda, Mollusca, Echinodermata, and Chordata and is found in some 33% of the presently known animal classes (351, 542, 578). Over the last dozen years, single-chain globins have been found in nonleguminous plants, algae, and a number of prokaryotes, ranging from bacteria to cyanobacteria. Chimeric Hbs comprised of an NH₂-terminal globin domain linked covalently to a flavoprotein have been found in bacteria and yeast. Very recently, an aerotaxis transducer in a bacterium and an archean was shown to have an NH₂-terminal globin domain (240).

Nonvertebrate Hbs ranging from monomers to giant multisubunit structures occur in widely different anatomical sites, either in the cytoplasm of specific tissues (muscle, nerve and glial cells, gametes, etc.) or red blood cells (RBCs) or freely dissolved in vascular, coelomic, or perienteric body fluids. In this review Hb refers to all classes of O₂ binding heme proteins ranging in size from single one-domain globins to the most complex ones, and myoglobin (Mb) is used nonexclusively to denote generally single one-domain Hbs occurring in muscle, nerve/glial, and other tissues as well as those found in unicellular organisms, including the IDO-like Mbs of abalone molluscs (525). Additionally, single one-domain globins occurring in symbiont-containing leguminous plants, in nerve tissue, and in cyanobacteria are denoted LegHbs, neuroglobins, and cyanoglobins, respectively. The term erythrocruorin used in the older literature for all extracellular Hbs and some cytoplasmic invertebrate Hbs is no longer employed. Chlorocruorin (Chl) refers to a subgroup of hexagonal bilayer (HBL) Hbs that are greenish red as the result of a modified heme group and occur in four marine annelid families (see sect. IIIC3). Chimeric Hbs comprised of covalently linked globin and flavoprotein domains and found in bacteria and yeasts (see sect. IIIA1) are named flavoHbs (FHbs).

Crystals of all the known vertebrate and nonvertebrate Hbs and Mbs exhibit a tertiary structure (the Mb fold) that consists of six to eight -helical segments connected by short loops (50, 343). This structure forms a three-on-three helical sandwich able to bind heme with high affinity within a cavity lined by hydrophobic residues. The amino acid sequences of nonvertebrate Hbs and Mbs, now more than 170, including the sequences of all the known globin chains comprising the large and more complicated invertebrate Hbs whose crystal structures are not known, can be aligned quite reliably with the over 600 sequences of vertebrate Hbs and Mbs using the known crystal structures, mostly of monomeric globins (36, 289, 343, 394). Although the percent of amino acid identity varies widely and can be almost random, two features are conserved: the invariant residues, Phe and His at positions CD1 and F8, respectively, and the characteristic patterns of hydrophobic residues in each of the -helical segments. The remarkable conservation of the Mb fold is consonant with the globin family having one of the highest conservation of residue-residue contacts among known protein families (82, 457). Phylogenetic trees based on the known sequences point to a common and quite ancient globin ancestor for all the known present-day globins (205, 394), possibly a primitive archebacterium that developed 3,500 million years ago (20).

Compared with the intensively studied vertebrate monomeric (17 kDa) Mb and tetrameric (64 kDa) Hb, nonvertebrate Hbs exhibit much broader variation in their primary and quaternary structures. Although nonvertebrates are phylogenetically more primitive than vertebrates, the high variability encountered in their Hbs reflects specialization and adaptation to a greater range of operating conditions than in vertebrates (630). However, compared with the vertebrate Hbs, much less is known about the relations between their physiological functions and their molecular structures at the atomic level.

The as yet incompletely investigated array of quaternary structures can be broadly subdivided into several distinct groups (618) (Fig. 1). 1) The monomeric, 17 kDa, one-domain, single-chain Hbs and Mbs, which can be intracellular (tissue or cytoplasmic or located within RBCs) or extracellular, form the largest group of nonvertebrate globins. In addition to the widely occurring muscle Mbs of molluscs, annelids, and nematodes and the monomeric Hbs in annelid RBCs, this group includes the "truncated" globins, which have some 30-40 fewer residues than normal globin chains. The latter comprise the 109-amino acid residue neuroglobin of a nemertean (602), the 116- to 121-residue protozoans Hbs (533), and the 118-residue bacterial cyanoglobin (438). 2) The second group contains dimers of bacterial Hbs (621), dimers and tetramers of intracellular RBC globins, such as the Hbs of the clam Scapharca (466), and higher complexes of single-chain globins, such as the polymeric intracellular Hb of Glycera (612). 3) The third group comprises large multisubunit Hbs with masses ranging from 200 to 800 kDa, comprised of two-domain globin subunits (~35 kDa), found in arthropods (251) and nematodes (44). 4) The fourth group contains multisubunit, multidomain Hbs, consisting of one or more chains of 4-20 covalently linked globin domains, encompassing the ~250-kDa Hb of brine shrimps (363), the 1,700- to 2,300-kDa snail Hbs (53), the largest known, polymeric Hbs (>8,000 kDa) found in clams (563), and the 124- and 153-kDa Hbs found in the hydrothermal vent polychaete Branchipolynoe (243). 5) The fifth group is the giant extracellular HBL Hbs (~3,600 kDa) of annelids and vestimentiferans comprised of 180-192 polypeptide chains of which about one-third are nonglobin linker proteins (339). In addition to a wide variation in molecular size, nonvertebrate Hbs exhibit a broad spectrum of O₂ binding properties. Their O₂ affinities that may be dependent or independent of pH (due to the presence or absence, respectively, of Bohr effects) cover over five orders of magnitude, and cooperativity coefficients vary over a ~10-fold range (Table 2).

View larger version (36K): [in this window] [in a new window]   Fig. 1. Schematic diagram of the main quaternary structural variations of invertebrate hemoglobins (Hbs) (see sect. I). Individual species may have a combination of Hb types.

Although the physiological functions of vertebrate Hb are the transport of molecular O₂ and a role in nitric oxide (NO) metabolism, those of nonvertebrate Hbs are much more diverse. In addition to O₂ transport (see sect. IIC3) and storage (see sect. IIC4), they include facilitation of O₂ diffusion (see sect. IIC5), reactions with sulfide and its transport (see sect. VB), complex and as yet incompletely elucidated roles in NO regulation and metabolism (see sect. IIIA1), maintenance of acid-base balance (see sect. VA), O₂ scavenging (see sect. IIIA2), O₂ sensing (see sect. VD5), oxidase and peroxidase activities, the latter related to detoxification (see sect. VD1), vitellogenin-like function (see sect. VD3) and roles as light-shading pigments (see sect. IIIA4) and regulators of the buoyancy of aquatic insects (see sect. VD4). A salient characteristic of invertebrate Hbs is heterogeneity in molecular and functional properties, which can be extensive and which is likely to be beneficial to the organism (see sect. IVA). The two best-characterized cases are the extracellular larval Hbs of the insect Chironomus (see sect. IIIC1) and the intracellular Hbs of the bloodworm Glycera (see sect. IIIB3). Hbs with different O₂ binding properties may also occur in different sites providing a basis for intersite O₂ transfer (see sect. IV). The diversity in function of cytoplasmic Hbs has been surveyed in a unique review by J. Wittenberg (664).

