Structural and Functional Properties of Class 1 Plant Hemoglobins
Abir U. Igamberdiev1, Natalia V. Bykova1and Robert D. Hill2
1Department of Biology, Memorial University of Newfoundland, St. John’s, NL, Canada A1B 3X9
2Department of Plant Science, University of Manitoba, Winnipeg, MB, Canada R3T 2N2
Nonsymbiotic class 1 plant hemoglobins are induced under
hypoxia. Structurally they are protein dimers consisting of two
identical subunits, each containing heme iron in a weak hexa-
coordinate state. The weak hexacoordination of heme-iron bind-
ing to the distal histidine results in an extremely high avidity to
oxygen, with a dissociation constant in the nanomolar range.
This low dissociation constant is due to rapid oxygen binding
resulting in protein conformational changes that slow dissocia-
tion from the heme site. Class 1 hemoglobins are characterized
by an increased rate of Fe31reduction which is likely mediated
by cysteine residue. This cysteine can form a reversible covalent
bond between two monomers as shown by mass spectrometry
analysis and, in addition to its structural role, prevents the mol-
ecule from autoxidation. The structural properties of class 1
hemoglobins allow them to serve as soluble electron transport
proteins in the enzymatic system scavenging nitric oxide pro-
duced in low oxygen via reduction of nitrite. During oxygen-
ation of nitric oxide to nitrate, oxidized ferric hemoglobin is
formed (methemoglobin), which can be reduced by an associ-
ated reductase. The identified candidate for this reduction is
monodehydroascorbate reductase. It is suggested that hemoglo-
bin functions as a terminal electron acceptor during the hypoxic
turnover of nitrogen, the process aided by its extremely high
affinity for oxygen.
? 2011 IUBMB
IUBMB Life, 63(3): 146–152, 2011
general bioenergetics; hemeproteins; hemoglobin; nitric
oxide; catalytic mechanism; electron transfer in proteins;
Hb, hemoglobin; MDHAR, monodehydroascorbate
reductase; NO, nitric oxide.
Class 1 hemoglobins are induced under hypoxic conditions
that plants experience, e.g., during flooding, at early stages of
seed germination, and in meristematic tissues rapidly depleting
oxygen. They are expressed at relatively low concentration of
0.1–0.3% of total protein content which is usually in the range
of 5–20 lM (1, 2). This concentration is two orders of magni-
tude lower than the concentration of leghemoglobin in root
nodules and three orders lower than hemoglobin in erythrocytes.
This indicates a different function for class 1 hemoglobins, con-
trary to leghemoglobins viewed as oxygen stores restricted to
symbiotic nodules. The induction of expression of class 1
hemoglobin gene under hypoxic conditions was first demon-
strated by Taylor et al. (3) in barley. It was demonstrated that
class 1 hemoglobin improves energy state and decreases reduc-
tion levels in hypoxic roots (4, 5).
Class 1 hemoglobins contain heme iron in the hexacoordi-
nate state. The heme prosthetic group in hemeproteins is most
often attached through coordination of either one or two
histidine side chains. Those hemoglobins with one histidine
coordinating the heme iron are called ‘‘pentacoordinate’’ hemo-
globins, a group represented by red blood cell hemoglobin and
most other oxygen transporters. Those in which the iron coordi-
nates with both the proximal and distal histidine are called
‘‘hexacoordinate hemoglobins’’ and have broad representation
among eukaryotes. While the first (proximal) histidine is bound
tightly to the heme iron, the coordination of the second (distal)
histidine in hexacoordinate Hbs is reversible and differs signifi-
cantly, allowing for binding of exogenous ligands like oxygen,
carbon monoxide, and nitric oxide. The common property of
non-symbiotic class 1 hemoglobins that distinguishes them from
other hemoglobins is a low value of the hexacoordination equi-
librium constant (KH), which is the binding constant of the distal
histidine, allowing an equilibrium of pentacoordinated and hexa-
coordinated species and facilitating the binding of ligands (6).
