AMER. ZOOL., 31:535-545 (1991)
Aspects of the Origin and Evolution of
Non-Vertebrate Hemoglobins1
AUSTEN F. RIGGS
Department of Zoology, University of Texas, Austin, Texas 78712
SYNOPSIS. Hemoglobins, found in members of almost all invertebrate phyla, display an
extraordinary diversity of form and function. Although some are intracellular with chains
and assemblies similar in size to those of vertebrates, others are giant extracellular proteins
with masses as large as 8,000 kilodaltons. Two very different strategies have evolved for
the stabilization of these large molecules. The first is the formation of both intra- and interchain disulfide bonds that effectively immobilize segments of the protein, and the second
is the evolution by gene duplication of multi-domain chains with from two to eighteen
myoglobin-like, heme-containing domains in a single polypeptide that may be as large as
~260 kilodaltons. The genes for vertebrate globins have a characteristic two-intron, threeexon structure. The gene encoding chain c of the hemoglobin of the earthworm Lumbricus
terrestris has precisely the same organization and splice junction positions. This shows that
these positions have been conserved for at least 600 million years, the estimated time of
divergence of annelids and the ancestor to chordates. The gene encoding a hemoglobin
(leghemoglobin) of higher plants shows exactly the same splice junctions as in the globin
genes of vertebrates except that the middle exon is split by an additional intron which is
believed to have been lost early in animal evolution. The occurrence of hemoglobins in
diverse higher plants suggests that they might be present in all plants albeit at very low
concentrations and perhaps serving an enzymatic function. Hemoglobin, broadly defined
as a heme-containing protein capable of reversible combination with oxygen, also occurs in
bacteria and fungi. The discovery of a bacterial hemoglobin that is 26% identical with lupin
leghemoglobin indicates a procaryotic origin. The possibility that hemoglobin may have
evolved from a cytochrome is suggested by the presence of hemoglobins in the yeast, Candida, and the bacterium, Alcaligenes, that contain both heme and flavin. They must therefore
have evolved by the fusion of the genes for two different proteins. However, the possible
homology of the heme domains with plant or animal hemoglobins remains to be determined.
The lactic dehydrogenase of yeast, cytochrome b2, is also a soluble flavoheme protein with
a heme domain that is homologous with mammalian cytochrome b5. Globin may have
evolved in part from a member of the cytochrome b5 family, but if so, the event must have
occurred so early that only a borderline perhaps random correspondence of amino acid
sequences remains.
INTRODUCTION
Hemoglobins provide a splendid opportunity to explore the origin and evolution
of a protein. The great diversity of the structures of non-vertebrate hemoglobins show
both the evolution of multiple solutions to
similar problems and the development of
unique functions. The discovery of O2binding heme proteins in bacteria and
diverse plants that are homologous with
known hemoglobins indicates a truly ancient
heritage and provides a basis for understanding the early phylogeny of the protein,
All hemoglobin polypeptides of 15-17
kDa for which X-ray structures exist have
1
From the Symposium on The Origins and Evolution ofMetabolic Pathways in Animals presented at the
Annual Meeting of the American Society of Zoologists,
27-30 December 1989, at Boston, Massachusetts.
a similar overall conformation, the "myoglobin-fold." Amino acid sequence comPansons leave little doubt that all hemoglobin
chains of this size have similar tertiary
structures (Dickerson and Geis, 1983;
Goodman et ai, 1988). The same conclusl0n holds for t h e
multi-domain hemoglobinof
Artemia (Moens et ai, 1988, 1989)
and presumably also for other multi-domain
hemoglobins found in arthropods and molluscs
- However, the assembly of mvertebrate
hemoglobins is both varied and
entirely different from that of vertebrate
hemoglobins which (except those of lamP re y s a n d hagfish) possess two types of chain,
a and |8, that form a^2 tetramers. The few
invertebrate hemoglobins for which X-ray
-•„,-•„„,«. a r P | , n n w n ofcnw rnmnletelv difstructures are Known snow completely a i i ferent subumt contacts than are present in
vertebrate hemoglobins.
