From the Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, Iowa 50011
Received for publication, October 11, 2000, and in revised form, November 30, 2000
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ABSTRACT |
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Plant nonsymbiotic hemoglobins are
hexacoordinate heme proteins found in all plants. Although expression
is linked with hypoxic environmental conditions (Taylor, E. R.,
Nie, X. Z., Alexander, W. M., and Hill, R. D. (1994)
Plant Mol. Biol. 24, 853-862), no discrete physiological
function has yet been attributed to this family of proteins. The
crystal structure of a nonsymbiotic hemoglobin from rice has recently
been determined. The crystalline protein is homodimeric and
hexacoordinate with two histidine side chains coordinating the heme
iron atom. Despite the fact that the amino acids responsible for the
subunit interface are relatively conserved among the nonsymbiotic
hemoglobins, previous work suggests that this group of proteins might
display variability in quaternary structure (Duff, S. M. G.,
Wittenberg, J. B., and Hill , R. D. (1997) J. Biol. Chem. 272, 16746-16752; Arredondo-Peter, R., Hargrove, M. S., Sarath, G., Moran, J. F., Lohrman, J., Olson, J. S., and Klucas , R. V. (1997) Plant Physiol. 115, 1259-1266). Analytical ultracentrifugation and size exclusion high
pressure liquid chromatography were used to investigate the
quaternary structure of rice nonsymbiotic hemoglobin at various states
of ligation and oxidation. Additionally, site-directed mutagenesis was
used to test the role of several interface amino acids in dimer
formation and ligand binding. Results were analyzed in light of
possible physiological functions and indicate that the plant
nonsymbiotic hemoglobins are not oxygen transport proteins but more
closely resemble known oxygen sensors.
Two classes of hemoglobins have been identified in plants. The
leghemoglobins were discovered many years ago in the root nodules of
legumes, where they play an important role in symbiotic nitrogen fixation (1). An effort to identify the evolutionary origin of
leghemoglobins led to the discovery of plant nonsymbiotic
hemoglobins (nsHbs)1 in
several plant species (2, 3). nsHbs have now been identified in many
mono- and dicotyledonous plants, including barley, soybean, rice,
Arabidopsis, chicory, and corn (NCBI accession number
AAF44664) (4-7). Homologous hemoglobins have also recently been
discovered in bryophytes, which leads to the conclusion that these
proteins are ubiquitous in the plant kingdom (8). The discovery of
nonsymbiotic hemoglobins was slowed by the fact that they are present
in very low concentrations inside plants. The details of their in
vivo expression and function are currently under investigation (9, 10).
The biophysical properties of several nsHbs have been characterized and
have provided important clues about possible physiological functions
for this family of proteins (4, 5, 11). nsHbs are unusual because their
heme prosthetic groups are hexacoordinate in the ferric and deoxy
ferrous states. Hexacoordination results from two His residues that
bind the heme iron at the fifth and sixth coordination sites; one
coordinates the proximal side of the heme iron, which is characteristic
of all hemoglobins, and a second coordinates the distal side, which is
traditionally the binding site for oxygen and other ligands.
Hexacoordination has been observed in hemoglobins from bacteria;
protozoa; several animal species, including humans; and an
oxygen-sensing heme protein from Escherichia coli (12-16).
Therefore, the mechanism of using displaceable coordination for
regulating heme protein function has been established. Despite this
potential inhibition of ligand binding, nsHbs reversibly bind oxygen
and other ligands with very high affinities (9). For this reason, it
has been suggested that they do not function as oxygen storage or
transport proteins but might be involved in plant metabolism under
oxygen-limiting conditions (2, 10).
The crystal structure of rice nsHb (rHb1) has recently been solved and
is currently the only structure of a nsHb (17). This structure reveals
a homo-dimeric protein in the asymmetric unit, indicating that it can
form a specific dimer independent of crystal packing. A ribbon model
and details of the rHb1 dimer interface are shown in Fig.
1. The interface is composed of the amino
acid residues at the beginning of the CD helix region and the G helix. The interaction between the two subunits consists of a hydrogen bond
between the side chain of Glu119 of one subunit and
Ser49 on the other. These two polar interactions surround a
hydrophobic core consisting of the side chains of Val46,
Val120, and Phe123 of each subunit. The amino
acid residues that make up this interface region are relatively
conserved among the nsHbs (9, 17), indicating that the quaternary
structures of other members of this family could be similar to
rHb1.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
The crystal structure of rHb1.
A, dimeric protein showing the interface region.
B, stereo picture of the amino acid side chains in the dimer
interface. The residues with gray bonds are in one subunit,
and those with black bonds are in the other. The symmetric
dimer interface is a hydrophobic core of Val and Phe amino acids that
is capped at either end by an electrostatic interaction between
Glu119 of one subunit and Ser49 of the other.
