From the Department of Biochemistry,
Faculty of Sciences and Engineering, Laval University,
Quebec G1K 7P4, Canada and the § Department of Physiology
and Biophysics, Albert Einstein College of Medicine,
Bronx, New York 10461
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ABSTRACT |
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We report the optical and resonance Raman
spectral characterization of ferrous recombinant
Chlamydomonas LI637 hemoglobin. We show that it is present
in three pH-dependent equilibrium forms including a
4-coordinate species at acid pH, a 5-coordinate high spin species at
neutral pH, and a 6-coordinate low spin species at alkaline pH. The
proximal ligand to the heme is the imidazole group of a histidine.
Kinetics of the reactions with ligands were determined by stopped-flow
spectroscopy. At alkaline pH, combination with oxygen, nitric oxide,
and carbon monoxide displays a kinetic behavior that is interpreted as
being rate-limited by conversion of the 6-coordinate form to a reactive
5-coordinate form. At neutral pH, combination rates of the 5-coordinate
form with oxygen and carbon monoxide were much faster
(>107 µM Three hemoglobin genes have been detected in the genome of the
green unicellular alga Chlamydomonas eugametos. Two of
these, LI637 and LI410, have been cloned (1).
Both are expressed at the onset of the light period in cells grown
under light/dark regimes. Photosynthesis is required for full
expression of LI637 but not for expression of
LI410 (1, 2). The LI637 and LI410 genes encode proteins of 164 and 171 amino acids, respectively, including at their N termini, leader sequences that serve to target the
proteins to the chloroplast. Within the chloroplast, they are
distributed between the proteinaceous ribulose-diphosphate carboxylase/oxygenase-rich pyrenoid and the thylacoid membrane regions
(1). Their discovery constitutes the first evidence that hemoglobin
occurs in chloroplasts. Chlamydomonas hemoglobins are
induced by light to a final concentration of approximately 130 nM within the chloroplast. As the internal pH of the stroma of the chloroplast is known to range from about pH 6.9 in the dark to
more than pH 8.0 in the light (3), in this investigation we examine
acid, neutral, and alkaline forms of Chlamydomonas chloroplast hemoglobin.
Phylogenetic analysis based on primary amino acid sequences suggests
that Chlamydomonas hemoglobin shares a small gene family with the hemoglobins of the cyanobacteria Nostoc commune (4) and Synechocystis sp. (PCC 6803) (5) and the ciliated
protozoa Paramecium caudatum (6) and Tetrahymena
(7). The three-dimensional structure has not been solved for any of
them. Multiple sequence alignment and comparison with myoglobin
suggests that these hemoglobins have a universally conserved proximal
histidine and a phenylalanine at position CD1 (1) (Fig.
1). All hemoglobins of this family are
predicted to have glutamine in the distal position instead of the more
usual histidine (1).1 Most
are predicted to have tyrosine at position B10 instead of the usual
leucine (1).1 N. commune with histidine at
position B10 is an exception. Optical spectra of ferric
Chlamydomonas (8) and Nostoc (9) hemoglobins at
neutral pH indicate the presence of a low spin 6-coordinate complex,
whereas those of Paramecium (10) and Tetrahymena
(7) hemoglobins are consistent with a high spin 6-coordinate
conformation reminiscent of the aquo ferric species of myoglobin and
hemoglobin.
1 s
1).
The dissociation rate constant measured for oxygen is among the slowest
known, 0.014 s
1, and is independent of pH. Replacement of
the tyrosine 63 (B10) by leucine or of the putative distal glutamine by
glycine increases the dissociation rate constant 70- and 30-fold and
increases the rate of autoxidation 20- and 90-fold, respectively. These
results are consistent with at least two hydrogen bonds stabilizing the bound oxygen molecule, one from tyrosine B10 and the other from the
distal glutamine. In addition, the high frequency (232 cm
1) of the iron-histidine bond suggests a structure that
lacks any proximal strain thus contributing to high ligand affinity.
