(Received for publication, December 23, 1996, and in revised form, May 2, 1997)
From the § Department of Physiology & Biophysics, Albert
Einstein College of Medicine, Bronx, New York 10461 and the
Department of Plant Science, University of Manitoba,
Winnipeg, Manitoba R3T 2N2, Canada
A cDNA encoding barley hemoglobin (Hb) has
been cloned into pUC 19 and expressed in Escherichia coli.
The resulting fusion protein has five extra amino acids at the N
terminus compared with the native protein, resulting in a protein of
168 amino acids (18.5 kDa). The recombinant Hb is expressed
constitutively. Extracts made from the bacteria containing the
recombinant fusion construct contain a protein with a subunit molecular
mass of approximately 18.5 kDa comprising approximately 5% total
soluble protein. Recombinant Hb was purified to homogeneity according
to SDS-polyacrylamide gel electrophoresis by sequential polyethylene
glycol precipitation and fast protein liquid chromatography. Its native
molecular mass as assessed by fast protein liquid chromatography-size
exclusion was 40 kDa suggesting that it is a dimer. Ligand binding
experiments demonstrate that 1) barley Hb has a very slow oxygen
dissociation rate constant (0.0272 s1) relative to other
Hbs, and 2) the heme of ferrous and ferric forms of the barley Hb is
low spin six-coordinate. The subunit structure, optical spectrum, and
oxygen dissociation rate of native barley hemoglobin are
indistinguishable from those obtained for the recombinant protein. The
implications of these kinetic data on the in vivo function
of barley Hb are discussed.
The recently discovered nonsymbiotic plant hemoglobins constitute a new class of protoheme proteins that are expressed at low concentrations, on the order of 1-20 µmol per kg wet weight tissue, in non-nodule, often rapidly growing or rapidly metabolizing tissues such as roots, stems, or germinating seeds of monocotyledonous (1) and dicotyledonous plants (2, 3).
Nonsymbiotic plant hemoglobins are functionally and genetically distinct from the familiar symbiotic plant hemoglobins. Symbiotic hemoglobins are expressed in the cytoplasm of bacteriocytes of nodules formed in symbiotic association between legumes and the bacterium Rhizobium (3, 4) or between the actinomyocete Frankia and variety of non-leguminous woody dicotyledonous plants. In a unique instance, the bacterium Rhizobium nodulates a non-legume Parasponia andersonii (3). Although symbiotic hemoglobins show large differences in their geminate reactions, all have in common rapid, almost diffusion-limited combination with oxygen and moderate rates of oxygen dissociation, so that they achieve very great oxygen affinity (5). Typically, nodule hemoglobins occur at up to millimolar concentration in the cytoplasm of the nodule cell, concentrations perhaps 105 times greater than that of free oxygen. These properties, taken together, are ideally suited to facilitate a large flow of oxygen to the symbiosome at free oxygen concentrations as low as 10 nM, the oxygen pressure obtaining in the nodule cell.
Nonsymbiotic plant hemoglobin genes of both monocots and dicots fall into a single coherent gene family, distinct from the family of genes that encode symbiotic hemoglobins (6). In the unique instance of the nodulating tree, P. andersonii, a gene in the nonsymbiotic hemoglobin family encodes a hemoglobin whose pattern of expression and functional properties are those of a symbiotic hemoglobin (5, 6). Since nonsymbiotic Hb has now been demonstrated to occur in two of the major divisions of the plant kingdom, it is likely that a Hb gene is present in the genome of all higher plants (7-10), and it is suggested that most symbiotic Hb genes arise by duplication of preexisting nonsymbiotic Hb genes (6).