These figures were prepared from Protein Data Bank
entry1d8u.
Quaternary structure plays an important role in the function of many hemoglobins by facilitating allostery and cooperativity for regulation of ligand binding. However, the solution quaternary structures of nsHbs have not yet been characterized in detail. Barley nsHb was reported by Duff et al. (11) to be dimeric, whereas rHb1 was initially reported to be monomeric (4). The quaternary structures of other nsHbs have not yet been investigated. The discrepancy between the quaternary structure of rHb1 in crystals and in solution and between the solution quaternary structures of the nsHbs from barley and rice are the rationale for the characterization of rHb1 described here.
In this work, the solution quaternary structure of rHb1 was
investigated along with its effects on the rate and affinity constants for ligand binding and the rate constants characterizing
hexacoordination by the distal His. In addition, site-directed
mutagenesis of several conserved amino acids that make up the subunit
interface was used to determine their effect on quaternary structure
and ligand binding. These structural and biophysical characteristics of
nsHbs are important for developing an understanding of the function of
these proteins in vivo.
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EXPERIMENTAL PROCEDURES |
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Protein Preparation--
All proteins were expressed and
purified using the methods described by Arredondo-Peter et
al. (4). Proteins were expressed in BL21(DE3) E. coli cells using the pET system from Novagen without amino- or
carboxyl-terminal tagging. This procedure results in bright red cell
pellets and a red supernatant following cell lysis. All mutant proteins
were generated using the Quickchange mutagenesis procedure available
from Stratagene. Extinction coefficients for different ligated forms of
wild type rHb1 were determined using the pyridine hemochromogen method
described by Riggs (18).
Quaternary Structure Determination--
Size exclusion analysis
was performed at room temperature with a TSK-GEL G2000SWXL HPLC column
from TOSOHAAS and a Varian Prostar HPLC system. 20-µl protein samples
at concentrations ranging from 1 to 800 µM were analyzed
at a flow rate of 1 ml/min, and retention time was defined as the
midpoint of the absorbance chromatogram observed at 410 nm. The buffer
used for all analyses was 100 mM potassium phosphate, pH
7.0, and 100 mM NaCl. The ligated states were prepared
using previously described methods (19, 20), except for the
Fe(III)CN sample that was in the above buffer and 5 mM NaCN
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Analytical ultracentrifugation was performed at 25 °C with a Beckman XLA analytical ultracentrifuge using absorption optics. Sedimentation profiles were collected for samples at 25,000 and 30,000 rpm, and over protein concentrations ranging from 10 to 50 µM. Each sample was loaded into all three sectors of an equilibrium centerpiece, and all three sedimentation profiles contributed to data analysis. Apparent molecular mass at each concentration was determined by combining data from each sector of the centerpiece and each angular velocity using the Multi Fit global analysis software available from Beckman. Solvent density and partial specific volume were calculated using previously described methods that have been summarized by Beckman (21, 22). These values for our buffer system and rHb1 were 1.0126 g/ml and 0.7488 ml/g, respectively. All other graphic analyses, fitting of data, and figure preparation were carried out with the program Igor Pro (Wavemetrics, Inc.). Estimates of error for final equilibrium dissociation constants were obtained from the absolute percent variation in at least three independent experiments.
Kinetic Measurements--
Oxygen and carbon monoxide association
rate constants and oxygen dissociation rate constants were
determined using previously described methods (19, 20). Measurement of
the rate constants for the association and dissociation of the distal
His and the flash photolysis apparatus used in all experiments have
been described by Hargrove (23).
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RESULTS |
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Concentration Dependence of the Apparent Molecular Mass of
rHb1--
Accurate analysis of the concentration dependence of
association requires absorption extinction coefficients specific to
each ligated state of rHb1. Fig. 2 shows
these absorbance spectra with the extinction coefficients plotted on
the ordinate axis, and Table I lists
specific extinction coefficients used for concentration determination
at different wavelengths. The extinction coefficients for the ferric
and deoxy proteins were identical to those previously reported (4).
These extinction coefficients are slightly smaller than other ferric
hemoglobins but comparable in the reduced forms (24).
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Fig. 3A shows the
concentration dependence of size exclusion HPLC retention time for
ferric rHb1. The retention time at a low protein concentration (~ 1 µM) was comparable to that of myoglobin (data not shown).
As protein concentration was increased, retention time dropped to a
value much smaller than that expected for monomeric rHb1 (18,418 Da).