Numerous reviews of specific groups of nonvertebrate Hbs and Mbs have appeared over the last three decades: Hbs in parasites (342), extracellular Hbs (13, 94, 351, 541, 609, 629), bacterial Hbs (434a, 647), crustacean Hbs (353), mollusc Hbs (53, 396, 445, 558), intracellular Hbs (351, 355, 463, 546, 558, 577, 629), symbiotic and nonsymbiotic plant Hbs (17, 18, 20, 21, 30, 167, 232), nematode Hbs (44) including Ascaris Hb (198, 199), Hbs in unicellular organisms (490, 533), cytoplasmic Hbs and Mbs (445, 664, 666, 668), Hbs of eukaryote/prokaryote symbioses (317a, 663, 666), extracellular Hbs (208, 342, 450, 451, 550, 578, 609, 618, 651) and Mbs (520). The respiratory functions of invertebrate Hbs have also been reviewed repeatedly (351, 353, 355, 356, 424a, 542, 550, 629, 630). The current knowledge of the crystallographic structures of the predominantly small nonvertebrate Mbs and Hbs has been reviewed by Bolognesi et al. (50).

In contrast to reviews on separate groups and types of nonvertebrate Hbs, we have attempted to provide here a comprehensive overview of their functional properties, structures, and adaptations and to identify the major adaptational and evolutionary strategies.

	II. BIOLOGICAL ROLE

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Although specific Hbs may be specialized for a particular function, a strict division between the roles in transporting O₂, storing O₂, and facilitating its diffusion is not feasible. In transporting O₂, Hb bridges wide and independent variations in O₂ tensions at the sites of O₂ loading and unloading, particularly in nonvertebrates subjected to highly variant ambient conditions. Although the O₂-transporting role of circulating Hbs can be readily established in larger organisms (from differences in O₂ saturations between the pre- and postbranchial/pulmonary circulations), doubt about their functional significance is provoked by the lack of correlation between its presence and the hypoxic/anaerobic tolerances in different species. However, apparent superfluousness under a given set of conditions does not exclude a vital role under another more stressful one (630). Furthermore, a capacity to live under anaerobic conditions does not exclude reliance on Hb, which "can function in O₂ transport only in the presence of O₂, not in its absence" (356). Several examples of the organismic role of Hb are described below.

The induction of Hb synthesis in many invertebrates under stressful conditions (hypoxia, temperature increase and CO poisoning) (59, 162, 307) attests to its role, as do inter- and intraspecific comparisons of animals with and without Hbs. Thus the mud-dwelling nematode Enoplus brevis that has pharyngeal Hb maintains higher feeding rates under hypoxia than the related, Hb-free E. communis (34). Analogously, CO blockade of Hb function drastically reduces filter-feeding in Hb-rich Chironomus plumosus larvae, but hardly affects that in Hb-poor Endochironomus albipennis specimens (623) and Hb-rich specimens of C. plumosus larvae survive progressive hypoxia longer than Hb-poor ones (624). Similarly, the Hb-bearing pulmonate snail Planorbis corneus shows greater diving potential, lower postdiving pulmonary O₂ tensions, and a greater exploitation of the pulmonary O₂ store than Hb-free Lymnaea stagnalis (274). At the tissue level, the biological significance of Hb is evident from a much longer duration of neural activity under anoxia (absence of free O₂) in cerebrovisceral tissues of the Hb-containing bivalve Tellina alternata than in the Hb-free tissues of Tagelus plebeius (144, 319). The biological advantages of hypoxia-induced increases in Hb concentration in the crustacean Daphia are discussed in section IIIC7A.

Another illustration is the reduction in O₂ consumption rates following "CO poisoning" that blocks O₂ binding without inhibiting mitochondrial function. Figure 2 illustrates the contribution of Hbs to aerobic metabolism. In the case of the coelomic RBC Hb of the polychaete Enoplobranchus sanguineus and the extracellular Hb of C. plumosus, which burrow in marine and freshwater sediments, respectively, the contribution increases with decreasing ambient O₂ tension. In contrast, the contribution of the coelomic Hb in the clam Noetia increases with increasing tension, while that of the extracellular Hb of Arenicola marina is greatest at intermediate (50-100 Torr) tensions. The role of O₂-transporting proteins may vary with endogenous factors. In the polychaete Sabella melanostigma, blocking Chl function by NaNO₂ showed that the role of the Chl increases with body weight (292).

View larger version (22K): [in this window] [in a new window]   Fig. 2. Depression of O2 uptake of invertebrates at different O2 tensions following CO-blockade of heme oxygenation, illustrating the role of Hbs in transporting O2. Data are from the following sources: Daphnia, Ref. 239; Chironomus, Ref. 152; Enoplobranchus, Ref. 362; Sabella, Ref. 153; Arenicola, Ref. 352; Noetia, Refs. 124a and 630.

CO-poisoning experiments require high values of the partition coefficient M (the ratio of CO to O₂ affinities), which is markedly dependent on the species (351, 630). Hb structure imposes restrictions on CO binding. Compared with M values of 200-250 and 20-50 characterizing mammalian Hbs and mammalian Mbs, respectively (12), the partition coefficient M varies widely in nonvertebrates, from 0.08 in perienteric Hb of Ascaris to ~20,000 in Glycera intracellular Hb (Table 1). Generally, the M values are low (2-50) in RBC and extracellular Hbs and even lower (<1) in cytoplasmic Hbs. The extremely high value (~20,000) found for the monomeric Hb of Glycera dibranchiata is a consequence of a correspondingly high CO-binding affinity (480). Analogously, the extremely low value for perienteric Ascaris Hb (191) tallies with its exceptionally high O₂ affinity, and the high M value for Branchiomma Chl (~540) (160) matches the very low O₂-binding affinities of Chls.¹

B.  Environmental and Endogenous Constraints on Hb Function

1.  Oxygen

The low capacities of nonvertebrates for regulating their "milieú intérieur" imply that the operating conditions of their Hbs vary markedly in parallel with ambient conditions, particularly in internal parasites and in aquatic and burrowing species that routinely are subjected to anoxia, hypoxia (O₂ shortage), or hyperoxia (O₂ tensions exceeding atmospheric levels resulting from plant photosynthetic activity). Nematodes from vertebrate intestines face O₂ tensions from zero in the lumen to 18-30 Torr at the mucosa (459). Higher tensions may prevail for Strongylus spp. that are embedded between crypts of richly vascularized duodenal villi supplied by O₂-rich portal circulation, while the tracheal parasites like Syngamus trachea will experience near-atmospheric tensions (121, 460a). On the other hand, the larvae of the insect Gastrophilus intestinalis face intermittent deficiency in O₂ within the semi-fluid contents of horse stomachs (294).

Internal O₂ tensions Pi_O2, however, depend not only on external values Pe_O2 but also on cuticular O₂ diffusion conductances (G_T), as evident from profound differences between the ambient O₂ tensions at which Hb transports O₂ in vivo (from CO-poisoning experiments; see sect. IIIA) and the O₂ tensions that half-saturate Hb in vitro (P₅₀ values). Given that O₂ uptake rate (MO₂) equals G_T(Pe_O2  Pi_O2) (229), O₂ transport by circulating Hbs is favored by ventilation and perfusion of the respiratory surfaces (gills, lungs, and skin).

Variations in salinity and pH in time and space may exert mandatory effects on Hb function in euryhaline invertebrates living in widely different salinities and lacking significant osmotic, ionic, and acid-base regulatory capacities. The in vivo body fluid pH values in polychaetes and other invertebrates studied range from 6.84 to 7.44 (360, 648) and predictably show greater variation under unfavorable conditions.