INTRODUCTION
Top
Abstract
Introduction
Appendix
References
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Fig. 1.
Multiple sequence alignment of
Chlamydomonas amino acid sequence with those of other
hemoglobins and myoglobins. The alignment was made with using a
progressive alignment method (1). Helix position refers to that in
sperm whale myoglobin. The position of the CD1 (*) phenylalanine, the
E7 (+), B10 (^) and F8 (#) residues and the C2 (~) proline is also
indicated. Sperm whale myoglobin (MBSW); human hemoglobin
-chain (HAHU); lamprey hemoglobin (Life Technologies,
Inc.); Aplysia kurodai myoglobin (MBAK);
earthworm hemoglobin Tylorrhynchus (HETY);
Lupinus luteus leghemoglobin (HBLL);
Casuarina glauca hemoglobin (HECA); E. coli flavohemoglobin (HEEC); Vitreoscilla
hemoglobin (HEVT); C. eugametos LI637
(HECH637); Tetrahymena pyriformis hemoglobin
(HETP); P. caudatum (HEPC);
cyanobacterium hemoglobin Nostoc commune
(HECY).
Chlamydomonas LI637 hemoglobin, when expressed in
Escherichia coli from the cloned gene, contains a
non-covalently bound protoheme and forms stable complexes with oxygen
and carbon monoxide (8). The ferric protein forms complexes with
cyanide and azide and also with the less usual ligands dithiothreitol
and -mercaptoethanol (8). Optical spectra of the 6-coordinate
ferrous and ferric forms of the protein suggest structures of the
distal ligand different from those usually encountered in hemoglobins.
In this study, we use optical, EPR, and resonance Raman spectroscopy to
probe the nature of the ligands to the ferrous heme iron of the
expressed protein and of single amino acid substitution mutants. We use stopped-flow spectroscopy to establish the kinetics of the reactions with oxygen, carbon monoxide, and nitric oxide. We present evidence that the bound oxygen molecule forms multiple hydrogen bonds with the
putative distal glutamine and tyrosine 63 (B10) residues that stabilize
the oxygenated structure.
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EXPERIMENTAL PROCEDURES |
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Chlamydomonas Hemoglobin-- In this investigation, we use the monomeric recombinant protein H21 whose size reflects that of the mature Chlamydomonas hemoglobin (16 kDa). This was created by removing the first 24 amino acids of the LI637 protein and by substituting a lysine residue for the unique cysteine residue found at position 41 of the parent protein (8). The first residue of this recombinant protein is thus Thr-25 of the wild type hemoglobin. The H21 protein, which we refer to as Chlamydomonas wild type recombinant hemoglobin and Chlamydomonas hemoglobin in this paper, was purified as described previously (8) with the following modifications. The overall yield of purified hemoglobin was increased 2-3-fold by using freshly transformed E. coli BL21(DE3) cells grown on Luria Bertani medium containing 200 µg/ml ampicillin instead of the previously used concentration of 50 µg/ml. Hemin was included in liquid cultures at a final concentration of 10 mg/liter. The hemoglobin was converted to the ferric form with ferricyanide and desalted over a P6DG column (Bio-Rad) prior to use.
Site-directed mutagenesis was performed on the parent plasmid to create
single amino acid substitutions by the method described previously (8).
The presumed distal glutamine was replaced by a glycine residue with
the primer 5' CTTGGAGCGCCCGACCTTCATGT 3' to create the
mutant hemoglobin Gln-84 Gly; tyrosine 63, presumed to be the B10
residue, was replaced by a leucine residue with the primer 5'
CGATCTTGTTGAGAAACTTGTCCA 3' to create the mutant hemoglobin
Tyr-63
Leu; the lysine 87 was replaced by an alanine residue with
the primer 5' GCGAACTGGGCGGAGCGCTGG 3' to create the Lys-87
Ala mutant; the methionine 81 was replaced by an alanine with the
primer 5' GCTGGACCTTCGCGTCTGTGTTG 3' to create the Met-81
Ala mutant, and the proximal histidine was replaced by a glycine
residue with the primer 5' CAGGTCCTTGCCTGCGGTGCGC 3' to
create the His-111
Gly mutant. The ratios of soluble to insoluble
recombinant proteins were the same for the mutant and the wild type
recombinant hemoglobins. The mutant hemoglobins were purified by the
same method described for the wild type recombinant hemoglobin (8).