For a homodimer, the concentration dependence of the fraction of
monomeric protein obeys the following equation, where
Kd is the equilibrium dissociation constant,
PT is the total protein concentration, and
fm is the fraction of protein that is monomeric.
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(Eq. 1) |
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(Eq. 2) |
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Having established that rHb1 is a partially associated homodimer at micromolar concentrations, analytical ultracentrifugation was used to determine the concentration dependence of the fraction of monomeric protein. Using absorption to detect equilibrium sedimentation profiles limits the absorbance of the sample to ~ 0.1-1.2 A. For nsHbs, if both the Soret and visible absorption bands are exploited, experiments can be carried out between ~1 and 50 µM in the Beckman equilibrium centerpiece.
Fig. 3B shows the dependence of apparent molecular mass on protein concentration for ferric rHb1. At very low concentrations, the protein appears monomeric, but the apparent molecular mass increases throughout the concentration range accessible in this experiment. The fitted curve in Fig. 3B is a nonlinear least squares fit to Equation 2 with molecular mass substituted for retention time. From these data, fitted values for the equilibrium dissociation constant and the molecular masses for the monomeric and dimeric proteins were 80 µM (±10%), 17,700 Da, and 35,200 Da, respectively. Fig. 3C shows the same data as Fig. 3B, but with the fitted curve extrapolated to higher protein concentrations, indicating the concentration range over which fractional subunit association occurs for ferric rHb1.
Dimerization Equilibrium Constants of Different Ligated States of
rHb1--
Analytical ultracentrifugation was also used to determine
the equilibrium dissociation constants of several different ligated and
oxidation states of wild type rHb1. These values for the
O2, CO, and Fe(III)CN forms of the protein
are listed in Table II.
Equilibrium dissociation constants for O2-, CO-, and
CN-bound rHb1 were slightly larger than that of the ferric protein.
However, deoxy rHb1 associates with a Kd >6-fold
higher than that of ferric rHb1, indicating a much weaker dimerization
interaction.
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Analysis of the Ferric rHb1 Dimer Interface-- In an effort to test the importance of the amino acid side chains in the dimer interface, several of these interface residues were replaced by different amino acids using site-directed mutagenesis. The Ser49 to Ala49 (S49A), Glu119 to Val119 (E119V), and double substitution (S49A/E119V) mutant proteins were used to determine the role in dimerization of the symmetric electrostatic interactions at each end of the subunit interface. The substitutions V46N, V120N, and V46N/V120N were used to determine the importance of the hydrophobic core in dimerization. The quaternary structures of the resulting mutant proteins were analyzed, and rate constants for oxygen and carbon monoxide binding were measured to test the impact of any quaternary structural changes on ligand binding.
Table III lists the observed molecular masses for the mutant proteins as determined by analytical ultracentrifugation. Each protein was assayed over a total protein concentration range of ~10 to ~50 µM. At these concentrations, the wild type protein exhibits a molecular mass significantly larger than that expected for monomeric protein. However, each of the interface disruption mutant proteins has the molecular mass expected for its respective monomers at all measured protein concentrations (Table III).
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Rate constants for O2 and CO binding are given in Table III for each of the dimer interface mutant proteins. There are no substitutions resulting in rate constants that deviate more than 2-fold from those of the wild type protein for any of the reactions measured. In addition, the reactions with wild type rHb1 were measured over a concentration range of 1-50 µM with no effect on rate constants for oxygen or carbon monoxide binding (data not shown).
Laser flash photolysis as a function of CO concentration can be used to
determine the rate constants for binding and dissociation of the
hexacoordinating His73 side chain (23). The rate constants
extracted from a two-exponent fit of CO rebinding after photolysis were
used to extract these values using the following equations (where
1 and
2 are the two rate constants
associated with rebinding).
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(Eq. 3) |
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(Eq. 4) |
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DISCUSSION |
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Quaternary Structure Determination-- rHb1 is a partially associated homodimer in the concentration range in which absorption spectroscopy is used for experimentation (~ 1-1000 µM; Fig. 3C). At a concentration of 0.8 mM, the protein is 90% associated, but at concentrations between 0.8 mM and ~ 5 µM, significant variation in apparent molecular mass is observed. If the nsHb from barley has a similar equilibrium dissociation constant, the data presented here explain the difference between the quaternary states initially assigned to it and rHb1. It is likely that the higher concentrations of barley nsHb used for size exclusion chromatography indicated a molecular mass closer to that expected for the dimeric protein (11). For example, if a protein concentration of 1 mM was used in these experiments, the apparent molecular mass would be ~ 32,000 Da. The first examination of rHb1 quaternary structure used analytical ultracentrifugation, which requires much lower protein concentrations (~ 3 µM). This experiment provided an apparent molecular mass of ~ 19,000 Da, suggesting a monomeric protein (4).