Barley (Hordeum sp., a monocotyledonous cereal) nonsymbiotic hemoglobin is expressed in seed and root tissues under anaerobic conditions (1). Since the level of hemoglobin in barley root or aleurone tissue is of the order of 20 µM, we have developed an expression system in Escherichia coli to produce a barley hemoglobin fusion protein that closely resembles the native protein but differs in having five extra amino acids at the N terminus. This report addresses the expression, purification, and characterization of the fusion protein, in particular its reactions with oxygen and carbon monoxide and the configuration of the heme pocket of the ferrous (deoxy) protein. Some properties of native barley hemoglobin are also described. Comparisons with other hemoglobins show that some of the binding properties of recombinant and native barley Hb with gaseous ligands are unique. Two, not necessarily mutually exclusive, functions have been proposed for nonsymbiotic hemoglobins, as sensors of oxygen concentration and as carriers in oxygen transport (6). The results presented here suggest a third possibility, namely electron transfer, possibly to a bound oxygen molecule. To our knowledge this report describes the first characterization of a nonsymbiotic monocot plant Hb.
pUC 19 plasmid (Canadian Life Technologies), into
which barley root Hb cDNA (1) was cloned, was used as the source of
the barley Hb cDNA. Linker-adapters were constructed by the
University of Manitoba DNA laboratory. E. coli strain
DH5- (Canadian Life Technologies) was used as the host for both
recombinant and nonrecombinant plasmids. Subcloning efficiency DH5-
competent cells (Canadian Life Technologies) were used for
transformation and growth of plasmids. Enzymes for DNA manipulation and
agarose were from Canadian Life Technologies. GeneClean II Kit was from
Bio-Can Scientific. All other biochemicals were from Sigma or Canadian
Life Technologies.
pUC 19 plasmid, into
which Hb cDNA had been subcloned from Bluescript (1) between the
restriction sites SstI and XbaI, was used as the
starting material. The procedure resulted in the removal of the
5-untranslated region from the cDNA to minimize the extra amino
acids of the final recombinant protein. The pUC19 ATG was used as the
starting codon. The insert was removed from the plasmid and then
reinserted into pUC19 in such a way as to remove the 5
region and have
the coding sequence in the correct reading frame.
The plasmid containing the insert (approximately 100 µg) was digested for 6 h with SstII (which cuts the insert at 7th and 10th base pair inside the coding sequence) (7) and then dephosphorylated with calf intestinal alkaline phosphatase. The 8-mer (single-stranded adapter, ATCGCCGG) was then phosphorylated and allowed to anneal with the 14-mer (single-stranded adapter, AGCTTAGCGGCCGC) to form a linker-adapter containing an SstII site at one end, HindIII site at the other end, and a NotI site in the middle. The adapter was then ligated to the insert end of the SstII-digested plasmid. The plasmid was then digested with SstI releasing the insert cDNA (with the linker-adapter ligated to it forming a HindIII site at the end). The insert was separated from the plasmid by agarose gel electrophoresis and purified using GeneClean II Kit.
Nonrecombinant pUC19 (approximately 100 µg) was double-digested with
SstI and HindIII and then dephosphorylated. The
insert was then ligated with the nonrecombinant pUC19, and the
resulting ligation mix was used to transform DH5- E. coli
cells.
DH5-
cells were transformed according to the instructions for the Canadian
Life Technologies subcloning efficiency competent cells. Blue-white
screening was unnecessary since all the colonies tested contained the
recombinant plasmid (efficiency = 100%). Colonies were picked
from the agar plates using a sterile loop and transferred to tubes
containing 10 ml of sterile LB (with 150 µg/ml ampicillin). The cells
were allowed to grow overnight. Plasmid DNA was prepared from the cells
using the small scale plasmid preparation protocol from Sambrook
et al. (11). Restriction enzyme digests of the resulting
plasmid DNA confirmed that Hb cDNA insert could be released as
expected by digests using SstI/NotI, SstI/SstII, or SstI/HindIII
(data not shown).
Cells containing the
recombinant and nonrecombinant (pUC 19) plasmids were grown for protein
expression in sterile LB media containing 150 µg/ml ampicillin, 100 µg/ml -aminolevulinic acid, with or without 1 mM
IPTG1 at 37 °C for 6-8 h. The bacterial
cells were collected by centrifugation and frozen at
80 °C until
used.