These compounds may block aerobic metabolism as well as Hb O₂ binding and their effects may compound the impact of other environmental stresses, as in the polychaete Arenicola marina and clam Astarte borealis from marine sediments, where hypoxia increases sulfide-induced Hb autoxidation and the simultaneous liberation of hydrogen peroxide (1). Sulfide-rich habitats range from inter- and subtidal muds to deep-sea cold seeps, seagrass beds, sewage outfalls, and deep-sea hydrothermal vents. Bivalves, vestimentiferans, and annelids like Alvinella pompejana from "white smoker" hydrothermal vents, encounter high concentrations of HS (up to 1 mM) as well as relatively high CO concentrations (148, 345, 544). The CO/O₂ partition coefficient M in Hbs from these animals appears not to have been measured. Many invertebrate groups living in sulfide-rich environments harbor symbiont chemoautotrophic bacteria that oxidize sulfide and fix CO₂. The Hbs of these organisms bind sulfide without covalent modification of the heme groups (unlike mammalian Mb and Hb) and may play a role in transporting sulfide or facilitating its diffusion and in protecting the tissues from sulfide poisoning (see sect. VB).

Variations in CO₂ tension may affect the O₂ binding properties through reversible CO₂ binding to the Hb (as carbamino compounds), or indirectly through pH changes that affect the O₂ binding affinity of the Hb (the Bohr effect). Compared with air breathers that are in a state of "compensated hypercapnic acidosis," i.e., a state where CO₂-induced pH decreases are compensated by high bicarbonate levels (133), aquatic species generally have low internal PCO₂ values due to the high CO₂ solubility in water. However, hypercapnia may occur in some aquatic forms, such as the gutless, deep-sea hydrothermal vent tube worm Riftia pachyptila which uses symbiotic chemoautolithotrophic bacteria for carbon fixing and is periodically exposed to CO₂ and sulfide-rich vent water, in which high internal PCO₂ values (up to 45 Torr) are associated with large base excesses in the coelomic and vascular fluids (580).

The decrease in O₂ affinity with rising temperature mandated by the exothermic nature of heme oxygenation (H ~50 kJ/mol O₂) may jeopardize O₂ loading to Hb in ectothermic invertebrates (whose body temperature coincides with that of the environment) under hypoxic conditions and rising temperature. The temperature effect is, however, reduced by endothermic processes, including oxygenation-linked dissociation of Bohr protons and other ions (646, 674). The Hb of the intertidal polychaete (lugworm) Arenicola marina, which functions under larger temperature variations than the Hb of the subtidal lugworm, Abarenicola claparedii, has a larger Bohr effect ( = 0.9 and 0.3, respectively) and a lower temperature effect (H = 22 and 66 kJ/mol, respectively) (627). Extreme thermal gradients occur in the deep-sea hydrothermal vent habitats resulting from random mixing of O₂-rich, cold (2°C) deep-sea and extremely hot (up to 320°C) anoxic vent waters (581). The hydrothermal vent polychaete Alvinella pompejana holds the record as the most thermotolerant known metazoan: in situ measurements of ambient temperature provided a mean of 68°C with spikes to 81°C (76). In this species, a high temperature sensitivity of O₂ affinity (H = 76 kJ/mol at pH 6.9) (Table 2, extracellular Hbs) appears compensated by an exceptionally high intrinsic O₂ affinity (P₅₀ = 0.3 Torr at 20°C) (581). Large thermal variations are also encountered by Hb-containing prokaryotes like the cyanobacterium Nostoc that extends from tropical to polar terrestrial environments (569).

C.  Intrinsic Structural and Functional Characteristics

1.  Molecular transitions

Hbs exhibit homotropic interactions (cooperativity between O₂ binding heme groups that causes the sigmoidal shape of the O₂ binding equilibrium curves and increase the O₂ turnover for a given change in PO₂) and heterotropic ones, like the Bohr effect (inhibitory interactions between proton binding sites and hemes that decrease O₂ affinity with falling pH and enhance O₂ unloading from Hb at the relatively acid pH in tissues). Tetrameric vertebrate Hbs, which accurately fine-tune O₂ transport to tissues through thermodynamic linkages between heme oxygenation and binding of a range of allosteric ligands, form a convenient reference point for reviewing structure-function relationships in nonvertebrate Hbs. The deoxygenated molecules occur in a low-affinity tense (T) conformation, constrained by intersubunit bonds that are disrupted upon oxygenation as the molecules shift to the high-affinity relaxed (R), oxygenated state. This shift is the basis for cooperativity and is reflected in the displacement between the lower and upper asymptotes of extended Hill plots (see sect. IIIC3D). In vertebrate Hbs, cooperativity requires the presence of two kinds of subunits associated as a tetramer (39), and the quaternary structural shift involves a 12-15° rotation of the ₁₁-dimer relative to the ₂₂-dimer, while the ₁₁- and ₂₂-contacts in the tetramer remain rigid (427). In vertebrate Hbs, proton binding responsible for the normal ("alkaline") Bohr effect occurs mainly at the NH₂-terminal amino acid residues of the -chains and the COOH-terminal histidines of the -chains (428, 449, 646), whereas 143(H21)His appears to be implicated in the reverse "acid" Bohr effect (increase in affinity with falling pH seen at low pH). Hb-O₂ affinity is moreover decreased by chloride and by anionic organic phosphates that bind stereochemically at specific cationic residues at the entrance to the central cavity between the two -chains of deoxyHb.² Accordingly, structural mutations that strengthen the T state or favor effector binding decrease O₂ affinity, and those that favor the R state increase affinity. In vertebrates as well as invertebrates, hyperventilation that promotes excretion of CO₂ and other acidic wastes raises O₂ affinity of Hbs with a normal Bohr effect. In mammalian and lower vertebrate Hbs (592, 639), increasing proton and organic phosphate levels lower O₂ affinity by decreasing the O₂ association constant of the T state (K_T) without significantly affecting that of the R state (K_R).

Although lacking quaternary transitions, some monomeric invertebrate Hbs show pronounced Bohr effects based on protonation-linked TR transitions. Thus conformation-linked proton dissociation and association, respectively, underlie the normal (alkaline) Bohr effect of the larval Hbs III and IV of the insect Chironomus (496, 637) and the pronounced, reverse (acid) Bohr effect ( = +0.8) in the Hb of the parasitic fluke from sheep liver Dicrocoelium dendriticum (500). Analogously, pH-linked conformational changes (138, 482) appear to be responsible for the pH and lactate effect in monomeric vertebrate Mbs (185), and tertiary level fluctuating T states may occur within individual monomeric constituents of human Hb (55). Monomeric nonvertebrate Hbs showing heterotropic effects provide an ideal opportunity to analyze the roles of individual Bohr groups compared with vertebrate Hbs, where these interactions are entangled with quaternary structural changes (500).

In mammalian Hbs, the "fixed-acid" Bohr effect resulting from proton binding is supplemented by a CO₂ Bohr effect attributable to oxygenation-linked binding of CO₂ at uncharged -NH₂ groups. Curiously, a specific, positive effect of CO₂ on O₂ affinity, opposite to that in vertebrate Hbs, has been demonstrated in the extracellular Hbs of the polychaetes Arenicola marina (333), Neoamphitrite figulus (654), the oligochaete Megascolides australis (634) and the crustacean Artemia franciscana (124). The regulatory significance of any of these effects remains obscure.