In this review we analyze the peculiarities of class 1 hemo-
globins structure, in particular taking into consideration the
extensively studied barley Hb, and link their structural features
Address correspondence to: Abir U. Igamberdiev, Department of
Biology, Memorial University of Newfoundland, St. John’s, NL, A1B
3X9, Canada. Tel.: 11-709-864-4567. Fax: 11-709-864-3018.
Received 30 December 2010; accepted 2 February 2011
ISSN 1521-6543 print/ISSN 1521-6551 online
IUBMBLife, 63(3): 146–152, March 2011
to ligand binding and catalytic properties of these proteins. We
show that class 1 Hb can serve in plant cells as a part of
efficient NO scavenging system operating under hypoxia. This
system can use oxygen as a terminal electron acceptor at
concentrations at which mitochondria do not effectively func-
tion. We also analyze how class 1 hemoglobin differs from
other NO scavenging systems operating from microorganisms to
higher plants and animals and show that this particular system
is well adapted to extremely low oxygen tensions appearing in
plants under low oxygen stress.
Oligomeric Structure of Class 1 Hemoglobins
The basic property of plant class 1 hemoglobins (weak hexa-
coordination) is determined by the structural orientation of
essential amino acid residues relative to the heme-binding site.
Class 1 hemoglobins exist as dimers made of two identical
subunits of molecular weight ?18 kDa and usually containing
one or two cysteine residues per molecule (7). Initially it was
considered that two subunits interact via non-covalent binding
similar to human hemoglobin. Blood hemoglobin is present at
millimolar concentrations in erythrocytes, while non-symbiotic
class 1 hemoglobins are expressed at micromolar concentra-
tions. The equilibrium dissociation constant for noncovalent
dimerization of ferric rice Hb is 86 lM (7), indicating weak
interactions compared with those for the a1b1dimer of human
hemoglobin and suggesting that the non-covalent interaction
between class 1 hemoglobin subunits may not be physiologi-
cally relevant. The dimer interface of a class 1 Hb is formed by
close contact between the G helix and the region between the B
and C helices of the partner subunit. The proximal and distal
histidine sidechains coordinate directly to the heme iron, form-
ing a hemichrome with spectral properties similar to those of
cytochrome b5(7). The main distinction of class 1 hemoglobins
from cytochromes is their solubility, with cytochromes being
anchored to membranes. Functionally, in terms of participation
in electron transport, class 1 hemoglobins resemble cytochromes
more than pentacoordinate hemoglobins such as blood hemoglo-
bin, myoglobin, or leghemoglobin.
Most investigated class 1 hemoglobins have a single con-
served cysteine residue located in the E-helix of the polypeptide
chain (7). This helix is bent toward the center of the porphyrin
ring due to a direct coordination of the distal His (located in the
same E helix as cysteine) with the iron atom. Arabidopsis class
1 hemoglobin has two cysteine residues per monomer (8)
located adjacent to one another in the same position where a
single cysteine is found in other class 1 hemoglobins. Most
plant leghemoglobins do not contain cysteines at all. Human
neuroglobin, which is hexacoordinated as plant class 1 hemo-
globins, contains three cysteine residues, which participate in
formation of intra- and intermolecular disulfide bonds depend-
ing on the redox state of the environment (9). Mutation of spe-
cific cysteines in neuroglobin or their reduction to break the S-S
bond resulted in a low oxygen affinity due to a decrease in the
histidine dissociation rate and provoking the release of oxygen
(10). In cytoglobin, an internal disulfide bond modifies the rate
of dissociation of the distal histidine and consequently leads to
different cytoglobin conformations altering the observed oxygen
affinity by an order of magnitude (11).