535
536
AUSTEN F. RIGGS
Several genetic processes underlie the
evolution of the structural and functional
diversity that occurs in invertebrate hemoglobins: (1) gene duplication, (2) acquisition
of the signal sequence necessary for secretion of extra-cellular hemoglobins, (3)
mutations resulting in substitutions, deletions and insertions, and occasionally (4)
loss of exons. Exchange of exons between
non-homologous genes ("exon shuffling"),
as suggested by Gilbert (1978), is certainly
possible but has not yet been demonstrated
in globin genes. Although insufficient information is available for complete assessment
of these processes, some of the major factors
in the evolution of invertebrate hemoglobin
can now be addressed. The accumulation of
a large array of amino acid sequences of
globins has made possible the construction
of a plausible phylogeny that is consistent
with the fossil record and with morphological information (Goodman et al., 1988).
The goal of the following discussion is to
examine some of the bacterial, fungal, plant,
and invertebrate hemoglobins and to consider factors in their origin and evolution.
imal histidine bound to the heme iron. A
second residue in the pocket, phenylalanine
at position CD1 in vertebrate globins, is also
conserved in all known hemoglobins. A third
residue, the distal histidine, is highly conserved in vertebrate hemoglobins; it is
replaced by glutamine in only a few species.
Although histidine is also the predominant
distal residue in invertebrate hemoglobins,
substitutions such as glutamine are common. Examples of alternatives are a distal
leucine in the hemoglobin of the annelid
Glycera dibranchiata (Imamura et al., 1972;
Arents and Love, 1989) and a tyrosine in
the hemoglobin of the parasiticflukeof sheep
liver, Dicrocoelium (Smit et al., 1990). The
occurrence of distal tyrosine is particularly
surprising because the corresponding substitution in the a chain of the human hemoglobin variant M-Boston results in detachment of the proximal histidine and bonding
of the Fe3+ to the phenolate group of tyrosine E7 so that the chain is unable to bind
O2 (Pulsinelli et ai, 1973). This observation
means that Dicrocoelium hemoglobin must
have other compensating differences that
allow reversible oxygen binding. Examination of many human hemoglobin variants
REVERSIBILITY OF O 2 -BINDING
has shown that single amino acid substiThe crucial requirement for all hemoglo- tutions can greatly modify the sensitivity to
bins is that the ferrous-heme-O2 bond must oxidation and can sometimes destroy the
be reversible. A second obvious require- capacity for reversible O -binding alto2
ment is that a pathway to the iron must gether (Bunn and Forget, 1986).
This sugexist, either continuously or as a result of gests that an evolutionary transformation of
conformational fluctuations. The latter is an O -reactive heme enzyme to one capable
2
shown by the presence of valine El 1 of the of binding
O2 reversibly might have required
human /3 chain which blocks the pathway only a few amino
acid substitutions. If this
in deoxy hemoglobin and must move for O2 were generally true, however, it would have
to gain entrance. The possibility that O2- been relatively easy for a hemoglobin to
utilizing heme enzymes might have evolved evolve repeatedly from a heme enzyme.
into hemoglobins has long been suggested Although no compelling evidence for this
(see Keilin, 1966). The distinction between possibility exists, some of the bacterial cytoreversibility and reactivity is not sharp: chromes o (see Jurtshuk and Yang, 1980)
many observations on hemoglobin show might be appropriate candidates for such a
that enzymatic activities do exist. Human transformation. These cytochromes have
hemoglobin, for example, can replace cyto- heme, combine with CO, react with O and
2
chrome P450 in many monoxygenase sys- are usually but not always membrane bound.
tems and NADH can directly and readily
reduce methemoglobin (Ferraiolo and
Mieyal, 1982; Ferraiolo et al., 1984). The
PLANT AND MICROBIAL HEMOGLOBINS
properties of the heme depend on the amino
Oxygen-binding heme proteins have now
acid environment of the heme pocket which been found in a wide range of distantly
is very hydrophobic. The only absolute related non-animal organisms. These
amino acid requirement may be the prox- include higher plants (leghemoglobins), fungi
ORIGIN AND EVOLUTION OF HEMOGLOBIN
and bacteria. All five kingdoms (Margulis
and Schwartz, 1988) have species with
hemoglobin or hemoglobin-like proteins.