SDS-PAGE was performed using a Bio-Rad Miniprotean II gel apparatus according to an established protocol (12). Final acrylamide monomer concentration in the 0.75-mm thick slab gels was 15% (w/v) for the separating gel and 4% (w/v) for the stacking gel. For the determination of subunit molecular mass, a plot of RF versus log molecular mass was constructed using the following protein standards: BSA (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (29 kDa), trypsin inhibitor (20 kDa), and lysozyme (14.3 kDa).
Protein DeterminationProtein concentration was measured by the method of Bradford (13) using the Bio-Rad prepared reagent and BSA as standard.
Extraction and Purification of Recombinant Barley HbAll
procedures were performed at 4 °C, and all chromatographic
separations used a Pharmacia FPLC system. All buffers were adjusted to
pH and degassed at 20 °C. Washed cells (5 g, wet weight) were
resuspended in 40 ml of extraction buffer (50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 10% sucrose (w/v), 1 mM
dithiothreitol, 1 mM EDTA, 14 mM
2-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml chymostatin, 10 µg/ml E-64) and
disrupted by three passes through a chilled French pressure cell at
approximately 20,000 p.s.i. The lysate was clarified by centrifugation
at 27,000 × g for 10 min. The supernatant fluid was
diluted to 50 ml with extraction buffer and fractionated with
polyethylene glycol 8000. The red colored 10-22% polyethylene glycol
pellet was redissolved in 30 ml of buffer A (50 mM
Tris-HCl, pH 8.5. 1 mM dithiothreitol, 1 mM
EDTA) and the clarified solution applied at a rate of 1 ml/min to a
column (1.5 × 6 cm) of Q-Sepharose (Pharmacia Biotech Inc.) preequilibrated with buffer A. After thorough washing at 1 ml/min, the
protein was eluted at the same flow rate with a 100-ml linear gradient
of 0-500 mM KCl in buffer A. The fractions eluting around 150-200 mM KCl contained most of the red color and were
pooled and diluted approximately 2.5-3-fold with buffer A. The pooled, diluted Hb sample was then applied at a flow rate of 0.5 ml/min to a
Mono Q HR 5/5 column equilibrated with buffer A. The Hb was then eluted
at a flow rate of 1 ml/min with a 50-ml linear gradient of 0-500
mM KCl in buffer A. The fractions eluting around 160 mM KCL were pooled. Ammonium sulfate was added to the
pooled sample to a final concentration of 30% (w/v) and dissolved. The
sample was then loaded onto a prepacked phenyl-Sepharose column
equilibrated with buffer A containing 30% (w/v) ammonium sulfate. Hb
was then eluted at a flow rate of 1 ml/min with a 50-ml linear gradient of 30-0% (w/v) ammonium sulfate in buffer A. The fractions eluting from the phenyl-Sepharose column were analyzed by SDS-PAGE, and the
most pure fractions were pooled and concentrated to a final volume of
approximately 200 µl and buffer exchanged into PBS (40 mM
KH2PO4/K2HPO4, pH 7.0, 150 mM NaCl) using a Centricon 10 concentrator. The
purified Hb was then either immediately used for analysis or stored at
80 °C until needed.
The native molecular mass of recombinant barley root Hb was determined by gel filtration on a prepacked Superose 12 HR 10/30 column using 0.2-ml sample volumes and 50 mM Tris-HCl, pH 9.0, 150 mM KCl as column buffer. Fractions (0.5 ml) were collected with a flow rate of 0.2 ml/min and assayed for A280 and A412 (absorbance maxima of barley HbO2). The native molecular mass of the fusion protein was determined from a plot of Kav (partition coefficient) versus log molecular mass for the following protein standards: alcohol dehydrogenase (150 kDa), BSA (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (29 kDa), and ribonuclease (14 kDa).