Oxygenation-linked dissociation, first described in lamprey Hb (62), represents the simplest forms of homo- and heterotropic interactions. The cooperativity (decrease in affinity with falling O₂ saturation) of this Hb and the effect of pH on its affinity is determined by an equilibrium between low-affinity oligomers and a high-affinity monomer. Because proton binding stabilizes the aggregate, the Bohr effect represents a cooperative uptake of protons upon deoxygenation. A structural basis for the dimerization and the Bohr effect was recently provided by the crystal structure of lamprey deoxyHb determined by Heaslet and Royer (227). Oxygenation-linked dissociation has been inferred to occur in nonvertebrate Hbs exhibiting oligomerization, including cytoplasmic Hbs from the nemertean Cerebratulus (602), neuroHb of the bivalve Tellina (319), tracheal cell Hbs from the insect Anisops (652), and RBC Hbs of the sea cucumber Molpadia arenicola (454), the polychaete Glycera dibranchiata (391) and the bivalve A. broughtonii (171). The state of oligomerization of the larval Gastrophilus Hb remains unclear (56).

Because the O₂ binding affinity is determined by the ratio of the O₂ association and dissociation rates (k_on and k_off, respectively), its variation can be due to alterations in only one or both rates. Table 3 shows the O₂ association and dissociation rates of a number of nonvertebrate Hbs and Mbs, compared with sperm whale Mb. The ligand binding kinetics of nonvertebrate Hbs are strongly influenced by the structure of the heme cavity, particularly the size and polarity of residues occupying the distal portion that exert steric and dielectric effects (50, 508).

Vertebrate Hbs and Mbs, with the exception of elephant Mb, have His and Leu at the distal positions E7 and B10, respectively, with the distal His able in some cases to hydrogen bond with the bound O₂, thus stabilizing the oxygenated structure (429). In many nonvertebrate globins, the E7His and B10Leu residues are replaced by Gln and Tyr, respectively, resulting in a tight cage for O₂ and much higher O₂ binding affinities relative to vertebrate Mb. Two types of kinetic mechanisms are now known to underlie the extremely high O₂ binding affinities of some nonvertebrate Hbs and Mbs. In one, exemplified by the perienteric Hb from the nematode parasite Ascaris suum, hydrogen bonding of the bound O₂ in the distal cavity to the B10Tyr is thought to be responsible for its very low O₂ dissociation rate, 4,500-fold smaller than in vertebrate Mb (k_off = 0.004 vs. 18 s¹, Table 3) (431). This low dissociation rate determines the extremely high O₂ affinity, which is 450-fold higher than Mb, even though the O₂ association rate of Ascaris Hb is 10-fold lower than Mb (k_on = 1.5 vs. 15 µM¹ · s¹, Table 3). The other type of kinetic mechanism found recently in trematode Hbs, where both distal residues at positions E7 and B10 are Tyr, combines a low dissociation rate (k_off = 0.03-0.4 s¹) with a high association rate (k_on = 100-200 µM¹ · s¹, Table 3) to produce even higher O₂ affinities (442). However, the Hb of another trematode, Dicrocoelium, in which one of the two distal tyrosines is turned out of the heme pocket, binds O₂ rapidly (k_on = 300 µM¹· s¹), but has an O₂ affinity only 10-fold higher than Mb due to a 2-fold higher dissociation rate (137, 341).

Symbiotic plant Hbs, which have E7His and B10Tyr (9) and very high O₂ binding affinities, >10-fold higher than vertebrate Mb (Table 3), appear to be intermediate between the nematode and trematode Hbs. In contrast to the nematode and trematode Hbs, the B10Tyr does not interact with the bound O₂ to stabilize it. X-ray crystallographic studies imply hydrogen bonding to E7His and close contacts with E11Val (Leu in soybean) (225). Although the association rates of the plant Hbs are comparable, their dissociation rates are appreciably higher than the other two groups, resulting in 30- to 300-fold lower O₂ affinity (192) (Table 3). The recently discovered nonsymbiotic plant Hbs (9), which seem to have the same distal E7His and B10Tyr residues, have O₂ binding affinities as high as the nematode and trematode Hbs, due primarily to very low dissociation rates (31, 120, 147). In this case, the very high O₂ affinities are likely due to stabilization of the bound O₂ via a hydrogen bond to the E7His inferred to exist in barley Hb (120). Furthermore, the HisE7Leu mutation in rice Hb increases the O₂ dissociation rate by more than 1,000-fold (31).

In several nonvertebrate globins, the distal E7His is replaced by a nonpolar residue. Such substitutions in vertebrate Mb result in 10- to 100-fold reductions in O₂ affinity (460). The monomeric Hb from the polychaete Glycera has a distal Leu and very high rates of association and dissociation (k_on = 190 µM¹· s¹ and k_off = 1,800 s¹, Table 3), which result in a ~10-fold lower affinity than Mb. Although the Mb from the gastropod mollusc Aplysia also has a nonpolar Val at E7, the bound O₂ appears to be stabilized by a hydrogen bond with the long and flexible E10Arg that rotates into the heme cavity; consequently, its O₂ dissociation rate is smaller than in Glycera Hb (k_off = 70 vs. 1,800 s¹, Table 3) and its O₂ affinity is higher (51, 661).

Although our understanding of the roles played by residues close to the distal heme cavity underlying the differences in ligand binding kinetics has increased very substantially over the last decade, due mostly to the efforts of Olson and co-workers, it is far from complete. Thus, while single, double, and even triple mutants of vertebrate Mb display the qualitatively correct alterations in ligand binding kinetics, the effects are not quantitative, i.e., they do not reproduce the properties of the wild-type nonvertebrate Mbs and Hbs. A triple mutant of sperm whale Mb designed to simulate the ligand binding properties of Aplysia Mb (ArgCD3Asp/HisE7Val/ ThrE10Arg) (499) has much higher association and dissociation rates, resulting in a fivefold lower affinity than the native Mb (k_on = 88 µM¹ · s¹, k_off = 2,300 s¹, K = 0.039 µM¹ vs. 15 µM¹ · s¹, 70 s¹ and 0.21 µM¹, respectively, Table 3). Another triple mutant designed to mimic Ascaris Hb (LeuB10Tyr/HisE7Gln/ThrE10Arg) has an O₂ dissociation rate of 1 s¹, >10-fold lower than Mb, but still 250-fold higher than the native Hb (694). The limited success achieved with engineered vertebrate Mb designed to simulate nonvertebrate Hb and Mb ligand binding properties, stands in contrast to the much higher level of success achieved with mutants of human Hb designed to mimic the O₂ affinity of high-altitude geese Hb (270, 640) and the bicarbonate effect of crocodile Hb (314). The obvious explanation is that the differences in tertiary structures between vertebrate Hbs are much smaller than those between mammalian Mb and nonvertebrate globins, reflecting the much higher percent identity of sequences and much closer phylogenetic relationship among the vertebrates.