Protein purity was monitored by SDS-polyacrylamide gel electrophoresis.
HbO22 was prepared by reduction of the ferric protein with a ferredoxin-based enzymatic reducing system (11). Ferrous Hb was formed by reduction of the oxy or ferric protein with sodium dithionite.
Optical Spectra-- A Cary model 3E spectrophotometer (Varian) equipped with a thermostatically controlled multicell holder or a modified Cary model 17 recording spectrophotometer (Aviv Associates, Lakewood, NJ), equipped with an Aviv data acquisition and analysis system were used to acquire optical spectra.
Resonance Raman Spectroscopy-- The concentration of protein samples used for the Raman measurements was typically 30-100 µM in 100 mM buffer. For deoxy samples, anaerobic Chlamydomonas hemoglobin was reduced by the addition of a freshly prepared dithionite solution to the degassed protein solution. HbCO was prepared by exposing dithionite-reduced samples to either 12C16O or 13C18O in tightly sealed Raman cells. 13C18O gas was a product of ICON (Mt. Marion, NY), and 12C16O was obtained from Matheson (Rutherford, NJ). HbO2 was prepared with an enzymatic reduction system (11) and separated from the proteins of the reducing system by chromatography over a ResourceQ column (Amersham Pharmacia Biotech). Absorption spectra were recorded before and after the Raman measurements to ensure the stability of the species studied.
The excitation source in the Raman studies was the 413.1-nm line of a
krypton ion laser (Spectra Physics, Mountain View, CA). The laser power
was maintained in the 0.5-2 mW range to minimize carbon monoxide (or
oxygen) dissociation. The sample cell was spun at 3000 rpm to avoid
local heating. The Raman scattered light was dispersed through a
polychromator (Spex, Metuchen, NJ) equipped with a 1200 grooves/mm
grating and detected by a liquid nitrogen-cooled CCD camera (Princeton
Instruments, Princeton, NJ). A holographic notch filter (Kaiser, Ann
Arbor, MI) was used to remove the laser scattering. Typically, several
10-s (or 1 min) spectra were recorded and averaged after removal of
cosmic ray spikes by a standard software routine (CCD spectrometric
multichannel analysis, Princeton Instruments, NJ). Frequency shifts in
the Raman spectra were calibrated using indene as the reference. The
accuracy of the Raman shifts was about ±1 cm1 for
absolute shifts and less than ± 0.25 cm
1 for
relative shifts.
Electron Paramagnetic Resonance Spectra-- EPR spectra were obtained at liquid nitrogen and liquid helium temperatures using a Varian E112 spectrometer equipped with a Systron-Donner frequency counter and a PC-based data acquisition program. The low spin ferric-cyanide complex was prepared in 20 mM CHES buffer, pH 9.3, containing 10 mM cyanide, and the spectrum was collected at 9 K. The ferrous nitric oxide complex was prepared in 20 mM CHES buffer, pH 9.3, and the spectrum was collected at 77 and 11 K.
Tryptophan Fluorescence-- Tryptophan fluorescence was measured using an SLM-Aminco 8000 fluorimeter with an excitation wavelength of 280 nm. Emission spectra were recorded from 290 to 450 nm.