Amino Acid AnalysisPurified native and recombinant hemoglobin underwent electrophoresis using the Bio-Rad minigel system (Ref. 12, see above) and was then transferred using the Bio-Rad system to polyvinylidene difluoride membrane (Bio-Rad). The membrane was then stained using 0.01% Coomassie Blue, 40% (v/v) methanol, and destained in 40-50% methanol until the hemoglobin band was apparent. The Hb band was excised and subjected to amino acid analysis using an Applied Biosystems Model 420 amino acid derivatizer system.
Optical SpectraThese were acquired using a modified Cary model 17 recording spectrophotometer (Aviv Associates, Lakewood, NJ) equipped with an Aviv data acquisition and analysis system.
Hemoglobin ConcentrationTotal heme was determined using a pyridine-hemochromogen assay (14). Hemoglobin concentration was determined by Bradford protein assay or by A412 measurements.
Buffer for Ligand Binding Experiments50 mM potassium phosphate buffer, pH 7.40, containing 0.5 mM EDTA was used throughout. The temperature was 20 °C.
Ligand Reaction RatesThese were measured using an Hi-Tech model SF 61 (Salisbury, UK) stopped-flow apparatus interfaced to an OLIS Data Acquisition/Computation System (On-Line Instrument Systems, Bogart, GA). Rates were computed using either the Olis system or Origins 4.1 (Microcal Software Northampton, MA). Most kinetic experiments used Hb purified by chromatography on Mono Q HR only, omitting the final hydrophobic interaction chromatography step.
Oxygen Combination RateA solution of ferrous barley Hb
(5.8 µM heme in buffer containing a 3-fold molar excess
of dithionite) was mixed rapidly with solutions of oxygen (250-1000
µM) in buffer, and the reaction was followed at 426 and
409 nm, respectively, a minimum and a maximum in the difference
spectrum: HbO2 minus ferrous Hb. Measurements were limited
to the range of 250 µM dissolved oxygen (before mixing)
because autoxidation reactions dominate at lower oxygen pressure.
Apparent first order rates were calculated assuming a single
exponential decay using the Origins 4.1 program.
Solutions of ferrous barley Hb (5.8 µM heme in buffer containing a 3-fold molar excess of dithionite) were mixed rapidly with solutions of carbon monoxide (25-1000 µM in buffer), and the reaction was followed at 428 and 415 nm, respectively, a minimum and a maximum in the difference spectrum: HbCO minus ferrous Hb. Apparent first order rates were calculated assuming a single exponential decay using the Origins 4.1 program.
Oxygen Dissociation RateA solution of barley Hb02 (3.5 µM HbO2; 27 µM free oxygen in buffer) was mixed rapidly with solutions of carbon monoxide (250-1000 µM in buffer containing 0-4 mM dithionite), and the reaction was followed at 419 and 404 nm, respectively, a maximum and a minimum in the difference spectrum: HbCO minus HbO2. To confirm the rate, the solution of HbO2 was mixed rapidly with 2 mM dithionite alone, and the reaction followed at 426 nm, a maximum in the difference spectrum: HbO2 minus ferrous Hb.
Carbon Monoxide DissociationSolutions of barley HbCO (3.2 µM HbCO, 5 µM free CO in buffer), prepared by equilibrating solutions of HbO2 first with CO at 1 atm and subsequently with CO in N2 (PCO = 3.7 torr), were mixed rapidly with solutions of oxygen (680 and 1300 µM in buffer), and the reaction was followed at 419 nm, a maximum in the difference spectrum barley HbCO minus HbO2. The rate was the same at lower HbCO concentration (approximately 1 µM, n = 3). To confirm the findings, solutions of HbCO containing a slight excess of dithionite were mixed rapidly with a solution of NO (1,000 µM, in buffer). The reaction was followed at 418 nm, a minimum in the difference spectrum nitric oxide ferrous hemoglobin minus HbCO.