Circulating Hbs occur commonly in metabolically active organisms. The conduits for O₂ transport in invertebrates vary widely: cellular and dissolved Hbs occur in blood (vascular fluids) and in hemolymph and coelomic and hemal fluids. Although invertebrates generally lack closed blood vascular systems, their extravascular fluids may be subjected to well-defined circulations that channel the internal distribution of Hb-bound O₂. In aquatic larvae of the insect Chironomus, a structured circulation of the Hb-rich hemolymph is secured by a well-developed system of internal septae that even extend into tubular appendages (425). Another interesting example is the scaleworm Branchipolynoe, which lives commensally in the mantle cavity of bivalves from deep-sea hydrothermal vents. In adaptation to its hypoxic, sulfide-rich microhabitat, its gills have very large surface areas and small O₂ diffusion distances. Unlike other polychaetes, its gills lack blood vessels but are perfused with Hb-rich coelomic fluid through the action of cilial and myoepithelial contractions (241).

Well-developed, closed vascular systems in invertebrates with distinct hearts, arteries, capillary networks, and veins that permit control of blood distribution are found only in annelids, vestimentiferans, and pogonophorans (377, 476). In large species such as the giant earthworm Glossoscolex giganteus and the polychaete Arenicola marina, blood propulsion is aided by periodic contractions of the blood vessels and gills (271, 277, 351). Unlike polychaetes and oligochaetes, the closed blood vascular system of hirudineans (leeches) is derived from the greatly reduced coelome (377). Although lacking coelomes, nemerteans routinely have a closed system of vessels that are lined with endothelia as in vertebrates (377). Echinoderms (starfish, sea urchins, and sea cucumbers) have coelomic, water vascular, and hemal fluid systems whose interrelationships are not well understood, although each may contain RBCs (355, 377, 476).

O₂ transport in closed circulations may be quantified by the Fick equation, MO₂ = V_b(c_a  c_v), where MO₂ is the rate of O₂ delivery to tissues, V_b is the fluid perfusion rate, and (c_a  c_v) is the difference in O₂ content between "arterial" (postrespiratory surface) and "venous" (prerespiratory surface) fluids. The O₂ content difference thus increases proportionally with O₂ carrying capacity (Hb concentration) and depends on the "loading" tension at the respiratory surfaces (skin, gills, or lungs) and "unloading" tension in the respiring tissues, the Hb-O₂ affinity, and the shape of the equilibrium curve. The O₂ content difference (c_a  c_v) is difficult to assess with available techniques in small and fragile invertebrates. For the lugworm Arenicola cristata, the pre- and postbranchial oxyHb saturation difference (0.06 ml O₂/ml blood), O₂ carrying capacity (130 ml/l), and O₂ consumption rate (0.158 ml · kg¹ · min ¹) (352) indicate a cardiac output of 2.7 ml · min¹ · kg ¹ at 22°C (575, 576).

Heme-bound O₂ forms vital O₂ stores in invertebrates subjected to intermittent O₂ supply, particularly in cyclic-ventilating animals and gut parasites, and in nerve and muscle tissues that exhibit intermittent high-level activities (33, 96, 664). The duration of the O₂ stores obviously increases with a reduction in metabolic rates under hypoxic conditions, as occurs in "oxyconforming" species (629, 630).

Several studies illustrate the significance of tissue Hbs as O₂ stores. In the minute gastrotrichan Neodasys, which is below the "Harvey size limit," the Hb-containing cells constitute 14% of the total body volume and are closely associated with nerve and muscle tissues; the heme concentration (18.5 mM) suffices for 17 min of O₂ consumption by an active animal under aerobic conditions (96).³ A detailed comparative electrophysiological study of several bivalve species with and without Hb in their nerve tissues (145, 319, 320, 322) showed that although there were no obvious electrophysiological differences between cerebrovisceral connectives with and without neuroHb, the connectives with neuroHb consumed much less O₂ during action potential conduction than connectives and other nerves without neuroHb. Thus the neuroHb-containing connectives may effectively use the neuroHb O₂ store to enable the organism to use continued neuromuscular activity under hypoxic conditions. O₂ bound to the neural tissue Hbs in the clams Tellina alternata and Spisula solidissima and the nemertean worm Cerebratulus lacteus can support the O₂ requirements of the nerves for up to 30 min during anoxic periods (145, 320, 602).

RBC Hbs may also serve as O₂ stores. In the phoronid Phoronis architecta, the coelomocyte Hb functions as an O₂ store for ~15 min (599), whereas coelomic PO₂ values in the echiuroid Urechis caupo indicate a longevity of the RBC Hb reservoir of up to 3 h (355, 439). Measurement of the rates of heat dissipation and O₂ consumption in two terebellid polychaetes, Enoplobrachus sanguineus with Hb-containing coelomocytes and Lysilla alba lacking them, revealed a difference in metabolic response upon return to normoxic conditions after exposure to anoxic conditions: a much higher O₂ consumption rate in the Hb-less species, indicating the repayment of an O₂ debt incurred during the hypoxic period (145). Similar experiments with two bivalves species showed a different effect: under hypoxic conditions the rate of heat dissipation of the neuroHb-containing Tellina alternata remained high, while the metabolic rate cycles of the neuroHb-lacking T. plebeius disappeared (145). These studies show that while the response of marine invertebrates to hypoxic conditions can be complex and variable, the presence of Hb may play a role in the partitioning of metabolic flux into aerobic and anaerobic processes.

Extracellular Hbs may also have O₂ storage roles. In the periodically ventilating tubiculous larvae of the insect Chironomus, the duration of the O₂ stores corresponds well with the ventilatory pauses (624), which they thus may permit. The O₂ carried in the blood and coelomic fluids of the hydrothermal vestimentiferan Riftia pachyptila (containing 3.5 and 1.9 mM heme, respectively) can support respiration at the constant and maximum rate for 35 min (87).

A special case of O₂ storage is found in the diving insects Buenoa and Anisops, where large Hb-laden tracheal cells that are penetrated by tracheoles appear to release O₂ in periods of O₂ paucity to help maintain buoyancy (385, 386, 652).

The diffusion of O₂ through tissues is enhanced through its participation in a second, equilibrium reaction with Mb-like proteins, given that the total O₂ flux is the sum of the fluxes of O₂ and protein (382). Facilitated diffusion requires O₂ loading at sites with sufficient O₂ tension and its release at sites with low tension. Large proteins will not contribute significantly since diffusion varies inversely with molecular weight, and significant facilitation requires a sufficient carrier concentration to create a gradient that supplies O₂ faster than the diffusion rate of free O₂ (232). Recent studies with vertebrate muscle reveal that the in situ cellular diffusion coefficient of Mb is much lower than earlier reported, that steric hindrance to Mb diffusion is dominated by cellular architecture rather than by overall protein concentration, and that significant facilitated diffusion requires very low PO₂ values and high Mb concentrations (279, 280). Monomeric Hbs may play important roles; although their diffusion rate is only 1/20 of that of free O₂, their concentrations in tissues may greatly exceed that of free O₂ (some 30-fold in working heart muscle and 10,000-fold in soybean root nodules) (668).

	III. OCCURRENCE AND FUNCTIONAL AND MOLECULAR PROPERTIES

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This section briefly reviews the phylogenetic and anatomical distribution of Hbs and focuses on the functional and structural properties of nonvertebrate Hbs. Although Hbs tend to occur more generally in organisms facing lack of O₂, their occurrence defies strict categorization in terms of phylogeny and environmental conditions. On the basis of their histological sites, quaternary structures, and physiological properties, nonvertebrate heme proteins can conveniently be categorized into 1) noncirculating cytoplasmic Hbs and Mbs that occur in unicellular organisms or in tissues of higher organisms, 2) RBC Hbs that occur in nucleated RBCs circulating in any fluid, and 3) extracellular Hbs that occur in solution in body fluids. The tremendous range in O₂ binding properties encountered in nonvertebrate Hbs is illustrated in Table 2 and Fig. 3.

