Oxygen-Binding Proteins (Molecular Biology)

Oxygen-binding proteins are defined as proteins that reversibly bind O2. They are exclusively metalloproteins.

1. Classification

The classification of oxygen-binding proteins and some of their properties and distribution are presented in Table 1. Protoheme IX is the common oxygen-binding site for myoglobins (Mb), IDO-like Mb, hemoglobins (Hb), compact Hb, and FixL. Dioxygen is bound at the ferrous ion at the center of the heme group (see Myoglobin). The vinyl group normally at position 2 of protoheme IX is replaced by a formyl group in the chlorocruoroheme carried by chlorocruorin (Chl). Hemerythrin (Hr), myohemerythrin (myoHr), and hemocyanin (Hc) are nonheme proteins. The oxygen-binding site for Hr and myoHr is a binuclear iron center, and that for Hc is a binuclear copper center.

Table 1. Classification of Oxygen-Binding Proteins

The hemoglobins in protozoa (Paramecium and Tetrahymena), green algae (Chlamydomonas), and cyanobacteria are not authentic hemoglobins because their polypeptide chains are about 20% shorter and without substantial amino acid sequence similarities. Several pieces of evidence suggest that the structures of these "compact hemoglobins" differ from the "globin fold" (see Globins), and it is generally considered that they arose from a different evolutionary ancestor (see Convergent Evolution). Likewise, IDO-like Mb evolved from a common ancestor of indoleamine dioxygenase, a heme-containing enzyme that degrades tryptophan. Based on their gene structures and amino acid sequences, myoglobins and hemoglobins are divided into three distinct evolutionary groups: (1) "universal globins", 17-kDa polypeptide chains, that have about 150 amino acid residues; (2) "truncated globins" or "compact globins", 13 kDa, that have about 120 residues per chain; and (3) "IDO-like Mb" (1). The second group has been known as "truncated globin", but "compact globin" is more appropriate because these proteins have no detectable amino acid homology with the universal globins.

2. Distribution, Structure, and Function

Figure 1 illustrates the distribution of oxygen-binding proteins in a simplified phylogenetic tree. Hr and Hc are distributed within certain animal phyla, such as Sipuncula, Brachiopoda, Annelida, Arthropoda, and Mollusca. Hb, the most widely distributed, occurs in bacteria to vertebrates, and even in plants, but only intermittently. The principal physiological function of the circulating proteins (vertebrate Hb, annelid Hb, Chl, Hr, and Hc) is to transport O2 from the lungs or gills to peripheral tissues. The function of the noncirculating proteins (Mb and myoHr), and probably also of IDO-like Mb, is oxygen storage and intracellular transport.

Figure 1. Phylogenetic distribution of oxygen-binding proteins. Abbreviations undefined in the text are Er, erythrocruor same as Chl (chlorocruorin). Erythrocruorin was the name used for what is now called annelid extracellular hemoglobin. hemoproteins of yeast, paramecium, and legume root nodules are designated as Mb in this figure, they are sometimes de: Hb, because they are not contained in muscle and Hb is a more comprehensive name of an oxygen carrier.

See Myoglobin for details of Mb and IDO-like Mb and Hemoglobin for details of Hb. The other oxygen-binding proteins are described here.

2.1. Compact Hemoglobin

Only a limited number of structure and functional studies of compact Hb have been carried out. The physiological role of these proteins is uncertain. It is proposed that Hb from the cyanobacterium Nostoc cummune is an oxygen scavenger in nitrogen fixation .

2.2. FixL

The FixL that occurs in Rhizobium meliloti and Bradyrhizobium japonicum is a chimeric protein consisting of a heme-containing domain and a protein kinase domain. Its proposed function is to sense oxygen through its heme domain and to transduce this signal by controlling the phosphorylation of transcriptional activator FixJ, which in turn induces the expression of the genes for nitrogen fixation, thereby providing oxygen-dependent control of nitrogen fixation (2). The heme domain has no significant sequence similarities to the globins, suggesting that it arose independently.

2.3. Chlorocruorin

Chl has only a limited phylogenetic distribution. It is found in the blood plasma of four families of polychaeate annelids, the Sabellidae, Serpulidae, Chlorhaemidae, and Ampharetidae. The protein structure of Chl is essentially the same as that of extracellular annelid Hb, except for the change in the heme group (3). The vinyl to formyl replacement at position 2 of protoheme produces the characteristic greenish color and low oxygen affinity of Chl due to the enhanced electron-withdrawing properties of the formyl group. Chl shows highly cooperative oxygen binding. As with annelid Hb, its oxygen affinity is pH-dependent (see Bohr Effect) and is increased by divalent cations like Mg2+ and Ca2+ (4).