Buffers-- Different buffers were used to cover different pH ranges as follows: sodium citrate, pH 4.8; sodium acetate, pH 5-5.5; MES-NaOH, pH 5.7-6.5; MOPS-NaOH, pH 6.7-7.5; TAPS-NaOH, pH 7.7-8.5; CHES-NaOH, pH 8.7-9.5; CAPS-NaOH, pH 9.7-11; and pyrophosphate at pH 10.0. All buffer concentrations were 50 mM and the solutions contained 50 µM EDTA. Except as noted, reaction kinetics and equilibria were determined at pH 9.5 and at a temperature of 20 °C.
pH Dependence of Optical Spectra-- A separate buffer solution at each designated pH was used to prepare the samples for optical measurements. Two concentrated protein stock solutions in 10 mM MES-NaOH, pH 5.8, and CAPS-NaOH, pH 10, were used to demonstrate the reversibility of optical titrations. Each individual diluted solution was reduced anaerobically with a 3-fold molar excess of dithionite to obtain the ferrous proteins. Spectra, recorded at a temperature of 20 °C, were monitored at 3-min intervals and were acquired when they showed no further change after 15 min. The midpoints of the titration curves, pKa values, and the optical spectra of individual components of mixtures containing multiple chemical species were calculated using Specfit software (Spectrum Software Associates, Chapel Hill, NC).
Ligand Reaction Rates-- Reaction rates were measured using a Hi-Tech model 61 (Salisbury, UK) stopped-flow apparatus interfaced to an OLIS Data Acquisition/Computation System (On Line Instruments Systems, Bogart, GA). Rates were computed using the OLIS system.
Oxygen Combination Rate-- Solutions of ferrous Chlamydomonas hemoglobin (5 µM heme in buffer containing a 3-fold molar excess of dithionite) were mixed rapidly with solutions of oxygen (250-1300 µM in buffer), and the reaction followed at 426 and 408 nm, a minimum and a maximum in the HbO2 minus ferrous hemoglobin difference spectrum, respectively.
Carbon Monoxide Combination Rate-- Solutions of ferrous Chlamydomonas hemoglobin (8.5 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 417, 428, and 557 nm, a maximum, a minimum, and a minimum in the HbCO minus ferrous hemoglobin difference spectrum, respectively.
Nitric Oxide Combination Rate-- A solution of ferrous Chlamydomonas hemoglobin (5.0 µM heme in buffer containing a 10-fold molar excess of dithionite) was mixed rapidly with solutions of nitric oxide (30-2000 µM in buffer), and the reaction was followed at 427 nm, a minimum in the HbNO minus ferrous Hb difference spectrum.
Oxygen Dissociation Rate-- Solutions of Chlamydomonas HbO2 (5 µM HbO2; 24 µM free oxygen in buffer) were mixed rapidly with solutions of carbon monoxide (250-1000 µM in buffer containing 0-2 mM dithionite), and the reaction was followed at 420 and 405 nm, a maximum and a minimum in the HbCO minus HbO2 difference spectrum, respectively. To confirm the rate of oxygen dissociation, the solution of HbO2 was mixed rapidly with 2 mM dithionite alone, and the reaction was followed at 420 nm, a maximum in the HbO2 minus ferrous hemoglobin difference spectrum.
Carbon Monoxide Dissociation Rate-- Solutions of Chlamydomonas HbCO (5 µM HbCO; 10 µM free carbon monoxide in buffer) prepared by equilibrating solutions of HbO2 first with carbon monoxide at 1 atm and subsequently with carbon monoxide in N2 (pCO = 3.7 torr), were mixed with solutions saturated with oxygen (1300 µM in buffer), and the reaction was followed at 420 nm, a maximum in the HbCO minus HbO2 difference spectrum.
Rate of Autoxidation-- The rate of autoxidation was determined in 50 mM TAPS buffer, pH 8.0, containing 50 µM EDTA, as described previously (8).
Partition of Chlamydomonas Hemoglobin between Oxygen and Carbon
Monoxide--
A solution of Chlamydomonas HbO2
(2.9 µM heme in buffer, pH 9.5) was equilibrated at 1 atm
total pressure with wet gas mixtures containing varying proportions of
oxygen and carbon monoxide. Autoxidation was minimal under these
conditions where the sum of the gas partial pressures was kept large.