View larger version (20K): [in this window] [in a new window]   Fig. 3. O2 equilibrium curves illustrating the enormous variation in O2 binding affinities encountered in nonvertebrate Mbs and Hbs. A.l., Ascaris lumbricoides (nematode) extracellular perienteric Hb at 37°C (191); G.m., Glycine max (soybean) root nodule LegHb at 20°C, pH 6.5-7.0 (15, 262); E.e., Explanatum explanatum (trematode) cytoplasmic Hb B at 37°C, pH 7.2 (443); S.e, Siboglinum ekmani (pogonophoran) extracellular Hb at 20°C, pH 6.5 (650); A.m.MbI and A.m.MbII, Arenicola marina (marine polychaete) MbI and MbII, respectively, at 15°C and pH 7.5 (644) (R. E. Weber, unpublished results); N.c., Nostoc commune (cyanobacterium) Hb at 20°C, pH 7.5 (569); A.m.Hb, Arenicola marina hexagonal bilayer (HBL) Hb at 15°C, pH 7.5 (574); B.g., Biomphalaria glabrata (gastropod snail) extracellular Hb at 25°C and pH 7.46 (64); U.c., Urechis caupo (marine echiuran) RBC Hb at 20°C, pH 7.5 (178); A.a., Anisops assimilis (aquatic insect) tracheal cell Hb at 25°C, pH 6.9 and 29 mM heme concentration (652); E.c., Eupolymnia crescentis (marine polychaete) HBL Hb at 10°C, pH 7.1 (365); E.v., Eudistylia vancouverii (marine polychaete) chlorocruorin (Chl) at 25° pH 7.1 (259).

Cytoplasmic Hbs and Mbs exhibit an even more intermittent phylogenetic distribution than circulating Hbs (520), occurring episodically in prokaryotes (bacteria), unicellular eukaryotes (yeasts, protozoans), flowering plants, and various tissue and cell types (muscles, nerves, gills, tentacles, and gametes) of phylogenetically diverse nonvertebrates animals. Although tissue Hbs are commonly 16- to 18-kDa monomers, dimeric 32- to 35-kDa Mbs occur in radulae of some gastropod mollucs (63) and in annelids (553). Smaller chain, 11- to 12-kDa "miniHbs" are encountered in cyanobacteria, protozoans, and nemerteans (264, 533, 602).

Mbs are encountered in body walls and probosces of annelids (179, 553, 557, 644) and in radular, body wall, adductor, and stomach muscles of molluscs (445, 515, 520, 554, 558). Nerve Hbs are scattered among different taxons including nematodes (Ascaris), annelids (Glycera and Aphrodite) (660), the echiuroid Urechis, lamellibranch molluscs (Tivela, Spisula), gastropod molluscs (Busycon and Aplysia) (319, 475), nemerteans (603, 605), and arthropods (Daphnia, where ganglial Hb concentration rises in response to hypoxic conditions) (163). In molluscs and annelids, it occurs at millimolar concentration in glial cells surrounding the nerve cord (318, 322, 323). As in vertebrates, the O₂ affinities of invertebrate Mbs are generally intermediate between those of circulating Hbs and mitochondrial cytochrome oxidase, thus suggesting that they form intermediate O₂ transfer stations.

The physiological roles of cytoplasmic Mbs have received less attention than those of circulating Hbs. This inattention was based on a lack of cooperativity and the perceived absence of functional heterogeneity, conformational transitions, and sensitivities to pH and allosteric effectors. Recent studies have shown these perceptions to be invalid (11, 185, 301, 442, 482) and have brought to light a variety of potential physiological roles for the cytoplasmic Hbs and Mbs.

A) FLAVOHEMOGLOBINS. In the last 10 years, members of a family of ~43 kDa two-domain ("chimeric") flavohemoproteins (FHP) or FHb, comprising an NH₂-terminal globin domain and a COOH-terminal flavin-binding domain, have been discovered in several microorganisms. Their functional significance is under intensive scrutiny. The Escherichia coli heme protein HMP was the first FHb to be sequenced by Poole and co-workers (604) in the course of an attempt to identify the genes encoding dihydropteridine reductase activity. Like the E. coli HMP, the FHb from the Gram-negative, hydrogen-oxidizing and denitrifying bacterium Alcaligenes eutrophus shows the highest sequence similarity to the homodimeric Hb from Vitreoscilla (107, 621); its COOH-terminal portion of ~250 residues appears to belong to the ferredoxin reductase-like family of FAD-dependent oxidoreductases, despite low sequence identity. Although there is high sequence similarity between the globin domains of E. coli and Alcaligenes FHbs and the single-domain Hb from the bacterium Vitreoscilla (see sect. IIIA1B), the crystal structure of Alcaligenes FHb (150, 413) contained a tightly bound phospholipid in the heme cavity that precluded any determination of protein-heme interaction at the distal side of the heme. Closely related FHbs have been found in other bacteria, Erwinia chrysanthemi (157), Salmonella typhimurium (109), Bacillus subtilis (336), and Mycobacterium tuberculosis (97, 246), as well as in the yeasts Saccharomyces cerevisiae (699) and Candida norvegensis (266). Membrillo-Hernandez and Poole (380) have used a primer based on the consensus sequence of the foregoing FHbs to search for Hb-like genes in other bacteria. Such genes were found in Campylobacter jejuni, Listeria monocytogenes, Rhizobium leguminosarum, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Staphylococcus aureus.

Although the kinetics of O₂ binding with Saccharomyces and bacterial FHbs have not been determined, the Candida FHb has a very high O₂ affinity [dissociation constant (K_D) = 0.02 µM] due to a high association rate and a low dissociation rate, 850 µM¹·s¹ and 17 s¹, respectively (416, 417).

E. coli HMP is expressed under aerobic and anaerobic growth conditions and has a high O₂ affinity (K_D ~2.6 µM); it has been suggested that it could serve as an O₂ sensor by combining with intracellular O₂, thus limiting flavin reduction in the aerobic state (435, 436). Because it is also able to reduce Fe(III), cytochrome c, and the Azotobacter regulatory flavoprotein NifL (348, 436), it might affect the redox status and contribute to the regulation of the fnr gene (fumarate, nitrate respiration) and the soxRS genetic locus.

The expression of FHbs appears to be sensitive to alteration in O₂ concentration; it is enhanced by hyperoxic conditions in Saccharomyces (110, 696) and induced by lack of O₂ in the bacteria Alcaligenes and Bacillus (336, 440). Although the FHb deletion strains of E. coli do not appear to be sensitive to superoxide (378, 379), a Saccharomyces FHb deletion strain was found to be sensitive to oxidative stress (696).