Leghemoglobin, first believed to be confined to the root-nodules of N2-fixing leguminous plants, has now been found in nonlegumes and in plants that do not fix N2 at
all. Bogusz et al. (1988) have consequently
suggested that all plants may have the globin
gene. The suggestion has been made that
the absence of hemoglobin in many plants
may be only apparent. If hemoglobin were
present only at the very low concentrations
of many intracellular enzymes, it would easily be missed. The same suggestion may be
made for invertebrates that apparently lack
hemoglobin: it might be present at such low
concentrations that it has escaped detection.
The X-ray structure of leghemoglobin
shows that it has the typical globin "fold"
even though the amino acid sequence shows
only a low correspondence to the sequences
of vertebrate globins (Vanshtein et al, 1975).
Leghemoglobin and animal hemoglobins
appear to have diverged from a common
ancestor more than 1 x 10' years ago. A
common origin is also suggested by the gene
structure. All the genes of vertebrate globins
have three coding regions (exons) separated
by two non-coding regions (introns). GO
(1981) discovered that the exon boundaries
correspond precisely to the positions of
compact units ("modules") found in her
analysis of a-carbon-a-carbon distances in
hemoglobin. The middle exon, however, had
two modules, so she predicted that the primordial globin gene of animals possessed a
third intron which was lost early in animal
evolution. The discovery of such an intron
in the leghemoglobin genes (Jensen et al,
1981) exactly where Go predicted greatly
enhances this possibility. However, the
intron might have been lost so early that it
does not occur in any present day gene of
an animal globin. Horizontal transfer of the
globin gene from an animal cannot be completely excluded but seems unlikely in view
of the widespread distribution of the genes
in plant families. If, however, a 3-intron
4-exon gene were to be found in a nematode,
for example, we would have to consider this
possibility further.
An early procaryotic origin of the globin
537
gene is strongly suggested by the discovery
of a hemoglobin (formerly considered a
cytochrome o) in the bacterium Vitreoscilla.
The amino acid sequence of this protein is
26% identical to that of lupin leghemoglobin; this value is higher than in any other
comparison (Wakabayashi et al, 1986).
Since leghemoglobin has the same overall
conformation as found for vertebrate globin
chains, we can conclude the Vitreoscilla
hemoglobin also has the "myoglobin-fold."
The correspondence between Vitreoscilla
and lupin globins is of particular interest
because Vitreoscilla belongs to the family
Beggiatoa, the gliding bacteria, that are
believed to have evolved from the photosynthetic cyanobacteria (blue-green algae).
The functional importance of Vitreoscilla
hemoglobin is indicated by the 50-fold
increase in its concentration when the bacteria are grown under hypoxic conditions
(pO2 < 15 torr). The Vitreoscilla globin gene
has been expressed in E. coli which thereupon grows faster and to greater cell densities (Khosla and Bailey, 1988). What does
the Vitreoscilla hemoglobin do? Facilitated
diffusion is one suggested function. The
intriguing work of Wittenberg and Wittenberg (1987) on isolated cardiac myocytes
suggests another possible function. They
report that myoglobin (Mb) not only facilitates diffusion but actually delivers oxygen
to the mitochondria. Presumably MbO2
binds transiently to an unknown protein in
the outer mitochondrial membrane which
then delivers oxygen directly to the space
between the two mitochondrial membranes. This, of course, raises the possibility
than an O2-binding protein in the intermembrane space might also exist to deliver
the O2 to the inner membrane. A "bucketbrigade" of cytochrome P450 molecules has
been proposed to serve this function (Longmuir, 1977). Another possibility is that Mb
and Vitreoscilla Hb might mediate electron
transport to oxygen.
Heme proteins that bind oxygen reversibly have also been reported from yeast,
Neurospora, and Penicillium among the
fungi (Keilin, 1953; Keilin and Tissieres,
1953) and from the bacteria Alcaligenes,
Azotobacter, Rhizobium and Chromatium
(reviewed by Jurtshuk and Yang, 1980).