Partition of Barley Hemoglobin between O2 and COA solution of barley HbO2 (4.7 µM heme in buffer) was equilibrated at 1 atm total pressure with wet gas mixtures containing varying proportions of O2 and CO. Autoxidation was minimal under this condition where the sum of the gas partial pressures was kept large. After equilibration at each gas composition was complete, optical spectra were acquired from 450 to 380 nm. Calculations were made from the sum of changes at 419 and 435.5 nm, respectively, a maximum, and an isosbestic point in the difference spectrum: barley HbCO minus HbO2.
Agar-LB plates of DH5- cells
transformed with the ligation mix produced many colonies. Cells grown
from these colonies when compared with those containing the
nonrecombinant pUC 19 were distinctly more red in color (not shown).
Restriction digests of plasmid DNA preparations from these cells
confirmed the existence of an insert that could be released by the
expected restriction enzymes and had the expected molecular weight
(approximately 800 base pairs, not shown). SDS-PAGE of crude protein
extracts from these cells revealed a major band at 18.5 kDa that was
found only in the lanes corresponding to the cells containing the
recombinant plasmid (Fig. 1A). Addition of 1 mM IPTG to the bacterial suspension had no significant
effect on the 18.5-kDa band (Fig. 1A).
A412 measurements of the crude extract (using a
crude extract from nonrecombinant bacteria as a blank) gave an
estimated concentration of extractable hemoglobin of 0.2 mg/g fresh
weight compared with 4 mg/g fresh weight extractable total soluble
protein. From these figures and visual analysis of SDS-PAGE (Fig.
1A), the fusion protein appears to be approximately 5% of
the total soluble bacterial protein.
Purification of Recombinant Barley Hb
The purification of barley Hb is shown in Table I. Recombinant barley Hb was purified 16.2-fold to a final purity of over 97% as assessed by A412 measurements with a yield of 5%. Purified recombinant Hb was over 95% pure as assessed by SDS-PAGE (Fig. 1B).
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The recombinant Hb has an expected N-terminal sequence of Met-Ile-Thr-Pro-Ser-Leu-Ala-Ala-Ala-Glu as compared with the native Hb N-terminal sequence of Met-Ser-Ala-Ala-Glu and therefore has five additional amino acids. SDS-PAGE of the final purified recombinant protein confirms that the expressed Hb has the expected molecular mass (Fig. 1B). Native molecular mass as assessed by size exclusion chromatography on Superose 12 was determined to be 40 kDa (±4 kDa (n = 3)). This suggests that barley hemoglobin is a homodimer.
The amino acid composition of the native2 and recombinant hemoglobin is shown in Table II, except for glycine which gave unreliable results probably due to the presence of glycine in the electrophoretic transfer buffer. The observed amino acid composition of the native and recombinant Hb were, within the error of the determination, generally in agreement with the expected, deduced composition, based on nucleotide sequence data. Methionine, aspartate, glutamate, and lysine were exceptions. Methionine is noted for being problematic in amino acid determinations. When asparate + asparagine and glutamate + glutamine levels are expressed in combination, the results (Table II) are in agreement with the expected levels. The differences between expected and observed values for lysine are not readily explainable, except to suggest that the differences could have resulted from modifications during the analysis process. It is notable, however, that the levels of lysine are in agreement between the native and recombinant barley hemoglobin. These results suggest that the native and recombinant proteins differ only in the additional five amino acids at the N-terminal end of the recombinant protein.
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The spectral properties of recombinant barley Hb are reported in Table III, and optical spectra are presented in Figs. 2 and 3. The spectra are essentially invariant from pH 6.0 to pH 9.0. To be certain that the spectrum of deoxy-Hb was independent of the route by which the compound was formed, deoxy-Hb was prepared in three ways: 1) reaction of ferric Hb with dithionite, 2) reaction of HbO2 with large excess of dithionite, and 3) removal of carbon monoxide from a solution of HbCO sparged with inert gas (dithionite was present to prevent ferric Hb formation). The optical spectrum was in each case the same. As discussed below, spectra of the ferric and deoxy ferrous forms suggest that these are low spin six-coordinate (6-C) species.
