For the most part biochemistry texts, for simplicity, tend to restrict their examination of the structure and properties of hemoglobins to these respiratory pigments found in higher vertebrates. Even when structural variants are considered, these are often confined to discussions of mutant human hemoglobins such as hemoglobin S, the form found in sickle cell anemia patients. But, in fact, hemoglobins are found in organisms as diverse as bacteria, yeast, insects, shrinp, and sea stars. A knowledge of the structure and properties of these non-vertebrate hemoglobins can help to inform us about how common protein folding patterns can be preserved throughout long periods of evolutionary history and also what tremendous variance there can be in the construction of these respiratory pigments that bind oxygen reversibly. The vertebrate myoglobins and hemoglobins are arguably the most studied of all proteins. They were the first proteins for which detailed X-ray crystallographic information was obtained and they have served as models for a variety of biochemical phenomena, such as cooperativity in ligand binding and modulation of biochemical properties by allosteric regulators. What all of these hemoglobins share, whether vertebrate or non-vertebrate, is a common module, typically of about 16,000 D, commonly termed the myoglobin fold. The lamprey hemoglobins, while tetrameric in the deoxygenated form, tend to dissociate to dimers upon oxygenation. Amongst the vertebrates we find the monomeric form, myoglobin, in tissues (particularly highly aerobic tissues such as heart muscle) while a tetrameric form, hemoglobin, is enclosed within circulating red cells. In most of the vertebrates the hemoglobin is tetrameric whether in the oxygenated or deoxygenated state. It is only when we get down to the lower roots of the vertebrate lineage, to the jawless fishes such as the lamprey, that we begin to see a change in behavior. Amongst the invertebrates the situation is much more extreme, with hemoglobin molecular weights ranging from monomeric myoglobin-like forms with molecular weights around 15,000 Da to multimeric forms with molecular weights in the millions. One can in fact generally classify the non-vertebrate hemoglobins into two broad categories, the intracellular hemoglobins which tend to be monomeric, dimeric, or tetrameric, and the extracellular highly polymeric forms which can range up to 10,000,000 or more Da in size. A few examples of the diverse array of non-vertebrate hemoglobins are discussed below, with emphasis on those forms for which detailed X-ray crystallographic information is available. A . Echinoderm hemoglobins Echinoderms, which include the starfish, brittle stars, sea urchins, sea cucumbers and sea lilies, are the most closely related group of the invertebrates, to the vertebrates, to express hemoglobins. Even here, the only members to produce hemoglobins are some of the sea cucumbers and a very few brittle stars. The sea cucumber Caudina arenicola can be taken as an example. This marine animal lives burrowed into the sand with only the head and rear end protruding. Surprisingly C. arenicola breathes through its anus, filling up a lung-like respiratory tree with water, from which the red cells in the coelomic cavity pick up oxygen. These nucleated coelomic red cells express four major types of globin chains, termed A,B, C and D, which combine to produce a variety of hemoglobin multimers. Further complexity arises from the fact that there are additional separate types of hemoglobins in a vein that wraps around the intestine and in red cells that inhabit a water vascular system located in the musculature of the head region. The isolated single globin types show a slight degree of cooperativity by themselves, but become quite cooperative in oxygen binding when there are multiple globin species present, as illustrated here. At least part of these cooperative properties comes from an unusual ligand linked dissociation of these hemoglobins. The deoxy forms are tetrameric, but binding of oxygen causes dissociation to dimers. These echinoderm globins also have an N-terminal extension of 9 residues compared with typical vertebrate hemoglobins. This is a feature that is observed in a number of other invertebrate hemoglobins . The intron structure of the sea cucumber globin genes is also a little unusual. In addition to Introns I and II which are found at almost invariant positions in vertebrate hemoglobins, there is a third (NA) intron which separates the N-terminal extension sequence from the rest of the chain. Analysis of the sequences of several of these sea cucumber globins suggested that they had similar helical regions to their