538
AUSTEN F. RIGGS
Animals
Gliding Bacteria
Beggiatoa:
Vitreoscilla
Cyanobacteria
(Blue-Green
Algae)
Oxidizing
Atmosphere
Reducing
Atmosphere
Purple Sulfur
Bacteria
Chromatium
H2O
Photosynthesis
\
H 2 S Photosynthesis
Ancestral Fermenting
Bacteria
FIG. 1. A phylogenetic tree showing relationships between various procaryotic and eucaryotic organisms that
have hemoglobin or hemoglobin-like proteins. Organisms with hemoglobin or hemoglobin-like proteins are
underlined. Pattern adapted from Alberts et al. (1989).
Perhaps some of these proteins are homologous to known hemoglobins. The broad
distribution (Fig. 1) suggests that hemoglobin and hemoglobin-like proteins may have
origins that date to the very early period not
long after water-splitting photosynthesis
began. It is surprising that the purple sulfur
bacterium, Chromatium, has an O2-binding
heme protein because this organism is normally considered to be an obligate anaerobe. However, it can grow and utilize O2 if
the pO 2 is sufficiently low. Although the 11
kDa O2-binding protein has been only partially characterized (Gaul et al., 1983), it is
probably not homologous with known globins; it has heme c (not protoheme IX) which
is covalently bound to the protein. Chromatium has the attributes of an organism
that might have existed during the billion
year period when O2 was present only as a
microconstituent of the atmosphere.
In view of the toxicity of O2, the earliest
O2-binding heme proteins may have served
primarily as detoxifying agents along with
superoxide dismutase. Later, electron flow
into the detoxifying O2-trap might have been
harnessed to the electron transport machinery. The need for O2-buffering may have
been met early in evolution by cooperative
ligand-binding. This is suggested by Vitreoscilla hemoglobin which is dimeric and highly
cooperative in CO-binding (Tyree and
Webster, 1978).
The Rhizobium bacteria that are responsible for symbiotic nitrogen fixation in the
root nodules of leguminous plants contain
an O2-binding heme protein distinct from
the leghemoglobin of the host plants. The
protein had been studied in free-living Rhizobium. Although it has the spectral characteristics expected of a cytochrome P450,
it may function in facilitated diffusion
(Appleby et al., 1975), or, as already suggested, as a protein that might deliver O2.
The possibility that cytoplasmic hemoglobins may function as terminal oxidases is
reviewed by Wittenberg and Wittenberg
(1990).
The hydrogen bacterium, Alcaligenes
eutrophus, has an unusual 43 kDa hemoglobin with domains for both flavin and
heme. It combines reversibly with O2 and
can be reduced directly with NADH (Probst
et al., 1979). The protein structure has not
yet been determined, but the location of the
gene on a megaplasmid should make the
determination straightforward (Weihs et al.,
1989).