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Overall Oxygen and Carbon Monoxide Combination Rates
Single
rates following apparent first order kinetics were seen at all ligand
concentrations. The rates with each ligand were independent of the
observing wavelength. Above 200 µM ligand concentration (after mixing), the rates of O2 and CO combination are
similar and independent of ligand concentration (Fig.
4). The CO combination rates decline monotonically
toward zero at zero ligand concentration. The maximum combination rate
for O2 (43 s1), determined graphically from
Fig. 4, is close to that found for CO (41 s
1).
Second Order Carbon Monoxide Combination Rate Constant
At the two lowest CO concentrations examined, 12.6 and
25.2 µM CO (after mixing), the observed combination rate
becomes small relative to the maximum rate (Fig. 4), and the initial
concentration of CO exceeds the Hb concentration by 4.3- and 8.6-fold,
respectively. Second order combination rate constants were approximated
as the observed first order rate divided by the ligand
concentration. The average value at these two CO concentrations
is k = 0.57 × 106
M1 s
1 (n = 10).
A single homogeneous kinetic event
(k = 0.0272 s1; n = 5),
independent of both CO and dithionite concentration, was observed in
the replacement of bound oxygen by carbon monoxide in the presence of
dithionite. A single but inhomogeneous rate (k = 0.0243 s
1; n = 2) was observed in the absence of
CO. To eliminate the possibility that dithionite was reacting with
HbO2 without prior dissociation of bound oxygen, the
reaction was followed in the presence of CO alone. The observed rate
(0.013 s
1 at 1000 µM CO) serves to
substantiate the rates observed in the presence of dithionite but is
about 2-fold less and approximately proportional to CO concentration.
Evidently CO does not compete effectively with dissolved oxygen in the
solution.
A single homogeneous rate
(k = 1.10 × 103 s
1;
n = 3) was observed in replacement of bound CO by
O2. In confirmation, using a less pure sample of HbCO, a
single but inhomogeneous rate (1.2 × 10
3
s
1; n = 2) was observed in the
replacement of bound CO by O2 and an inhomogeneous rate
(1.4 × 10
3 s
1; n = 5)
in replacement of bound CO by NO.
A plot of the ratio HbO2/HbCO against the
ratio oxygen partial pressure/carbon monoxide partial pressure was
linear over the range examined (Fig. 5). The partition
coefficient, M, given by the reciprocal of the slope of this
relation, is M = 1.48.
Second Order Oxygen Combination Rate Constant
This was
calculated from the value of the partition coefficient and the three
already established rate constants. The partition coefficient,
M, expresses the relative affinities of ferrous Hb for
O2 and CO: M = KD/LD, where
KD and LD are the equilibrium
binding constants for O2 and CO, respectively.
KD and LD in turn express the
ratios of the respective dissociation and combination rate constants:
KD = koff,O2/kon,O2 and
LD = koff,CO/k
on,CO. Entering
the known values into the expression for M, we obtain an
approximate numerical value for the second order rate constant for
combination of O2 with 5-C ferrous Hb
(k
on,O2 = 9.5 × 106
M
1 s
1).
Native barley Hb was
purified from seeds germinated on Petri dishes for 3 days.2
The native barley Hb was a dimer with subunit molecular mass approximately 18 kDa. The optical spectrum of the native Hb is indistinguishable from the recombinant protein (Table III, Fig. 1). The
oxygen dissociation rate for the native Hb was 0.022 s1
which is virtually identical to the recombinant
protein3 (Table
IV).
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Recombinant barley hemoglobin has been purified to homogeneity and was found to be a homodimer with subunit molecular mass of 18.5 kDa, in contrast to most symbiotic hemoglobins that are monomers (3, 10). The distinct red color of the recombinant bacteria and the purified protein, as well as its absorbance spectra, is strong evidence that the bacteria can properly produce a monocot globin protein capable of binding heme and constitute definitive evidence that the cloned Hb cDNA does in fact represent a Hb gene.