vertebrate counterparts and this was born out by subsequent x-ray analyses which showed that they have the typical myoglobin fold. You can use the CHIME program to explore this protein in more detail by clicking below. Here and elsewhere that you utilize CHIME please explore the various display and color options to get a full appreciation of the overall structures of the molecules. (Then use the back button on the browser to return to this narrative) As noted above, the functional forms of these sea cucumber hemoglobins are multimeric. Surprisingly, X-ray analysis of a C. arenicola dimer has shown that the subunit interfaces are very different from vertebrate hemoglobins and involve close intersubunit contacts of the E and F helices . This serves to bring the hemes in close proximity (below left). This is more dramatically illustrated in a close-up of the dimer interface region (below right). Contrast this with the hemes of a typical vertebrate hemoglobin:   You can explore the sea cucumber hemoglobin dimer in more detail with CHIME by clicking here: What about other closely related sea cucumbers and other echinoderms that do not express Hb? Do they have the Hb genes but in silent form? Genome sequence searches should soon give us the answer. B. Clam hemoglobins A well studied hemoglobin from the blood clam Scapharca inaequivalvis bears a strong overall resemblance in term of subunit interactions to the sea cucumber hemoglobin (left). As shown in more detail below, this resemblance extends to the close contacts of the E and F helices which bring the heme groups close together (right).   It is instructive however, to compare the sea cucumber (top) and clam (bottom) hemoglobin interfaces shown below: Even though they look superficially the same, if one looks closely, there are some marked differences in both the amino acid side chains lining the two interfaces and in the dimensions of the interface pocket. You can examine the clam hemoglobin more thoroughly in three dimensions using the CHIME plugin.   While one might think that this type of dimer was setting a trend for invertebrate hemoglobins, it is instructive to note that a hemoglobin from the innkeeper worm, Urechis caupo, has globin interfaces much more reminiscent of those found in vertebrates, as illustrated below. The detailed structure of this dimeric hemoglobin can be explored with the link below. C. Insect hemoglobins Even though most insects vastly exceed the size where one might expect oxygen to be delivered to tissues by direct diffusion, but many insects get around the general 2 mm limit by an extraordinary system of pores and tunnels, termed spiracles, throughout the body that bring air close to the tissues that need the oxygen. There are, however, a few insects which utilize hemoglobin for oxygen transport and storage. Some of these, like the larvae of the horse botfly live in very low oxygen environments. The most extensively studied of the insect hemoglobins are those from a group of the true fruit flies, the chironomids. These flies express a surprisingly large array of globins. For example, Chironomus thummi thummi has at least a dozen globin genes. For the most part these hemoglobins exist as monomers and dimers, which exhibit little cooperativity in oxygen binding. In general structure these insect hemoglobins have the typical myoglobin fold. You can explore the structure of one of these insect hemoglobins in more detail using CHIME. The most unusual aspect of nearly all the Chironomus globins is the fact that their genes are intronless. In contrast to the vertebrate hemoglobins which have two introns at highly conserved positions, or the plant leghemoglobins which have an additional central intron, these globin genes have none. This raises the issue of how introns are gained and lost and indeed what functions they serve. Interestingly one of the hemoglobin-expressing chironomids has globin genes with the typical vertebrate pattern of introns and this insect lies in a phylogenetic position intermediate between species with intronless globin genes. D. Shrimp hemoglobins Up to this point we have explored hemoglobins that when they aggregate into dimers, tetramers, etc., do so by non-covalent interactions of the globin chains. By contrast, brine shrimp hemoglobins consist of multiple globin units joined covalently by short peptide linker regions, rather like a string of pearls. These high oxygen affinity hemoglobins typically consist of 9 separate heme units each different from one another. There are two primary types of these nine-mer assemblages. E. Yeast hemoglobins As far back as 1953 unusual hemoglobins, possessing both heme and flavin groups and capable