HEMOGLOBINS OF ASCOMYCETOUS FUNGI
Keilin and colleagues early obtained spectral evidence in living cells for the presence
ORIGIN AND EVOLUTION OF HEMOGLOBIN
of hemoglobin in Saccharomyces cerevisiae,
Neuwspora and Penicillium (Keilin, 1953;
Keilin and Tissieres, 1953). Well-aerated
cells showed a sharp absorption band at
approximately 583 nm which disappeared
when aeration was stopped or replaced with
N2 or CO. Keilin and Tissieres (1953) made
an aqueous extract of the mycelium ofNeurospora crassa strain C117 and found
absorption bands at 545 and 583 nm when
the solution was aerated. The bands disappeared with Na2S2O4 or N2, reappeared
in air, and vanished entirely with ferricyanide. The bands shifted with CO: 545 ->
539 nm and 583 -• 574 nm. No cytochrome
had bands in these positions; cytochromes
display sharp bands when reduced but not
when oxidized. They therefore identified the
protein as hemoglobin. They estimated the
concentration of Neuwspora hemoglobin to
be 0.02% (w/w). Such a concentration, only
0.06% of that in the human red cell, might
readily be missed in looking at tissues of
invertebrates. The amount of the hemoglobin in Saccharomyces increased greatly if
the electron transport system was inactivated with acriflavine or antimycin A (Keilin, 1956). Spectroscopic evidence for
enhanced hemoglobin has also been reported
in deletion mutants of Saccharomyces cerevisiae that lack subunits Va or Vb of cytochrome oxidase (Chance et ai, 1988). The
yeast, Hansenula, synthesizes a new 36 kDa
protein when subjected to either antimycin
A or cyanide (Yoshimoto et ah, 1989) that
may be related to the hemoglobin of Saccharomyces. These data show that hemoglobin biosynthesis in yeast is enhanced by
any interference with the effective functioning of the normal mitochondrial electron
transport system. The hemoglobin might
function in facilitated diffusion, oxygen
delivery to an alternative cyanide-insensitive electron transport system or perhaps as
a terminal oxidase. Alternative cyanideinsensitive electron transport systems have
been reported in plants, fungi and protists
and are associated with plant thermogenesis
(Meeuse, 1975; Raskin et ai, 1989). The
nature of these alternative pathways has not
been determined. The isolation of an O2binding hemoglobin from the yeast Candida
mycoderma provides some insight. This
protein, like that from the bacterium Alca-
539
ligenes, is a soluble flavo-heme protein with
a similar molecular weight (Oshino et ai,
1973). The Alcaligenes flavohemoglobin
catalyzes reduction of cytochrome c by
NADH. It seems reasonable to suggest that
the Candida hemoglobin might have a similar action. The presence of domains for flavin and heme in both Alcaligenes and Candida hemoglobins indicates that both were
ultimately derived by the fusion of two
genes.
What might be the origin of these yeast
hemoglobins? Saccharomyces has a soluble
flavo-heme protein, cytochrome b2, that is
a lactic dehydrogenase in the mitochondrial
intermembrane space. The flavin (FMN),
reduced by lactate, can readily reduce the
heme because flavin and heme are only 9.7
A apart and mobile aromatic residues lie
between them (Xia and Matthews, 1990).
Cytochrome b2 then reduces cytochrome c.
Cytochrome b 2 is evidently part of a secondary electron transfer system. However,
it does not react with either CO or O2 and
is not a terminal oxidase; oxygen cannot
reach the iron. Its sequence and X-ray structure have been determined: the heme
domain is homologous with microsomal
cytochrome b 5 from mammalian liver and
with the heme domain of the yeast sulfite
oxidase, a molybdenum-heme protein
(Guiard et al, 1974; Guiard and Lederer,
1979; Xia and Matthews, 1990). Does any
relationship exist between members of the
cytochrome b5 family and globin? Runnegar
(1984) has proposed just such a relationship
and has suggested that globin is partly
derived from a cytochrome b5-like protein.
He proposes that the second exon of the globin gene is partly derived from the region
of the cytochrome b 5 gene encoding the distal histidine. However, the proximal histidine of cytochrome b5 does not correspond
to the proximal histidine of globins. He suggests instead that a different, unknown protein contributes the segment containing the
proximal histidine of globin. Existing
sequence data, however, do not make this
complex rearrangement compelling. The
actual sequence correspondence of any one
cytochrome b5 with any one globin is not
clearly outside what might be expected on
a random basis. The three-dimensional
structures of cytochromes b 5 and b 2 do not
540
AUSTEN F. RIGGS
resemble that of globin closely even in
selected parts. However, correspondence
might not be evident if the divergence time
was 1.5 to 2.0 x 109 years. The determination of the sequences of the hemoglobins
of yeast and Alcaligenes may provide essential links in determining whether cytochrome b 5 and the heme-domain of cytochrome b 2 are phylogenetically related to
known hemoglobins. The remarkable discovery that C-phycocyanin from the cyanobacterium, Nastigocladns laminosus, has the
three-dimensional structure of a globin,
raises the possibility that they may share a
distant common ancestry (Schirmer et al,
1985).