Properties and FunctionThe isolated, recombinant barley hemoglobin displays an optical spectrum in the visible and Soret regions which is very similar to those of many oxygenated hemeproteins (Table III, Fig. 2). An equilibrium oxygen affinity cannot be demonstrated; deoxygenation generates a mixture of species in which the ferric protein predominates. However, reversible replacement of bound oxygen by carbon monoxide to generate a species with the unmistakable and characteristic spectrum of carbon monoxide hemoglobin (Table III, Fig. 2) establishes the identity of the putative oxygenated species.
Ferric barley hemoglobin exhibits an optical spectrum (Table III, Fig. 3) reminiscent of many low spin 6-C ferric hemeproteins such as ferric myoglobin cyanide, isocyanide, sulfide, or hydroxide (15). The optical spectrum is distinct from that expected of a 5-C species (16). The ferric oxidation state is confirmed by dithionite reduction to the low spin 6-C ferrous protein.
Ferrous barley hemoglobin displays an optical spectrum with prominent maxima in the visible region at 529 and 563 nm (Fig. 3). This spectral pattern is strongly reminiscent of a number of 6-C low spin ferrous derivatives with oxygenous, nitrogenous, or sulfurous ligands in the distal ligand position. These include ferrous hemochromogens, ferrous protoheme cytochromes including cytochrome b, ferrous cytochrome c, ferrous myoglobin or horseradish peroxidase cyanides (17), ferrous myoglobin (18), or leghemoglobin (19) nicotinates, and ferrous hemeproteins in which the ligand atom immediately adjacent to the heme iron is oxygen (17).
Ligation of a distal nitrogenous residue to the heme iron to generate 6-C species has precedents in other hemoglobins. For instance, ferric bis-histidyl kidney and soybean leghemoglobins are in temperature-dependent equilibria with their aquoferric forms (20). More dramatically, oxygenated glial hemoglobins of the nerves of the clams Spisula (21, 22) and Tellina (23), both in vivo and in vitro, are in freely reversible equilibrium with oxygen and low spin 6-C species. Normal deoxy derivatives are not seen.
The and
bands of the ferrous barley hemoglobin optical spectrum
are each double, with well resolved maxima at 555 and 563 nm and 527 and 535 nm, respectively (Fig. 3). The Soret band at 426 nm, however,
is narrow with the half-bandwidth (20 nm) expected for a single
species. Such four-banded visible spectra have been encountered in the
ferrous hemochromogens formed reversibly by reaction of mammalian
myoglobins with moderate concentrations of the nitrogenous ligands
hydrazine, pyridine,
-picoline, and especially, nicotinic acid
(pyridine 3-carboxylic acid) (18). The nicotinic acid-myoglobin complex
contains one molecule of nicotinic acid per atom of heme iron (18), and
the pyridine ring nitrogen is ligated to the sixth ligand position of
the heme iron atom of both ferrous and ferric leghemoglobin nicotinates (20, 24). Accordingly, a simple but not unique explanation of the
four-banded spectrum may be that a nitrogenous ligand, most probably
the side chain of an amino acid of the backbone polypeptide chain, may
bind to the heme in two different, slowly interconverting
conformations: one conformer giving rise to absorbance bands near 527 and 555 nm and the other giving rise to the 535- and 563-nm paired
bands (18). Sequence alignment suggests that histidine occupies the
position distal to the heme iron (1), but it remains entirely possible
that other nearby residues may contribute the actual ligand.
Very recently Lamb et al. (25) have prepared a derivative of
low spin ferrous sperm whale myoglobin with a water molecule in the
distal position at 20 K. This species exhibits an optical spectrum very
similar to that of ferrous barley hemoglobin, with a Soret maximum at
428 nm and conspicuously split and
bands near 560 and 530 nm,
respectively.4 This suggests that ferrous
barley hemoglobin may be a low spin 6-C species with water (or similar
oxygen-containing ligand) in the sixth coordination position.