Several recent results have indicated the involvement of FHbs in the metabolism of nitrogen oxides. Marked upregulation of E. coli HMP by NO was observed under aerobic and anaerobic conditions (435), and Bacillus subtilis FHb was found to be induced by nitrite (336). Crawford and Goldberg (109) showed that Salmonella FHb has a specific role in the protection from nitrosoglutathione, and presumably NO, that is independent of O₂. They also demonstrated that under anaerobic conditions, the inducible FHb protects Salmonella from acidified nitrite or an NO donor compound (108, 109). Likewise, Poole and co-workers (378, 379) showed that E. coli HMP expression was upregulated in the presence of NO and provided protection against oxidative stress. Anaerobic growth of an Alcaligenes FHb strain on nitrite was found not to accumulate nitrous oxide as a transient intermediate (107). Gardner et al. (177) isolated an O₂-dependent and cyanide-sensitive NO dioxygenase activity from NO-resistant E. coli and identified it as HMP, a result confirmed by Hausladen et al. (226). These results and more recent ones (512a) suggest that FHbs are protective against nitrosative stress. It is likely that FHbs have an additional function, since the O₂-dependent NO dioxygenase activity cannot explain the protection FHb affords against nitrosoglutathione in anaerobic Salmonella (109) or NO gas in anaerobic E. coli (177). Nor does it explain the benefit of increased FHb expression in anaerobic E. coli or B. subtilis grown with NO, nitrate, or nitrite (176, 336, 440). Kim et al. (299) have demonstrated that the E. coli FHb has NO reductase activity and have suggested that this activity may account for the anaerobic protection by FHbs.

A very interesting point made by Gardner et al. (177) is that the agreement between the estimated origins of primeval Hb, blue-green algae, and free atmospheric O₂ at ~1.8, ~2.3, and ~1.8 billion years ago, respectively (699), suggests a plausible coevolution of Hb and NO dioxygenase functions. Linkage is possible because the dioxygenase activity of FHbs requires O₂ and because toxic NO is produced by an O₂-dependent oxidation of nitrogen compounds.

B) BACTERIAL HBS. The Gram-negative bacterium Vitreoscilla, an obligate aerobe, contains a dimeric Hb (621) whose sequence is more similar to the globin domains of FHbs and plant Hbs than other invertebrate Hbs (31). Its expression is upregulated at low O₂ levels (139, 140), and ~40% of it occurs in the periplasmic space (296). The Hb occurs as a stable oxy form, has a normal O₂ association rate and an unusually large dissociation rate of 5,600 s¹ (414), and may play a role in the transfer of O₂ to the terminal oxidases under hypoxic conditions by facilitated diffusion (664, 668). The crystal structures of the ferriHb and of its azide complex have suggested hydrogen bonding of the distal B10Tyr hydroxyl via a water molecule to the O₂ ligand (537). The discovery of a NADH-dependent flavin reductase in Vitreoscilla suggests that the heme binding and flavin binding domains have separated in this bacterium during evolution (267).

Vitreoscilla Hb gene was found to be strongly expressed in E. coli, where its presence promotes growth under microaerobic conditions (295). In the absence of the two E. coli terminal cytochrome oxidases, cytochromes o and d, the expressed Hb behaves as a terminal oxidase (141). E. coli mutants lacking the fnr gene product were unable to activate the Vitreoscilla Hb gene under microaerobic conditions (275). Bailey's group (283, 586) has documented the involvement of Vitreoscilla Hb in enhancing the activity and efficiency of the electron transport chain in E. coli under hypoxic conditions. The beneficial effect of the heterologous Vitreoscilla globin gene expression in improving O₂ utilization by the host cells and their increased growth under hypoxic conditions is not limited to E. coli: it has been found to occur in other bacteria, in yeast, in a mammalian cell line (284), and in transgenic plants (237).

Two Hb genes glbN and glbO occur in the genome of Mycobacterium tuberculosis (97). The deduced amino acid sequences are similar to those of algal, protozoan, and cyanobacterial Hbs (106). Spectroscopic studies of the expressed HbN indicate the distal residue to be Leu, with Tyr in the B10 position (680). It has a substantial cooperativity (n₅₀ = 2), probably due to self-association of the deoxy form and an extremely high O₂ binding affinity (P₅₀ = 0.013 Torr at 20°C), resulting from fast association and slow dissociation rates of 25 µM¹·s¹ and 0.2 s¹, respectively (106). Mutation of the B10Tyr to Leu and Phe resulted in an increase in the dissociation rate constant by more than two orders of magnitude (45 and 30 s¹, respectively) (106), indicating the importance of the stabilization of the bound O₂ through hydrogen bonding with the Tyr hydroxyl. It is likely that the oxyHb has a role in protecting the Bacillus from NO similar to the role of FHb in E. coli (176, 177).

C) TRUNCATED CYANOGLOBINS. Cyanoglobin is a single-chain Hb of 118 residues found in the photoautotrophic cyanobacterium Nostoc commune, which is capable of aerobic nitrogen fixation (438), and in Synechocystis (286). Both sequences have substantial identities with the truncated Hbs from the protozoans Paramecium and Tetrahymena, 116 and 121 residues long, respectively. Nostoc cyanoglobin synthesis is induced by nitrogen starvation and anaerobic incubation; the positioning of its gene between two other genes essential for nitrogen fixation suggests it is involved in the latter function, perhaps as an O₂ scavenger (10). Because protozoa and cyanobacteria often occur together and symbiosis between the two groups is known to occur, it is likely that protozoan Hbs are of cyanobacterial origin (569). A recent study (315a) showed that, in contrast to the Hb genes of Paramecium, those of Tetrahymena and Nostoc lack introns. Cyanoglobin binds O₂ with a rate (390 µM¹·s¹) (570) that is among the highest known (Table 3); however, a fairly high O₂ dissociation rate gives cyanoglobin an affinity (P₅₀ ~0.55 Torr at 20°C; Fig. 3) intermediate between Mb and the LegHbs (570). Cyanoglobin has a higher rate of autoxidation than sperm whale Mb and a ~100-fold faster rate of hemin loss, due probably to the absence of a D helix evident from sequence alignment, which is known to destabilize the heme-apoMb complex (658).

D) TRUNCATED PROTOZOAN HBS. Although the presence of heme proteins in ciliated protozoans was shown many years ago (293), little was known about them except for Paramecium Hb, which exists as a monomeric globin with several different isoforms (6 and 12 components in P. caudata and P. aurelia) and an O₂ affinity comparable to mammalian and Nostoc Mb (P₅₀ ~0.6 Torr at 20°C) (233, 263, 501, 510, 588, 593-596). Shikama and co-workers (264, 265, 534) have sequenced the Hbs from Paramecium caudatum, Tetrahymena pyriformis, and T. thermophila and found them to have 116-121 residues, with deletions occurring in the A- and D-helical as well as in the CD-interhelical regions. Tetrahymena Hb has an O₂ affinity (~0.2 Torr) (316) comparable to that of the other truncated Hbs. Curiously, its rate of autoxidation is ~10-fold slower than that of Paramecium Hb, and similar to that of sperm whale Mb. It is possible that the few additional residues relative to Paramecium Hb and the even shorter, 109-residue nerve tissue Hb from the nemertean Cerebratulus Hb (602), could be the reason for the greater stability of Tetrahymena Hb.