Three-dimensional structures are known for
the homodimeric and heterotetrameric
hemoglobins of the red cells of the clam
Scapharca inaequivalvis (Royer et al, 1985)
and the tetrameric hemoglobin of the echiuroid Urechis caupo (Kolatkar etai, 1988).
These hemoglobins all have subunits with
the typical architecture of vertebrate
chains—the myoglobin fold—yet they are
assembled entirely differently. In all of these
hemoglobins the E and F helices form intersubunit contacts—a role played by the G
and H helices in vertebrate hemoglobins.
The homodimeric Scapharca hemoglobin is
highly cooperative in oxygen binding. This
cooperativity has evidently evolved completely independently of that in vertebrate
hemoglobins because the mechanism is
INVERTEBRATE HEMOGLOBINS
Members of the major invertebrate phyla entirely different. The two hemes of the Scahave pursued evolution separately for at least pharca homodimers are separated by about
500 million years. The common ancestor to 10 A with a mobile tyrosine between them
protozoa and other invertebrates may date (Royer et al, 1989). Thus the hemes can
several hundred million years earlier. It communicate directly with each other withshould come as no surprise, therefore, that out large conformation changes. The E and
the blood hemoglobins from organisms in F helices of the two-domain hemoglobin of
the different phyla show an immense diver- the clam Barbatia reeveana are ~70% idensity in amino acid sequences, three-dimen- tical with those of the Scapharca homodisional structures and function. This diver- mer (Riggs et al., 1986; Riggs and Riggs,
sity has been extensively reviewed (Chung 1990). Almost all the intersubunit contact
and Ellerton, 1979; Ilan and Daniel, 1979; residues in the E and F helices are identical
Vinogradov, 1985). Our goal here is to in the two species. This suggests that the
describe some of the major factors that may two-domain hemoglobin also has E/F interbe responsible for the diversity of extracel- subunit contacts. These observations indilular hemoglobins and those of specialized cate why the E and F helices bear little
erythrocytes. The functions of myoglobin resemblance to those in vertebrate hemoand other tissue hemoglobins are reviewed globins. In the latter the hydrophilic resiby Wittenberg and Wittenberg (1990). Vi- dues of the E and F helices interact largely
nogradov (1985) has described a useful clas- with solvent but residues in the same posisification of invertebrate hemoglobins into tions provide intersubunit contacts in the
four categories in terms of the structures and two clam hemoglobins. The tetrameric
assemblies: (1) single domain, single heme, hemoglobin of the echuiroid Urechis caupo
single subunit hemoglobins, ~ 16 kDa; (2) also utilizes the E (but not the F) helix for
two-domain, multisubunit hemoglobins of intersubunit contacts, but the contacts are
250-800 kDa with 30-40 kDa chains each different and fewer. Unlike the Scapharca
with 2 hemes; (3) multidomain, multisub- homodimer, Urechis hemoglobin has little
unit hemoglobins, 2-18 domains per chain or no cooperativity of oxygen binding (Garey
and assemblies of 240-8,000 kDa; and (4) and Riggs, 1984) as would be appropriate
single-domain, multisubunit hemoglobins, for its apparent function in storing oxygen
chains of 15-17 kDa, some of which are during the anoxic periods of low tides.
disulfide linked.
The mechanism by which extracellular
Invertebrate blood hemoglobins may be hemoglobins are assembled and secreted in
either intracellular or extracellular. Most vivo is unknown and constitutes a major
intracellular hemoglobins are small with challenge. The extracellular hemoglobins
molecular weights no greater than 65,000. and the hemocyanins together include the
ORIGIN AND EVOLUTION OF HEMOGLOBIN
largest secreted globular proteins known, and
it will be important to determine where they
are assembled and how they are secreted.
Among the giant extracellular hemoglobins
that of the oligochaete Lumbricus terrestris
has been the most studied. Although an
X-ray crystallographic structure is not yet
available, the sequences of the major hemecontaining chains have been determined, the
functional properties have been measured
in detail, and the complete nucleotide
sequence of the gene encoding one of the
chains is known. These observations provide some insight into the assembly, function and evolution of this hemoglobin.