To determine the possible role of cysteine in the structure
and function of class 1 Hb, barley hemoglobin was mutated
(Cys79replaced by Ser) (12). Nano-electrospray ionization mass
spectrometry analysis of the native intact wild type and mutated
barley Hb showed that the mutated barley Hb was more readily
dissociated into its monomer subunits and was more susceptible
to denaturation and autoxidation than the native form. Using
tandem mass spectrometry analysis it was possible to demon-
strate that Cys79participated in intermolecular S–S bond forma-
tion. It was concluded that the cysteine residue is an important
contributor to the quaternary and tertiary structure of barley
hemoglobin. This study demonstrated that the single Cys79is
important for the dimeric organization of barley Hb, via disul-
fide bond formation that stabilizes its quaternary structure and
contributes to the maintenance of the heme iron in the ferrous
The stability of the oxyferrous compound against autoxida-
tion is provided by the interaction of distal histidine with the
bound ligand through a strong hydrogen bond (13). The large
increase in autoxidation upon cysteine mutation could be related
to the disruption of these interactions in combination with sol-
vent entry into the distal heme pocket. Cysteine may contribute
to the maintenance of distal heme pocket through the formation
of a stable conformational state. In human myoglobin, the
cysteine residue may participate directly in reduction of heme
iron, which results in intermolecular disulfide bond formation
(14). This bond is not permanent and appears as a part of the
reduction–oxidation cycle of the myoglobin molecule. This
could be an explanation for the role of this cysteine in barley
and other class 1 plant hemoglobins.
Figure 1 presents a model describing the participation of cys-
teine in the reduction of ferric heme and assumes the existence
of class 1 Hb in a monomeric state. Reductants can reduce iron
either directly (reaction 1) or via reduction of the disulfide bond
(reactions 2 and 3), followed by dimerization (reaction 4). The
direct reduction of iron can be achieved by the reductants (such
as ascorbate) that preferably transfer electrons to the heme iron
directly, while cysteine can be reduced by the reductants (such
as glutathione or thioredoxins) having primarily aiming to main-
tain thiols in the reduced state. In this mechanism, cysteine is
important as an additional means to maintain heme iron in a
reduced state. A certain amount of monomeric Hb is always
present in the solution (12), making this reaction feasible. With-
out added reductant, the monomeric concentration is only a few
percent, after reduction with tris(2-carboxyethyl)phosphine, its
concentration increases to about 50% at Hb concentration of
12 lM and near 90% at 24 lM [Fig. 2 in (12)]. The hypothesis
predicts that barley Hb will always be in the equilibrium
between dimeric (with oxidized cysteine) and monomeric (with
147STRUCTURAL AND FUNCTIONAL PROPERTIES OF PLANT HEMOGLOBINS
reduced cysteine) forms, with the equilibrium shifting, depend-
ing upon redox conditions.
Bis-histidyl coordination of iron itself in hexacoordinated
hemoglobins greatly increases the rate of Fe31reduction when
compared to pentacoordinate hemoglobins, while the autoxida-
tion rate is low (13). In myoglobin and leghemoglobin, reduc-
tion is limited by the kinetic rate constant for electron transfer,
whereas in the hexacoordinate hemoglobins reduction is limited
only by bimolecular binding of the reductant (13). The tertiary
structure of barley Hb is resistant to autoxidation only when the
cysteine residue is present. The mutated Hb (without cysteine)
was oxidized to the ferric state approximately 103times faster
than the non-mutated form, which was the consequence of cys-
teine substitution but not of the formation of monomer itself
(12). This may indicate its dual role in the protection against
autoxidation and in the tertiary structure maintenance linked to
the direct participation in reduction of the heme iron. Dimers
involving disulfide bond formation would require interaction of
the E helix in each subunit, which may also stabilize non-cova-
lent interaction between the monomers. The hydrophobic disul-
fide bond contributes to the folding, stability, and reactivity of
barley Hb. Cys79may, therefore, play a role in the maintenance
of the heme in a reduced state either via direct electron transfer
to the heme iron or through the maintenance of Hb in its most
stable conformational geometry.