The 6-C nature of the ferrous derivative has interesting consequences for the reactions of barley hemoglobin with ligands. A priori, one would expect a leaving ligand to leave behind a 5-C species and only subsequently could a ligand from the distal pocket attack the heme iron atom to generate a 6-C form. Conversely, an attacking ligand would be expected to react only with the 5-C form, implying prior dissociation of the sixth ligand from the 6-C iron atom.
At the largest attainable concentrations of attacking ligand (0.5 atm,
504 µM CO, 675 µM O2, after
mixing), the rates of combination of CO or O2 with 5-C
ferrous Hb (Reaction 2) may exceed the rate of formation of 5-C
from 6-C ferrous Hb (k+1 of Reaction 1). In this
event, dissociation of the sixth ligand (Reaction 1) will limit the
overall combination rate (k+3 of Reaction 3). This kinetic model indeed fits the facts. At high ligand
concentration, the rates of combination of oxygen and carbon monoxide
with ferrous barley hemoglobin each become independent of ligand
concentration and, within the constraints of the experiment, are
essentially the same (kO2 = 43 s1;
kCO = 41 s
1) (Fig. 4). This
implies a common rate-limiting step, which, we suggest, is conversion
of 6-C to a 5-C ferrous Hb (see Reaction 1).
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The rate of deoxygenation of barley HbO2 is among the
slowest known, exceeded only by Ascaris Hb (Table III). Slow
oxygen dissociation implies stabilization of the bound ligand, commonly
by hydrogen bond formation from a nearby or distal residue to the bound
oxygen, as in soybean leghemoglobin or whale myoglobin (Table III; Ref. 24). In the absence of such stabilization, dissociation is fast, exemplified by Mb (His(E7) Gly), Table IV). Very slow oxygen dissociation may imply further stabilization, for instance in Ascaris Hb (Table IV) by interaction of the bound oxygen
with both the distal residue and a tyrosine in position B10 (26-29). Phenylalanine introduced into position B10 may also interact, decreasing the dissociation rate about 10-fold (30). Sequence alignment
(1) places phenylalanine at position B10 of barley hemoglobin. Indeed
all plant hemoglobins have either phenylalanine or tyrosine at this
position (6), but none appear to interact with the bound ligand.
Parasponia Hb, a nodule hemoglobin encoded by a gene in the nonsymbiotic Hb family, invites comparison with barley nonsymbiotic Hb. Both Parasponia and barley Hb amino acid sequences fall within the cluster of nonsymbiotic hemoglobins (6). Both are homodimers (31). Yet Parasponia hemoglobin, isolated from the root nodule, displays chemical reactivities characteristic of symbiotic hemoglobins (24, 31), which are entirely different from those described here for barley Hb. We consider that subtle and precise local structural changes near the heme may dictate large differences in chemical behavior with conservation of the globin fold and of the particular structural arrangement dictated by the nonsymbiotic amino acid sequence.
Extraordinarily slow dissociation of oxygen from barley HbO2 (t1/2 = 25 s) would appear to rule out reaction paths involving reversible oxygen dissociation. This, in itself, does not rule out facilitation of oxygen diffusion because ligand dissociation from Ascaris myoglobin and from two cytoplasmic hemoglobins in the gill of the clam Lucina, which are believed to facilitate diffusion of their ligands, is also very slow (14, 32). Alternatively, the 6-coordinate conformation of ferrous and ferric barley Hb may suggest a role of barley hemoglobin in electron transfer, possibly to a bound oxygen molecule.
Small amounts of the native Hb were purified, and its subunit structure, optical spectrum, and oxygen dissociation constant were compared with those of the recombinant protein. Similarly, the amino acid compositions of the native and the recombinant Hb were in agreement with their expected deduced compositions based on the nucleotide sequences. All of these properties of the native protein were indistinguishable from those of the recombinant protein, indicating that the characteristics of the recombinant protein adequately reflect those of native barley hemoglobin.
We are grateful to Drs. Brian Fristensky, Joel Friedman, Cyril A. Appleby, and Alberto Boffi for helpful and stimulating discussions and to Doug Durnin for technical assistance and to Dr. A. W. MacGregor for the use of his facilities.