Molecules of Lumbricus hemoglobin have
a mass near 3,800 kDa and contain approximately 200 subunits of at least 7 different
kinds. The chains include 4 major hemecontaining chains, a, b, c, and d together
with additional chains of 33-38 kDa some
of which are required for assembly of the
full sized molecule (Kapp etal., 1982; Vinogradov et al., 1986). Chains a, b, c and d
each have an intra-chain disulfide bond
which joins a cysteine near the NH2-terminus with one in the middle of the H helix
(Fushitani et ai, 1988). The effect of this
bond is to freeze the relative positions of
the A and H helices and so presumably to
enhance thermal stability. Chains b and c
are joined to chain a to form a functionally
important trimer. Since all of these disulfide
bonds are also found in exactly the same
positions in the corresponding chains of the
marine polychaete Tylorrhynchus heterochaetus (Suzuki and Gotoh, 1986) the evolution of these stabilizing links must have
evolved before the divergence of polychaetes and oligochaetes. The disulfide bond
between chains a and c of Lumbricus hemoglobin appears particularly important
because it puts the NH2-terminal extensions
of chain a (H2N-Ala-Asp-Asp-Glu-AspCys-)near to that of chain c (H2N-Asp-GluHis-Glu-His-Cys-). This remarkable cluster
of 7 negative charges from two different
chains appears to form a prime binding site
for calcium ions which are known to be
essential modulators of oxygen binding.
Oxygen binding measurements indicate that
one Ca2+ ion is bound and two protons
released for each O2 bound (Fushitani et al,
1986). The two processes are tightly linked:
541
the pH dependence of O2 binding is zero in
the absence of calcium. Thus it appears likely
that a physiologically important calcium
binding site has evolved that requires the
participation of two different chains for
manifestation of the pH dependence of oxygen binding. Three additional chains, D1A,
DIB and D2, of Lumbricus hemoglobin are
deficient in heme. The two-layered hexagonal molecule cannot form in their absence
(Vinogradov et ai, 1986). The masses of the
chains, 33-38 kDa, suggest that they may
be composed of two heme-like domains.
This idea has been confirmed by Suzuki et
al. (1990) who have determined the amino
acid sequence of a corresponding "linker"
chain from the hemoglobin of the related
worm, Lamellibrachia sp. The 224 residue
chain apparently resulted from gene duplication followed by loss of both the first exon
of the first domain and the last exon of the
last domain. The second domain appears
incapable of binding heme because it lacks
a proximal histidine. A plausible evolutionary sequence of events is to suppose that a
fully functional two-heme, two-domain
chain evolved first and became essential for
the structural integrity of the molecule. Thus
it had two quite different functions, oxygenbinding and as a linker, either of which might
be evolutionarily important. The structural
requirement allowed the evolutionary loss
of heme binding but ensured retention of
the gene.
THE GLOBIN GENE
The structures of globin genes from only
two invertebrates have been determined so
far: those of the insect Chironomus (Antoine
and Niessing, 1984; Antoine et al., 1987)
and the earthworm Lumbricus terrestris
(Jhiang etal., 1988; Jhiang and Riggs, 1989).
A major unexplained feature of the Chironomus system is the extraordinarily high
number of components: at least twelve
unique hemoglobins have been found.
Although the multiple globin genes of Chironomus all lack introns, the one Lumbricus
gene sequenced has exactly the same intronexon organization as in the globin genes for
vertebrates. The absence of introns in the
Chironomus globin genes might have
resulted from reverse transcription from
mRNA. The two introns of the Lumbricus
542
AUSTEN F. RIGGS
globin gene contain a series of tandem
repeats that are very similar to parts of genes
in a variety of unrelated organisms. The
extensive simple repeats in the gene cluster
of rabbit globin (Margot et al., 1989) appear
to have resulted from transposon-based
insertions. We believe that the extensive
correspondence of the tandem repeats in the
Lumbricus globin introns to sequences in
other organisms may have a similar explanation. It seems likely that blocks of repetitive sequences can be transferred in and out
of the Lumbricus introns readily.