Binding of Ligands
Class 1 plant hemoglobins are characterized by extremely
high oxygen avidity and extremely low values of the dissocia-
tion rate constant. Duff et al. (15) first determined oxygen dis-
sociation rate constants of plant class 1 Hb (barley Hb). The
most striking observation was the extremely slow dissociation
of oxygen (t1/2?25 s or koffless than 0.03 s21) which in com-
bination with very fast oxygen binding resulted in a value of kD
of less than 3 nM. This means that Hb remains in the oxygen-
ated state at concentrations of oxygen two orders of magnitude
lower than the concentrations required for the operation of cyto-
chrome c oxidase. This remarkable property is determined by
the structure of the heme binding site of plant class 1 Hbs. Con-
trary to other hexacoordinated hemoglobins, like human neuro-
globin and cytoglobin, plant class 1 hemoglobins are character-
ized by distinctly low hexacoordination constants (KH), in the
range of two-three orders of magnitude lower than for neuroglo-
bin and cytoglobin (16). For class 1 rice Hb the value of KHis
1.9, for class 1 maize Hb it is 0.9 with moderate rate constants
(in the range of tens s21) for both (penta- vs. hexacoordinate)
transitions, whereas for neuroglobin the value of KH is more
than 1,000 and for cytoglobin it is 860, with three orders of
magnitude faster transition to hexacoordination than to penta-
coordination (16). At low KHthere is a much higher portion of
heme pentacoordination in the equilibrium (1/3 for rice and 1/2
for maize class 1 Hb calculated from the values of KH), allow-
ing binding of ligands that exhibits a conspicuous multiphasic
character. This allows extremely rapid binding of oxygen to
deoxygenated Hb, even at nanomolar oxygen concentrations
Figure 2. Participation of class 1 hemoglobin in nitric oxide
dioxygenase reaction. Hemoglobin in ferrous (Fe21) state binds
oxygen and oxygenates nitric oxide to nitrate. The resulting fer-
ric (Fe31) hemoglobin is reduced by ascorbate with formation
of monodehydroascorbate which is reduced back to ascorbate
by monodehydroascorbate reductase (MDHAR) using NADH or
NADPH as a reductant. [Color figure can be viewed in the
online issue, which is available at wileyonlinelibrary.com.]
Figure 1. The oxidative-reductive cycle of class 1 hemoglobin
and possible role of cysteine. While reduction of ferric (III)
heme in dimer can take place directly (reductant AH, reaction
1), cysteine can also participate in keeping heme in the ferrous
(II) state being reduced by another reductant (BH, reactions 2,3)
followed by dimerization (reaction 4). Reduced (ferrous) heme
binds oxygen (reaction 5) and converts nitric oxide (NO) to ni-
3) (reaction 6). Thus cysteine participates in the sup-
porting mechanism for keeping iron in the reduced state and
preventing autoxidation. [Color figure can be viewed in the
online issue, which is available at wileyonlinelibrary.com.]
148 IGAMBERDIEV ET AL.
(17). The coordinating distal histidine might spend only a small
fraction of its time locked into a tighter bond with the heme
iron (16). The reversible coordination of the heme iron by a
distal histidine side chain located in the heme pocket competes
with exogenous ligand binding and causes multiphasic relaxa-
tion time courses, altering a steady-state relationship between
hexacoordination and exogenous ligand binding. Hexacoordina-
tion plays a role in regulating affinity constants for ligand bind-
ing through competition between an internal ligand (distal E7
histidine) and external ligands. Weak binding of distal histidine,
a function of the hexacoordination constant, shifts the equilib-
rium towards external ligand binding. Even in the heme hexa-
coordinate state, class 1 Hb is still able to bind O2rapidly and
with unusually high affinity compared with most other hemoglo-
bins and myoglobins (7) due to rapid dissociation of the heme
iron from the distal histidine, generating pentacoordinate
species. The distal histidine remains sufficiently close to stabi-
lize bound O2through formation of strong hydrogen bonds with
hydrogen atoms of the imidazole sidechain. This mechanism
requires significant changes in the orientation of sidechains in
the active site (16). The distal histidine equilibrium between
two conformations has far-reaching consequences for ligand
binding and substrate conversions. In the case of dehaloper-
oxidase hemoglobin, for example, it contributes to allowing or
forbidding the enzyme to react with H2O2in the 6-th coordina-
tion position (18).