After equilibration at each gas composition was complete, optical
spectra were acquired from 500 to 380 nm. At equilibrium, HbCO and
HbO2 were the only forms present at significant
concentration. Calculations were made from the sum of changes at
420 and 500 nm, wavelengths of the maximum and minimum change in the
HbCO minus HbO2 difference spectrum, respectively.
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RESULTS |
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pH Dependence of the Ferrous Optical Spectrum--
The properties
of the acidic, neutral, and alkaline species may be deduced from their
optical and resonance Raman spectra. Fig.
2 shows that the optical spectra of
ferrous Chlamydomonas hemoglobin show significant changes
over the pH range 5 to 9. When the protein is reduced at pH 5.0, there
is a time-dependent conversion of an initial 5-coordinate
species (max 428 and 556 nm, with a minor contribution
near 520 nm, Fig. 3) to a stable 4-coordinate species (see below) (
max 421, 551, and 580 nm, Figs. 2, 3, and 5a). The optical spectrum at neutral pH
(
max 423, 529 shoulder, and 556 nm, Figs. 2 and
5a) is primarily that of a 5-coordinate species. The optical
spectrum at alkaline pH (
max 424, 528, and 557 nm, Figs.
2 and 5a) is that of a 6-coordinate low spin species.
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In order to obtain the pKa values of the spectral transitions and the pure spectra of the individual species giving rise to these spectral changes, spectra, determined over the pH range 5 to 10.5, were deconvoluted using Specfit software (Figs. 4 and 5). The best fit was obtained using a model comprising three different spectral entities and two pKa values. Trials using a single pKa gave a poor fit to the data, and adding a third pKa did not improve the fit. Fig. 4 shows the fit of the experimental points to the theoretical relation at three different wavelengths. Two inflections are evident in the data taken at 424 nm (Fig. 4a) and at 557 nm (Fig. 4c). The inflection at pH 8.5 is well resolved at these two wavelengths. Both inflections are clearly resolved at 435 nm (Fig. 4b). The midpoints were calculated as pKa = 8.5 and pKa = 6.4. Because the two pKa values are well separated, optical spectra obtained by deconvolution of composite spectra recorded during the course of the titration corresponded closely to those taken at pH 5.0, 7.5, and 9.5 (Figs. 2 and 5a). The calculated proportions of the three ferrous species as a function of pH are given in Fig. 5b.
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We considered the possibility that the protein could be denatured at the pH extremes of our measurements. To test this possibility, we measured the tryptophan fluorescence and observed no significant changes over the pH range studied in this work. This indicated that there was no significant loss of heme from the protein and no significant unfolding of the secondary structure. In addition, we found that the changes in optical absorption spectra of the ferrous protein were completely reversible on cycling the protein over the pH range studied. Furthermore, at pH 7.5, the optical spectrum showed the same mixture of predominantly 5-coordinate species (85% with the balance 6-coordinate and 4-coordinate species), in all buffers used (Hepes, Tris, or phosphate), demonstrating that the buffers have no effect on the heme coordination. This was confirmed at high pH where the same 6-coordinate low spin spectrum was obtained with CAPS, TAPS, CHES, and pyrophosphate buffers.
High Frequency Resonance Raman Spectra of Ferrous Chlamydomonas
Hemoglobin--
The high frequency region (1200-1800
cm1) of the resonance Raman spectra of hemeproteins is
comprised of porphyrin in-plane vibrational modes that are sensitive to
the electron density in the porphyrin macrocycle and also to the
coordination and spin state of the central iron atom (12, 13). The
resonance Raman spectra of the ferrous protein in the high frequency
region was recorded as a function of pH (Fig.