A similar situation has recently been observed for Vitreoscilla hemoglobin. The crystal structure reveals a dimeric protein to which had been attributed the ability to bind ligands cooperatively (25). However, a recent investigation of the solution properties of this protein using analytical ultracentrifugation indicates that it is monomeric when present in micromolar concentrations (26). These experiments have important implications with respect to cooperative ligand binding and potential protein function. The results presented here for rHb1 indicate that it could not possibly bind ligands cooperatively unless it is present at concentrations >1 mM inside plant tissue. It is unlikely that this is the case, as very little nsHb has ever been extracted from plant tissue (9). However, it is difficult to completely rule out the possibility of high local concentrations of protein compartmentalized in small intracellular areas.
The Roles of Quaternary Structure and the Dimer Interface-- Understanding the physiological function of the nsHbs has been the focus of much research over the past several years. Current hypotheses result from the roles of hemoglobins in other systems in combination with biophysical and structural data for nsHbs that limit some of these possibilities. It has been suggested that extremely high oxygen affinities and low dissociation rate constants preclude the nsHbs from facilitating the diffusion of oxygen in the absence of some factor that could increase the dissociation rate constant at least 100-fold (5, 17). The results of the work presented here indicate that quaternary structural changes do not significantly affect ligand binding kinetics or rate constants associated with hexacoordination. Therefore, a change in quaternary structure is not a factor that could implicate the nsHbs as oxygen transport proteins.
Table IV is a sequence alignment of the interface region of rHb1 with several other plant hemoglobins, including Parasponia hemoglobin, which is dimeric (27, 28), and soybean leghemoglobin (Lba), which is monomeric. The residues which create the dimer interface in rHb1 are also found in the nsHbs from soybean, barley, corn, Arabidopsis nsHb1, and Parasponia Hb. However, Arabidopsis contains a second nsHb that is predicted to be incapable of dimerization with respect to the criteria established here for rHb1 because it contains an Ala residue at position 49. The mutation creating a similar interface in rHb1 (S49A) results in a monomeric protein. Presumably Lba dimerization is prevented by the presence of hydrophobic residues at positions 49 and 119.
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Implications for Possible Physiological Function-- The phylogenetic conservation of the dimer interface in nsHbs indicates that it probably has some physiological role. It is possible that, in vivo, the interface serves as a binding site for another molecule and that the low affinity interaction resulting in dimerization is simply the consequence of the single, symmetric binding site on each protein molecule. This would explain why the interface residues are conserved among the nsHbs, but dimerization is a physiologically unlikely event.
The plant nsHbs must undergo conformational change upon ligand binding (17), and are probably capable of binding a partner molecule via the interface region investigated in this work. Therefore, it is possible that nsHbs are molecular sensors that use heme ligation to trigger other physiological events. There are many examples of heme proteins that use coordination and redox properties to affect the behavior of partner molecules. Many of these proteins are heme-based sensors of small diatomic ligands like oxygen, carbon monoxide, and nitric oxide (29). FixL, CooA, soluble guanylate cyclase, and ECDos are examples of heme proteins that undergo a distal ligand displacement that produces conformational changes that affect covalently bound partner molecules (16, 29-31).
There is little change in association equilibrium for
Fe(III)CN, CO, and O2, but there is a 6-fold
increase in the Kd of the Fe(II)deoxy protein
compared with the ferric form. This could indicate that the interface
region of deoxy rHb1 is less likely to interact with another protein
and that ligation of the deoxy protein might trigger binding of a
partner molecule. A similar redox and ligand-dependent
quaternary structure has also been observed in the hemoglobin from
Caudina arenicola, one form of which exhibits
reversible hexacoordination (32, 33).
The possibility of forming heterodimeric nsHbs has not yet been
investigated but is a potentially very important direction of research.
If the and
subunits of human hemoglobin had first been
identified genetically and studied individually as recombinant proteins, quaternary structure and cooperativity would not have been
observed until the subunits were studied together. This limitation of
working with recombinant proteins must be considered when trying to
identify physiological roles of the nsHbs.
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FOOTNOTES |
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* This work was supported by United States Department of Agriculture Award 99-35306-7833.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 515-294-2616;
Fax: 515-294-0453; E-mail: msh@iastate.edu.
Published, JBC Papers in Press, December 5, 2000, DOI 10.1074/jbc.M009254200
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ABBREVIATIONS |
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The abbreviations used are: nsHb, plant nonsymbiotic hemoglobin; Hb, hemoglobin; rHb1, rice nsHb1; HPLC, high pressure liquid chromatography.
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