2.4. Hemerythrin (Hr) and Myohemerythrin

Hr has been found only in four phyla of marine invertebrates: Priapulia, Sipuncula, Brachiopoda, and one family of annelida, Magelonidae. It is contained in red cells called "hemerythrocytes" that float in the coelomic fluid. A noncirculating Hr is found in muscle fibers of sipunclids and is called "myoHr", in analogy to myoglobin. Coelomic Hr is found predominantly as the octamer, but also as tetramers, trimers, and dimers. The octamer is composed of two different subunits that have different amino acid sequences (5). The octamer has a mass of 108 kDa, the shape of a square doughnut, and consists of two rings of four subunits each whose rings are associated in a face-to-face, isologous manner (see Quaternary Structure). Each 13.5-kDa subunit consists of four parallel alpha-helices twisted in a left-handed bundle (6) (see Four-Helix Bundle Motif). The tertiary structure of myoHr (13.9 kDa) is similar to that of one Hr subunit. MyoHr and each subunit of Hr contains a site that has two iron atoms, which bind one O2 molecule. The iron atoms are bound directly to seven amino-acid side chains of the protein. One of the O2 atoms binds to one of the iron atoms, and the other oxygen atom forms a hydrogen bond with the oxo bridge that connects the two iron atoms.

The oxygen affinity of myoHr is higher than that of coelomic Hr of the same species. Octameric Hr from two brachiopods (Lingula unguis and Lingula reevii) show notable cooperativity in oxygen binding and have Hill coefficients of 1.8 to 2.2 (see Hemoglobin), whereas other Hrs are essentially noncooperative, irrespective of their state of aggregation. It has been suggested that the allosteric unit of L. unguis octameric Hr is the entire molecule (7). Effects of intracellular cofactors, such as

on the oxygen affinity have been observed with some Hrs, but their physiological significance is not clear.

2.5. Hemocyanin

Hc is found in the blood plasma of two phyla, arthropod and mollusk. The oxygen-binding site of Hc is composed of a pair of copper ions, each of which is liganded by three histidine residues of the protein. It was long thought that O2 binds as a peroxo ion, serially bridging the two Cu(II) ions, but now there is direct evidence that the mode of O2 binding is a m-h h coordination, in which both oxygen atoms are bound to both of the copper ions and the O-O axis perpendicular to the Cu-Cu axis.

Arthropod and mollusk Hc exhibit similar optical and ligand-binding properties, but their molecular architectures are quite different. All arthropod Hcs are composed of hexamers. Each 75-kDa polypeptide chain carries one oxygen-binding site. These aggregate into 1, 2, 4, 6, or 8 hexamers, depending on the species, and no intermediate aggregations states are observed in each case. With this mode of assembly, one Hc molecule has from 6 to 48 oxygen-binding sites and a molecular mass ranging from 450 to 3300 kDa. Each hexamer is a "trimer of dimers", and the shape is a trigonal antiprism. Each polypeptide chain consists of three domains. The first and second are a-helical, whereas the third has an irregular structure that contains a 7-strand b-barrel (see Beta-Sheet). The O2-binding site is in the second domain.

In contrast to the arthropod Hc, those from mollusks are organized entirely differently. The molecule is composed of one or more cylindrical units, each containing 10 subunits. Each subunit is a long polypeptide chain of 350 to 440 kDa composed of seven or eight globular folded regions, each of which binds one O2 molecule. It is not known whether each such region is composed of more than one domain, so it is called instead a "functional unit." Its three-dimensional structure is not yet known.

The quaternary structures of both arthropod and mollusk Hc are stabilized by many calcium ions. The Hc oxygen affinity varies, depending on the species, especially in the Arthropoda, and reflects adaptations to the great variety of life styles and environments of the animals. Most Hcs demonstrate a distinct Bohr effect and varying degrees of cooperativity in oxygen binding. The Hill coefficient ranges between 1 and 12. A higher degree of aggregation is not necessarily accompanied by greater cooperativity. The oxygen dissociation curves of Hcs that have large multimeric structures are not compatible with the classic allosteric concerted model of Monod, Wyman, and Changeux but are described best by a "nesting" model (8). This assumes that the allosteric units capable of transitions between two states, r and t, are in turn nested within larger structural units that are in equilibrium between two global states, R and T. In addition to the structural calcium ions, other calcium ions, plus magnesium ions, regulate the oxygen affinity.

There is very little sequence similarity between arthropod subunits and molluscan functional units. This and their very different molecular architectures indicate that they probably evolved independently.