6 and Table
I). In the alkaline form (pH 10.5), the
frequency for the electron density marker line,
4, is
1361 cm
1 and that of
3, which is
coordination-sensitive, is 1492 cm
1. These frequencies
are typical of the Fe(II) oxidation state in the low spin form of
hemeproteins in which the sixth coordination position is occupied by
ligands other than oxygen and carbon monoxide, which are
electron-withdrawing ligands. The neutral form (pH 7.5) shows a
downshift in frequency of
4 to 1355 cm
1,
typical of a deoxy form with no sixth ligand. The frequency of
3 is now 1468 cm
1, also characteristic of
a 5-coordinate high spin heme. In the acidic species (pH 5.0), the
frequencies of
4 and
3 are 1373 and 1499 cm
1, respectively. The high frequency spectrum of the
acidic species is similar to that reported for 4-coordinate ferrous
model compounds (14-16) and for ferric 6-coordinate low spin species
(17). For both coordination states, the iron is expected to reside in a planar heme as there are no non-bonded interactions from axial ligands
in the 4-coordinate case, and they are balanced in the 6-coordinate
case. Consequently, for an intermediate (S = 1) or low
spin planar configuration of a 4-coordinate species with an unoccupied
dx2
y2 orbital, the
frequency of
3 would be similar to that of a
6-coordinate low spin heme complex. Raman spectra of model 4-coordinate
ferrous intermediate spin heme complexes (14-16) are, in fact, very
similar to those observed for ferric 6-coordinate low spin complexes.
To determine the oxidation state of the heme, imidazole was added to
the sample, and the resonance Raman spectrum was measured. A spectrum
was obtained that was characteristic of a ferrous 6-coordinate low spin
heme indicating that prior to the addition of the imidazole the heme
was in its ferrous oxidation state. Thus, we assign the acidic form of
Chlamydomonas hemoglobin as a ferrous 4-coordinate form.
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Low Frequency Resonance Raman Spectra of Ferrous, Oxy, and
Carbonmonoxy Chlamydomonas Hemoglobin--
The low frequency region of
the resonance Raman spectrum of hemeproteins is comprised of several
in-plane and out-of-plane vibrational modes of the heme including heme
propionate modes, vinyl modes, and ligand vibrational modes (13). The
axial ligand vibrational modes arise from electronic coupling of the
ligand to the metal electronic orbitals. The assignment of a ligand
vibrational mode is extremely useful as it directly identifies a
particular ligand and the nature of its interactions with amino acid
residues in the heme pocket. In particular, the Fe-His (proximal)
stretching mode (Fe-His) can be identified in the
5-coordinate deoxy forms of hemoglobins in the 200-250
cm
1 region. The
Fe-His mode of deoxy
Chlamydomonas hemoglobin at pH 7.5 is assigned to the line
at 232 cm
1 (Fig. 7). This
mode is not seen in 6-coordinate ferrous and ferric forms of
hemeproteins. The observation of the Fe-His stretching frequency at
neutral pH indicates the presence of histidine as the proximal ligand
of a 5-coordinate heme. The absence of this line in the resonance Raman
spectrum of ferrous Chlamydomonas hemoglobin at high pH
(Fig. 8) is consistent with
hexa-coordination of the
heme.3
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The low frequency regions of the resonance Raman spectra of the
alkaline form of ferrous Chlamydomonas hemoglobin in
H2O and in D2O are presented in Fig. 8. The two
spectra are very similar except for the band at 335 cm1.
Although this particular mode cannot be assigned to a specific vibration, it may be a porphyrin internal mode or a mode from an amino
acid residue closely coupled to the heme. In D2O, the peak
position of this line undergoes a 6-cm
1 downshift in
frequency as well as a drastic loss in intensity. It appears that the
band at 335 cm
1 experiences resonance enhancement due to
coupling with heme vibrations, but the mode becomes uncoupled in
D2O due to the frequency shift, and it thereby loses its
acquired intensity. Intensity enhancement and a frequency shift of
Raman bands due to vibrational coupling of the heme porphyrin
macrocycle with adjacent chemical species indeed have been shown in the
past (18). We invoke a similar vibrational coupling mechanism in
Chlamydomonas hemoglobin.