A requirement for the secretion of the
extracellular hemoglobins is that the globin
genes encode a signal sequence. How might
such a signal sequence have evolved? A
secretory signal sequence typically has about
16 hydrophobic amino acid residues. The
sequence might be acquired by the insertion
of the appropriate sequence from some other
gene by genetic recombination. Alternatively, the signal sequence might evolve by
a series of mutations that produce a gradual
increase in the hydrophobicity of the N H r
terminal segment of the protein. Perhaps
the second process is occurring in Vitreoscilla where the hemoglobin is partially
secreted (Khosla and Bailey, 1989). The
NH 2 -terminal sequence of Vitreoscilla
hemoglobin is hydrophobic, but it is not a
"good" signal sequence because it contains
a charged lysyl residue in the middle, and
is not proteolytically cleaved. It is also possible that a selective advantage occurs to
Vitreoscilla if hemoglobin is present both in
the cytoplasm and in the periplasmic space.
No information is available on the genes
for the multi-domain hemoglobins, but their
protein structures raise some intriguing
questions. What mechanism will put as
many as 18 globin genes, produced by gene
duplication, coming to be immediately
adjacent to one another? In the cDNA
encoding the two-domain globin of the clam
Barbatia reeveana, no more than 1-2 residues separate the two domains (Riggs et al.,
1986).
Another process that is probably involved
in the evolution of the globin gene is exon
loss. We have already described the apparent loss of exons from the gene encoding
one of the chains required for assembly
of Lamellibrachia hemoglobin. The extremely short 119 residue hemoglobin from
the protozoan Paramecium caudatum
(Iwaasa et al., 1989) may result from a similar loss.
Comparison of many globin sequences by
Zharkikh et al. (1984) shows that globin
evolution is largely the result of point mutations which are approximately 50 times
more frequent than insertions and deletions
which disrupt helices. They found that
insertions and deletions are almost four
times more frequent in interhelical regions
that within helices. These conclusions
depend on the correctness of the alignments
and on the assumption of common tertiary
structures with the same helical segments.
However, the X-ray structures of only six
non-vertebrate small hemoglobins are
known (from lupin, Chironomus, Aplysia,
Scapharca, Glycera and Urechis) and none
is yet determined for any of the large extracellular hemoglobins. Thus substantial variations on the theme of the myoglobin-fold
may well exist. Such variations exacerbate
the problems of alignment and appear likely,
at least in part, to be responsible for the
"implausible residues" reported by Bashford et al. (1987). They aligned and compared 226 globin sequences and found certain "implausible" residues which they
attributed to sequencing errors. For example, glutamine at position F5 in Lumbricus
chain b (=chain AIII) was deemed implausible because a hydrophobic residue was
expected. However, a complete redetermination of the sequence confirmed the identification (Fushitani et al., 1988). Furthermore, arginine and lysine are found in this
position in several other globin chains of
extracellular annelid hemoglobins (Fushitani et al, 1988; Suzuki and Gotoh, 1986).
The most likely explanation therefore is that
the tertiary structures differ rather than that
a sequencing error has occurred.
CONCLUSION AND PROSPECTS
A survey of hemoglobin-like, O2-binding
proteins among widely diverse procaryotic
and eukaryotic organisms suggests that proteins homologous with hemoglobins may be
ubiquitous in virtually all eukaryotic organisms and in many procaryotes. The asco-
ORIGIN AND EVOLUTION OF HEMOGLOBIN
mycetous fungi and the hydrogen bacterium
Alcaligenes possess flavohemoglobins that
appear likely to be homologous with known
hemoglobins and to members of the cytochrome b family. If so, we could trace hemoglobin ancestry to the earliest aerobic organisms. The determination of amino acid
sequences, three-dimensional structures and
the nucleotide sequences of the genes of the
flavohemoglobins should hold the key to
understanding the relationship.
ACKNOWLEDGMENTS
Original work on invertebrate hemoglobins in the author's laboratory has been supported by National Institutes of Health
Grant GM35847, National Science Foundation Grants DMB 85028587 and DMB
88-10828, and Welch Foundation Grant
F-0213.1 thank Dr. Thomas Vandergon for
valuable discussions.
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