of reversible oxygen binding, with high affinity, had been identified in several different types of yeast, especially when grown under oxygn deprived conditions. The amino acid sequences of such chimeric molecules from Saccharomyces and Candida species have recently been determined. In both cases they are nearly 440 amino acid residues in length and the heme bindin region is in the N-terminal part of the sequence, while the flavin binds closer to the carboxyl terminus. F. Leghemoglobins of plants Hemoglobins have long been known to be associated with the nitrogen fixing nodules of leguminous plants where these high oxygen affinity proteins are though to protect the functions of the oxygen sensitive nitrogenases. These hemoglobins have been termed leghemoglobins. More recently hemoglobin genes have been identified in a wide range of non-leguminous plants from species as diverse as rice and Arabidopsis. The function of the hemoglobins in these non-leguminous plants, especially where no symbiotic relationship with another organism exists, is not fully understood. G. Bacterial hemoglobins The prototypical bacterial hemoglobin was first identified as a gene sequence in the Gram-negative bacterium Vitreoscilla in the mid-1980's. This gene has measurable homology with both plant and animal globins. The protein resides in the periplasmic space and there is now considerable evidence that the hemoglobin plays a physiologically active role in oxidative metabolism. It is of interest that this bacterial hemoglobin is now being investigated for a biotechnology role. Researchers at the Illinois Institute of Technology have spliced the Vitreoscilla gene into Pseudomonas with the intent of providing the latter organism with increased oxidative potential for breaking down potentially carcinogenic breakdown products of TNT in soil deposits. H. Earthworm hemoglobins By contrast to the small size of the intracellular hemoglobins, the extracellular hemoglobins found in many annelid species are giant molecules indeed, with molecular weight ranging up to several million daltons. In electron micrographs these hemoglobins have a characteristic hexagonal bilayer structure. In the common earthworm, Lumbricus terrestris, the quaternary structure consists of 24 (a,b,c,d)2 units held together by 24 linker chains. These hemoglobins exhibit highly cooperative ligand binding, with Hill coefficients of up to 7 having been recorded. Summary As the above discussion of non-vertebrate hemoglobins attests, these respiratory pigments have a diverse array of structure and properties. One of the more intriguing features about the non-vertebrate hemoglobins is a very scattered phylogenetic distribution. The organisms in which myoglobin and hemoglobin are found are scattered throughout the biological spectrum. Hundreds of millions of years appear to have gone by in biological lineages without noticeable expression of hemoglobins, until they pop up again almost unchanged in structure. Such continuities of structure and properties, despite lack of expression, and thus evolutionary pressures over extremely long periods, raise serious questions about the maintenance and integrity of silent genes. By contrast, there is always the possibility that at least some of the hemoglobins seen in this scattered lineage may have achieved their status through horizontal gene transfer. The new techniques of genomic molecular biology can help to provide us with some answers to these questions. Web Links and Literature Sources Sea Cucumber - more esoteric info on sea cucumbers Hemoglobin Evolution - A general recent discussion of hemoglobin evolution Biotechnology use of bacterial hemoglobins Protective effects of inducible bacterial hemoglobins Plant hemoglobins and resisting stress Protein dynamics and ligand binding of myoglobins and hemoglobins Papers covering the properties of a variety of non-vertebrate respiratory pigments: S.N. Vinogradov et al. Comp. Biochem. Physiol. Vol 103B No 4, pp 759-773 (1992) S.N. Vinogradov et al. Comp. Biochem. Physiol. Vol 106B, No 1, pp 1-26 (1993) A recent paper covering the self association and cooperativity of hemoglobins: Riggs, A.F. J. Exp. Biol. Vol 201, 1073-1084 (1998) Thanks to Dr. David Mitchell and Dr. Marvin Hackert for the use of some invertebrate hemoglobin illustrations and to the New York State Department of Environmental Conservation for the use of the lamprey photograph.   Copyright © 1999, Harcourt College Publishers, A Harcourt Higher Learning Company. All rights reserved. Use of this site indicates acceptance of the Terms and Conditions of Use. For problems or suggestions concerning this service, please contact Chemistry Webmaster.