Commentary a Brief History of Hemoglobins: Plant, Animal, Protist, and Bacteria Ross C
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Proc. Natl. Acad. Sci. USA Vol. 93, pp. 5675-5679, June 1996 Commentary A brief history of hemoglobins: Plant, animal, protist, and bacteria Ross C. Hardison Department of Biochemistry and Molecular Biology, The Center for Gene Regulation, The Pennsylvania State University, University Park, PA 16802 Porphyrins, Hemes, Oxygen, and Electrons fold (2). Further studies have found hemoglobins in jawless vertebrates and in diverse invertebrates ranging from flies The utility of metal-bound porphyrin rings for the transfer of (arthropods) to earthworms (annelids) to nematodes (3-5). electrons was established early in evolution, as witnessed by the These invertebrate hemoglobins are clearly related to those of ubiquitous cytochromes that pass electrons down an electro- the vertebrates in primary structure, although in some inver- chemical gradient in the respiratory chain, eventually reducing tebrates, the large extracellular hemoglobins are fusion pro- 02 to H20 and yielding energy in the form of ATP in the teins composed of multiple copies of the familiar monomeric process. Other hemoproteins catalyze a variety of oxidations, globins. As hemoglobins are found in more and more distantly with roles as diverse as protecting cells from peroxides and related species, the estimated time for the last common breaking down the very stable wood polymer, lignin. Even in ancestral hemoglobin gene moves further back to at least 670 photosynthesis, where solar energy is used to take electrons million years ago in the case of the invertebrate/vertebrate from a "low-energy" donor such as H20 (evolving 02) and pass divergence (6) (Fig. 1). them down an ATP-yielding electron transfer chain, the porphyrin rings in chlorophylls serve to harvest the quanta of Plant Hemoglobins: Symbiotic and Nonsymbiotic light energy and eventually feed them to the photosynthetic reaction center. Protoporphyrin IX with an iron ion coordi- Plants not only make oxygen during photosynthesis, but they nately bound in the middle of its flat, planar structure (known also use it for respiration through the electron transfer chain as a heme molecule when the iron is in the 2+ oxidation state) in mitochondria. Recent studies show that they use hemoglo- is used in proteins as diverse as cytochromes and ligninases for bins to bind and transfer that oxygen. The first plant hemo- just such electron transfers and oxidations, with the iron globins were discovered in the root nodules of legumes (for switching cyclically between oxidation states. Other hemopro- review, see ref. 9). These nodules are a symbiosis between teins have been adapted to allow the reversible binding of rhizobial bacteria and the plant to allow fixation (reduction) of oxygen to the heme, with the iron staying in its 2+ state. The atmospheric nitrogen into a usable form, eventually appearing proteins that carry the heme and facilitate the reversible in amino acids and other building blocks for the cells. Reduc- binding of oxygen are called hemoglobins. Hemoglobins are tion of nitrogen consumes large amounts of energy, and the usually thought of as the major proteins in erythrocytes nodules have an abundant, plant-encoded hemoglobin, called circulating in the blood of vertebrates, carrying that oxygen leghemoglobin, that facilitates the diffusion of oxygen to the generated by photosynthesis to the far reaches of respiring respiring bacteriods in the root nodule (9). In addition, the tissues in the body. Indeed, hemoglobins were first found in binding of oxygen to leghemoglobin may help sequester the blood simply because they are so abundant, with a concentra- oxygen away from the nitrogen-fixing machinery, which is tion in normal blood of 15 g per 100 ml. However, it has readily poisoned by oxygen (2). Although the amino acid become clear that hemoglobins are very widespread in the sequences of leghemoglobins differ from those of vertebrate biosphere and are found in all groups of organisms including globin genes at about 80% of the positions, leghemoglobin prokaryotes, fungi, plants, and animals. Recent papers have folds into the same three-dimensional structure as the animal clarified some issues about the evolution and possible func- globins (3). Thus, it was clear that some plants had hemoglo- tions of these hemoglobins, although many issues remain bins, but initially they appeared to be limited to legumes. unresolved. So how could a selected subset of plants have a homolog to the vertebrate hemoglobins? Was a hemoglobin gene planted Animal Hemoglobins in a legume genome by horizontal gene transfer (10), possibly through an insect or nematode vector? Or are the leghemo- The hemoglobin that can be readily isolated from the blood of globins a specialized product of divergence from an ancient any vertebrate is a heterotetramer of two a-globin and two plant hemoglobin gene-a gene that is itself descended from 13-globin polypeptides, with a heme