The observed facilitation of reduction of ferric hexacoordi-
nate hemoglobins in the presence of external reductants can be
explained by differences in the reorganization energy for reduc-
tion between hexacoordinate and pentacoordinate hemoglobins
(19). Again, the reduction of ferric plant nonsymbiotic hemo-
globins is facilitated due to a substantially lowered histidine
coordination affinity, which is imposed by the protein matrix of
these macromolecules (20). Interaction of the second (distal)
histidine in the heme centre with the bound ligand through a
strong hydrogen bond supports the stability of the oxyferrous
compound against autoxidation (21). Consequently, the large
increase in autoxidation upon cysteine mutation can cause loss
of these interactions in combination with solvent entry into the
distal heme pocket. In addition to the above mentioned role of
cysteine, the conserved phenylalanine residues are important for
the maintenance of these interactions (22). The stabilization of
the oxyferrous compound through interactions between PheB10
and HisE7 that induce a strong hydrogen bond between the dis-
tal histidine and the bound ligand can be important for NO
scavenging activity (22).
There are also important consequences for NO binding to
plant class 1 hemoglobins. Although it is difficult to measure
binding constants with NO, it is likely that NO binding to
ferrous deoxygenated heme is complicated by the structure of
the molecule. Different energetic barriers encountered by oxy-
gen and NO (23) can explain preferential binding of oxygen to
ferrous Hb and the oxygenase mechanism of the reaction. In
this mechanism, binding of oxygen results in its activation via
sharing an electron with iron, making possible the attack of
other ligands such as NO. The steric orientation of hexacoordi-
nated histidine, important for O2binding, surprisingly plays no
apparent role in ligation with NO, which occurs at a very low
rate (17). The internal cavities in the molecule, as in the case of
myoglobin, may serve as storage sites for NO. Binding of oxy-
gen facilitates the formation of a pathway through the protein
leading from the distal cavity to the bulk solvent (17). This
pathway could enable NO to settle in site C, approach the
bound O2, and react to form nitrate. The discrete docking sites
in which ligands can temporarily be stored before rebinding to
the heme at different times were shown for Arabidopsis class
1 Hb AHb1 (24). The reduced thermodynamic stability of such
open conformations appears to be compensated by the capabil-
ity of controlling ligand diffusion through the protein matrix to
the active site, possibly by stocking more than one reactant
molecule in selected sites. This stability is conferred by a single
cysteine residue and dimeric structure (12) and by phenylala-
nine residues that can line the migration pathways for transport
of ligands (21). While two docking sites appear to be available
for transient storage of photodissociated ligands, the direct
channel connecting the distal cavity with the solvent points to a
facilitated exchange of ligands with the oxygenated protein.
This may therefore mediate NO entry and reaction with the
The denitrosylase mechanism (25) would involve NO bind-
ing first either to cysteine or to the deoxygenated heme. This
bound NO would then react with oxygen to form nitrate. As
discussed earlier, nitrosylation of cysteine is not a plausible
mechanism because this cysteine participates in covalent inter-
molecular binding of monomers in Hb molecule and in support-
ing the conformation that prevents the heme autoxidation (12).
NO binding to the heme is also unlikely because it is relatively
slow and is facilitated by binding of oxygen which is very tight
(17). It could function at high NO/oxygen ratios but usually the
NO dioxygenase reaction is inhibited under these conditions
(for cytoglobin more than 1:500 (26) and for flavohemoglobins
more than 1:100 (27)) arguing against the denitrosylase mecha-
nism. The HbNO complex is inactive in the case of flavohemo-
globins (27), and it is also inactive for cytoglobin that follows
from NO inhibition (26), and, in the same manner, it is likely
to be inactive in plant hemoglobins. There is no evidence to
indicate whether NO dioxygenase activity associated with plant
Hbs can be inhibited by increased NO/O2 ratio but if this
happens, it may actually be a limiting factor for NO scavenging
As can be seen from the Table 1, barley Hb can slowly scav-
enge NO in the presence of NADH or NADPH but this reaction
is strongly increased, by three orders of magnitude, with the
addition of monodehydroascorbate reductase (MDHAR) and
ascorbate (28). This confirms that the limiting step in the NO
149 STRUCTURAL AND FUNCTIONAL PROPERTIES OF PLANT HEMOGLOBINS
dioxygenase reaction is reduction of methemoglobin (13). The
complete NO dioxygenase reaction can be described by the
2NO þ 2O2þ NADðPÞH ! 2NO?