E) ALGAL HBS. Recently, Guertin and co-workers (102) discovered three globin genes in the genome of the green unicellular alga Chlamydomonas eugametos; two genes were cloned, only one of which requires photosynthesis for expression (102). They code for 164- and 171-residue globins, which occur in the chloroplast at concentrations of up to 130 nM and are distributed between the proteinacious ribulose diphosphate carboxylase-rich pyrenoid and the thylakoid membrane regions. Although the sequences exhibit the highest similarity to the Paramecium (264), Tetrahymena (265), Nostoc (438), and Synechocystis (286) Hbs, they do not have the deletions associated with the truncated globins. The algal ferroHb forms stable complexes with O₂ and CO, and the ferriHb forms complexes with thiols as well as with azide and cyanide (104). Spectroscopic and kinetic studies have established that the bound O₂ forms multiple hydrogen bonds with the putative distal E7Gln and B10Tyr, residues, which are found predominantly in high O₂ affinity invertebrate Hbs (104). The most recent spectroscopic study of Chlamydomonas Hb shows it to differ from the other E7Gln- and B10Tyr-containing Hbs, in that it is the B10Tyr and not the E7Gln that ligates the heme iron in the ferri form, aided by a strong interaction with E10Lys (119). The occurrence of a Hb in Chlamydomonas chloroplasts raises the question of its functional role in such a high O₂ tension environment. Because of its small concentration, chloroplast Hb is unlikely to have a storage or facilitated diffusion function. In addition, the O₂ dissociation rate being one of the slowest known (half time = 49 s), it is unlikely to support any function that requires dissociation of the bound O₂. Chlamydomonas Hb has many ligand binding properties in common with the nonsymbiotic plant Hbs: slow dissociation of O₂, a 6-coordinate low-spin Fe(III) form, ligand binding to a 6-coordinate low-spin Fe(II) form which requires prior dissociation of the sixth ligand, and fast autoxidation (103, 119).

The three-dimensional structures of Paramecium caudatum Hb and of a truncated form of Chlamydomonas eugametos Hb have been recently solved by X-ray crystallography by Bolognesi (M. Bolognesi, personal communication). Both proteins display very similar tertiary sructures, consisting essentially of helices B, C, E, G, and H of the conventional globin fold. Only one -helical turn is found in the expected A- and F-helix regions. Particularly, almost the whole F helix is substituted by an extended protein loop, which may reflect specific sequence motifs (including Gly residues) in this molecular region. TyrB10 and GlnE7 residues are properly positioned to form direct hydrogen bonds to the distal site ligand.

In contrast to Hbs from the animal kingdom that have been known at least since Cain (First Book of Moses, chapter 4), "symbiotic" plant LegHbs in soybean root nodules were first described in 1939 by Kubo (334). The last decade has witnessed the discovery of another class of plant Hbs: the "nonsymbiotic" plant Hbs that are expressed at low concentrations in nonnodular, rapidly growing and metabolizing plant tissues and are widely distributed in both mono- and dicotyledonous plants (9, 31, 147, 232).

A) SYMBIOTIC HBS. LegHbs occur in nitrogen-fixing nodules formed as the result of infections of legume roots with one of four genera of prokaryotes, called rhizobia (17, 18); they belong to multigene families and comprise the most abundant soluble protein in the cytoplasm of the root nodules at local concentrations as high as 2-3 mM (167). The crystal structures of lupin (225) and of soybean LegHbs (222) have established the presence of His at both the proximal and distal heme sites.

Due to the combination of high O₂ association rates and normal dissociation rates, 100-300 µM¹·s¹ and 5-30 s¹, respectively (192, 665), LegHbs have extremely high O₂ affinities: P₅₀ = 0.04-0.07 Torr at 20°C for three components of soybean Hb (Table 2, Fig. 3), and intermediate for a mixture of the components, indicating the absence of specific interactions, at least in dilute solutions (16). Direct equilibration measurements (A. Rashid and R. E. Weber, unpublished results) show a slightly lower affinity (P₅₀ = 0.14 Torr at 25°C).

The kinetics of oxygenation of soybean LegHb distal histidine mutants support the hypothesis that the high affinity is determined mainly by enhanced accessibility and reactivity of the heme group (222). Contrary to the persistent notion that LegHbs act as O₂ scavengers to prevent inhibition of nitrogen fixation, it is now clear that LegHbs function to provide an adequate supply of O₂, albeit at low concentrations, to the terminal oxidases of the symbiotic bacteroids (17). This occurs at a stabilized free O₂ concentration sufficient for the functioning of the oxidase but low enough to prevent inactivation of the nitrogenase enzyme also located in the bacteroids (18). The only nonlegume plant known to form a symbiosis with rhizobia is Parasponia andersonii, where a single gene appears to encode a Hb found in roots and root nodules (49). Although its O₂ binding kinetics are similar to LegHbs (669), indicating a similar function, its sequence differs from other legHbs and is more similar to the nonsymbiotic Hbs. Symbiotic (actinorhizal) association between Frankia, a member of moldlike bacteria (actinomycetes) with a diverse range of dicotyledonous plants such as Casuaria glauca and Myrica gale, also results in the formation of nitrogen-fixing and Hb-containing root nodules (424, 498). The sequence of Casuarina Hb has been determined; its O₂ affinity is similar to that of LegHbs (159).

B) NONSYMBIOTIC HBS. The nonsymbiotic Hbs of plants occur in the roots, stems, or germinating seeds of mono- and dicotyledonous plants at low concentrations, ~1-20 µM/kg wet tissue, and have amino acid and gene sequences quite distinct from those of LegHbs (9, 18, 29, 30, 232). Except for barley (539), two or more globin genes are expressed in diverse tissues of soybean (9), Arabidopsis (583), and rice (31), with the highest levels of expression occurring in metabolically active or stressed tissues (30).

Appleby et al. (20) were the first to propose an O₂ sensing role for nonsymbiotic Hbs. The latter occur at very low concentration, ~100 nM, and could trigger an anaerobic response via formation of the deoxy form under lower than normal O₂ concentrations. Based on the expression at moderate levels of nonsymbiotic Hb genes in soybean and Casuarina, Andersson et al. (9) proposed that the principal role of the nonsymbiotic Hb could be to facilitate intracellular diffusion of O₂ to the mitochondria in metabolically active cells to meet an increased demand for oxidative respiration. Although Rhizobium-containing nodules (legumes or Parasponia) that fix nitrogen in the absence of cytoplasmic Hb have not been observed, some Actinorrhizal symbioses do not require it for survival. Thus, in the former case, a facilitated flux of O₂ via the Hb appears to be a necessary part of such symbioses (C. A. Appleby, personal communication). The kinetics of ligand binding determined recently with native and recombinant nonsymbiotic Hbs have shown that the high O₂ binding affinities (7-1,800 µM¹) are due to a moderate association rate (1-620 µM¹s¹) coupled with a very slow dissociation rate (0.028-51 s¹) exceeded only by Ascaris Hb (31, 120, 147, 222, 583), probably due to stabilization of the bound O₂ through hydrogen bonding to the distal E7His (120). These results do not provide support for either of the two proposed Hb roles: O₂ sensing and facilitation of O₂ transport.

Arredondo-Peter et al. (30) have proposed that nonsymbiotic plant Hbs could be involved in more than one metabolic pathway. The possible roles include, 1) O₂ scavenging suggested by their high O₂ affinity; 2) participation in electron transfer via interaction with a flavoprotein by analogy with the bacterial and yeast FHbs; 3) binding of O₂, NO, and CO, which are known to be ligands, as part of a sensing mechanism including involvement in the regulation of cellular metabolism in response to fluctuation in the ligand level; and 4