tightly bound to a pocket a hemoglobin gene in the last common ancestor to plants and in each globin monomer. The movements and interactions animals, and hence is still widespread in plants? between the a- and p-globin subunits lead to the cooperative The discovery of hemoglobins in a large variety of plants binding of oxygen to this heterotetramer, allowing it to pick up strongly supports the latter model. A hemoglobin distinct from oxygen readily in the lungs and to unload it efficiently in the leghemoglobin was initially discovered in root nodules of the peripheral respiring tissues. The amino acid sequences of the nonleguminous plant Parasponia andersonii (11). This is a a-globins and 3-globins are about 50% identical, regardless of relative of the elm tree that is nodulated by strains of Rhizo- which vertebrate species is the source, arguing that these two bium, and it is likely that the Parasponia hemoglobin also plays genes are descended from a common ancestor about 450 a role in oxygen transport during symbiotic nitrogen fixation. million years ago, in the ancestral jawed vertebrate (1). Both [Subsequent work indicates that this single protein has both a a-globins and P-globins are about equally divergent from the symbiotic and a nonsymbiotic role (12)]. But the discovery of monomeric myoglobin, an oxygen storage and delivery protein a hemoglobin in a non-nodulating relative of Parasponia, found in many tissues. Myoglobin lacks the exquisite cooper- Trema tomentosa, suggested that hemoglobins are in fact ativity of the blood hemoglobins, but its relationship to them widespread in plants and can carry out more generalized roles is clear both from the primary sequence and from the virtually besides those in nodulation (13). Indeed, hemoglobins have identical three-dimensional structures-the classical globin now been found in many plants, not only in the dicots just 5675 Downloaded by guest on September 28, 2021 5676 Commentary: Hardison Proc. Natl. Acad. Sci. USA 93 (1996) phycocyanins X cytochrome b562 cytochrome b5 ? other hemoproteins K Ancestral Hb gene I - *or or - Bacterial Hb genes .- 1800 Myr ,-5FAD_ Fungal flavohemoglobin gene Alcaligenes FAD Yeast Saccharomyces Vitreoscilla 35 E14 F9 Hb E Algal gene I z Chlamydomonas F2 Protozoan Hb gene B12 E? G6 Paramecium 1500 Myr ^ Plants Animals B12 E14 G6 B12 E8 G6 * E * UEI- Nematode central intron 3.^J 670 Myr k Annelid ^650 Myr nonsvmbiotic symbiotic Hb gene /6 MVertebratey Hb gene lose all / |-L introns/ < 450 Myr AP 150 Myr Monocot 0'0 -globin gene -gobin I LI + Dicot * ** ^^H ca-globin gene P-globin I j gene Moncot ULCOtI Insect Mammal or Earthworm nonsymbioticI symbiotic Chironorr1US Homo Lumbricus Hordeum Glycine Nematode Mammal P barley Hb Dicot soybean Hb Pseudoterranova Homo nonsymbiotic Glycine soybean Lb FIG. 1. Some key events in the evolution of hemoglobin genes. Three different phases are shown at increasing levels of resolution. The top three lines indicate that several contemporary hemoprotein genes may be derived from a common ancestral gene, and some may be descended from others, such as the suggested derivation of hemoglobin genes from a cytochrome b5 gene. The next set of lines illustrates the descent of hemoglobin genes in bacteria, fungi, and protists from a common ancestral globin gene, following the cladogram in ref. 7. Globin exons are darkly shaded boxes, introns are open boxes, and the FAD-binding domain is a lightly shaded box. The predicted or known position in the a-helical globin fold of the amino acid whose codon is interrupted or followed by an intron in the gene is shown above the intron, e.g., B5 is the amino acid at the fifth position of helix B. Although current data do not address whether the ancestral globin gene contained introns, it is clear that introns were present in the hemoglobin gene in the last common ancestor to plants and animals. The events depicted in the "Plants" section summarize the data and cladogram in ref. 8. Selected events in the evolution of invertebrate and vertebrate hemoglobin genes are shown in the "Animals" section. The dates and branching patterns are taken from refs. 6 and 8. The lightly shaded diamonds represent gene duplications, the striped circles represent speciation events. Representative contemporary examples of the hemoglobin genes in plants, invertebrates, and vertebrates are given at the bottom of the figure. Myr, millions of years ago; Hb, hemoglobin. mentioned but also in the monocot cereals Hordeum (barley), This would suggest that all plants should have the nonsym- Triticum (wheat), and Zea (corn) (14). Thus, two different biotic hemoglobin, but previous studies with legumes have types of hemoglobin have been discovered in plants: (i) a revealed only the abundant leghemoglobins. In this issue ofthe nonsymbiotic type that is widely distributed among species and Proceedings, Andersson et al. (8) report the isolation of the (ii) a symbiotic type that is induced on nodulation of at least "missing" nonsymbiotic hemoglobin gene from legumes. two families of plants.