3þ NADðPÞþþ Hþ
MDHAR is the only reductase identified to date that can
operate in conjunction with non-symbiotic hemoglobins (6).
Figure 2 shows a putative mechanism of the NO scavenging
reaction involving MDHAR. In this mechanism, ferric Hb
(methemoglobin) is reduced by ascorbate and the formed mono-
dehydroascorbate (ascorbate free radical) is immediately scav-
enged by MDHAR. Low turnover rate of reaction in the pres-
ence of ascorbate and absence of MDHAR (apparently 0.03 s21
heme21, calculated from the rate of heme reduction by ascor-
bate, Igamberdiev, unpublished), which is one order of magni-
tude higher than with NADH but one order of magnitude lower
than for cytoglobin (26), demonstrates that the removal of
monodehydroascorbate from the proximity of the heme consti-
tutes the rate-limiting step and is crucial for NO scavenging
that involves plant Hb.
The NO scavenging system that involves non-symbiotic
plant Hb differs strongly from other NO scavenging reactions
(Table 2). Bacterial and yeast flavohemoglobins, that include a
reductase flavodomain in the molecule, have a very high turn-
over number but operate at high oxygen, with a Kmthat is at
?40% of ambient O2, and with relatively high micromolar and
submicromolar NO concentrations (26). They also have rela-
tively low affinity to the reductant (NADH or NADPH). Similar
characteristics, with even a higher turnover number but a lesser
affinity for NO, have been demonstrated for dihydrolipoamide
dehydrogenase (30) and cytochrome c oxidase, with the latter
being an NO oxidase and forming nitrite (31). A protein, with
marked similarity to dihydrolipoamide dehydrogenase, was
shown to be able to reduce ferric leghemoglobin (29, 32). Cyto-
globin is the hexacoordinate hemoglobin with properties similar
to plant class 1 hemoglobins but a higher extent of hexacoordi-
nation and hence lower oxygen avidity. Cytoglobin demon-
strates significant NO scavenging activity in the presence of
ascorbate but needs the presence of tens micromolar concentra-
tions of oxygen, which is close to the oxygen saturation levels
of mitochondrial oxidative phosphorylation (26). Cytoglobin is,
therefore, likely not efficient as an NO scavenger at low oxygen
and its function may be different. The NO affinity of cytoglobin
is also lower (40 nM) than that of plant hemoglobins (26). All
this suggests that only plant class 1 hemoglobins and similar
proteins, such as Ascaris Hb, are able to effectively scavenge
NO to extremely low levels in an almost anoxic atmosphere.
The reported concentration of Hb of 5–20 lM in hypoxic tis-
sue (1, 2) along with a maximum turnover number of 6 s21per
heme (Table 1, calculated from data in (28)) suggests that NO
scavenging in hypoxic tissue can be in the range of 10 lmol
NO per g fresh weight per second. This rate can be achieved at
concentrations of oxygen well below the oxygen saturation
levels of cytochrome c oxidase, with NO being scavenged down
to its very low levels. In fact, a fourfold difference between the
NO levels in Hb overexpressing and underexpressing transgenic
alfalfa root cultures has been demonstrated in anoxic alfalfa
roots (33). This strongly supports the NO scavenging function
of the plant class 1 hemoglobin. Rates of NO scavenging will
Nitric oxide dioxygenase activity (rates calculated per heme)
involving barley hemoglobin
Barley Hb 1 MDHAR 1
Barley Hb 1 MDHAR
Nitric oxide dioxygenase activity (rates per active site) involving barley hemoglobin, yeast flavohemoglobin, and
Barley Hb 1 MDHAR 1
NA, not applicable.
150IGAMBERDIEV ET AL.
depend on different factors, and one of them may be the actual
NO/O2ratio in the tissue, elevation of which could inhibit the
dioxygenase reaction of the system involving plant Hb in the
same manner as it inhibits flavohemoglobins (27) and cytoglo-
bin (26). The measured NO scavenging activity in plant extracts
is comparable with the rates determined in vitro with MDHAR
(5, 28), however the in vivo NO turnover is likely to be much
lower. Using spin trap techniques, Dordas et al. (33) showed
that NO accumulation in alfalfa roots reached 120 nmol per g
of fresh weight after 24 h incubation, being 50% lower in the
line overexpressing Hb and 1.5 times higher in the line under-
expressing Hb gene. Under aerobic conditions, NO accumula-
tion was at least two orders of magnitude lower. This is in
agreement with the data on physiological NO concentrations in
animal tissues, where the amounts in aerobic tissue (rising from
the nanomolar to submicromolar and micromolar under stress
conditions) (31). The rate of NO production from nitrite at the
catalytic site of cytochrome c oxidase was determined at
17 nmol per min per mg protein for the isoform Va of the cyto-
chrome c oxidase which is hypoxically inducible (34). The rate
of NO production at the site of complex III was of the same
order of magnitude at 15 nmol min21mg21(34). These values
are comparable to the rates of NADH and NADPH oxidation by
anaerobic barley and rice mitochondria (35).
Operation of the hypoxically induced plant hemoglobin in
conjunction with production of NO by mitochondria and with
reduction of nitrate by nitrate reductase has been defined as a
hemoglobin/ nitric oxide cycle (36). Operation of this cycle
involves nitric oxide turnover between mitochondria and
cytosol, thus contributing to NADH and NADPH oxidation and
ultimately to ATP synthesis. This turnover represents hemoglo-
bin-dependent respiration at very low oxygen, under conditions
where cytochrome c oxidase is inoperative, and its possible
contribution to the maintenance of ATP levels is comparable to
glycolytic fermentation (37). It can also be efficient in recycling
the glycolytic NADH and turning metabolism from ethanol to
alanine production (38).
Class 1 plant hemoglobins have an extremely high avidity to
oxygen and extremely slow oxygen dissociation properties,
which means that they exist in plants in an oxygenated form
even at oxygen concentrations in the nanomolar range. These
properties, unique for class 1 hemoglobins, are the result of the
weak hexacoordination of heme iron involving the globin distal
histidine. Oxygen-bound ferrous Hb acts as a soluble electron
transport protein in a reaction converting NO to nitrate, with
the formation of ferric Hb (methemoglobin). The limiting step
in this reaction is the reduction of ferric heme thath is strongly
facilitated by the presence of ascorbate and MDHAR. The NO
dioxygenase enzymatic system in plants likely consists of class
1 hemoglobin and its associated reductase. Hemoglobin in this
system functions as a water-soluble electron carrier protein
(analogous to a cytochrome) and oxygen is the terminal electron
acceptor just like in respiration, but the affinity to oxygen is
much higher than in two terminal mitochondrial oxidases (cyto-
chrome c oxidase and alternative oxidase). The system uses
portions of the mitochondrially electron transport chain, in con-
junction with the class 1 hemoglobin-driven NO dioxygenase to
replace aerobic mitochondrial electron transport, providing
essential oxidative capability at anaerobic oxygen levels incapa-
ble of supporting aerobic respiration.
The authors thank S. Dewilde and L. Moens for an extensive
discussion of the structure and functional roles of oxygen-bind-
ing proteins at the O2BIP meeting in Antwerp. This work was
supported by grants from the Natural Sciences and Engineering
Research Council of Canada.
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