To investigate further the nature of the heme pocket of
Chlamydomonas hemoglobin, the carbonmonoxy derivative was
also studied. The Fe---CO stretching frequency is sensitive to the
nature of distal interactions with carbon monoxide and is also
dependent on the nature of the proximal ligand. An added advantage of
studying the carbonmonoxy derivative is that the ligand can be
photolyzed to yield a transient population of 5-coordinate deoxy
species. Fig. 9 shows the resonance Raman
spectrum of the CO adduct in which the line at 491 cm1 is
assigned as the Fe---CO stretching mode and the line at 572 cm
1 is assigned as the Fe---C---O bending mode. Each of
these assignments was confirmed by isotope
(13C18O) substitution experiments (491/482 and
572/559 cm
1, respectively). The C---O stretching mode,
also determined from isotope substitution experiments, is assigned at
1957 cm
1 (1868 cm
1 for
13C18O, data not shown).
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Partial photodissociation of bound carbon monoxide from
Chlamydomonas hemoglobin was achieved with high laser power
(400 mW). It was observed that the intensity of the line at 232 cm1 increases with increasing laser power. Thus, this
line is seen in the deoxy form of the protein produced either during
photodissociation or upon chemical reduction. This observation is
consistent with our assignment of the 232 cm
1 line as the
Fe-His stretching mode of the 5-coordinate deoxy heme. In contrast to
the observed photolability of the carbon monoxide adduct of
Chlamydomonas hemoglobin and the photolability of most oxy
complexes of hemoglobins, we find that in the presence of high incident
beam powers (~600 mW of power at 413.1 nm), the spectrum of the high
frequency region (
4, 1370 cm
1 and
3, 1500 cm
1) of the oxy derivative of
Chlamydomonas hemoglobin shows no formation of a
photodissociated (5-coordinate, deoxy) species. We conclude that this
oxyhemoglobin is not photolabile under our experimental conditions.
Electron Paramagnetic Resonance-- At alkaline pH and in the presence of 10 mM cyanide (~0.8 mM protein), a signal was observed at g = 3.19, consistent with that of a ferric heme cyanide complex with a trans-histidyl ligand. This signal is likely the gmax for the cyanide complex and indicates a low spin form with anisotropy similar to that of myoglobin cyanide (19) but greater than that for the cyanide complexes of horseradish or cytochrome c peroxidases (20). This suggests that the endogenous axial ligand of ferric Chlamydomonas hemoglobin is likely to be a neutral imidazole as in the hemoglobins, rather than one with imidazolate character as in the peroxidases (21).
Upon binding of NO to the ferrous protein at alkaline pH, a rhombic spectrum typical for a 6-coordinate heme-NO complex was obtained (Fig. 10, upper), showing a triplet splitting pattern, characteristics of hyperfine interaction with 14N (I = 1), near g = 2. A derivative (22) of this spectrum (Fig. 10, lower) shows that the hyperfine pattern is a triplet (21-22 G) of triplets (6-7 G). This hyperfine pattern is indicative of interaction of the unpaired electron with two I = 1 nuclei and strongly suggests the presence of two axial 14N ligands to the ferrous iron of the NO complex of Chlamydomonas hemoglobin. The magnitudes of the two sets of 14N hyperfine splittings are comparable to those found for NO-bound ferrous hemoproteins with axial histidine (23, 24). The larger hyperfine coupling (21-22 G) arises from the 14NO nitrogen, whereas the smaller coupling (6-7 G) is from the proximal histidyl imidazole nitrogen. The EPR of Chlamydomonas HbNO is therefore consistent with histidine as the proximal ligand as suggested by resonance Raman studies of the deoxy ferrous and FeCO proteins and by the EPR study of the ferric cyanide protein.
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Oxygen Dissociation--
A single homogeneous kinetic event,
independent of the observation wavelength or of the presence or absence
of dithionite, was recorded upon replacement of bound oxygen by carbon
monoxide. The rate was the same in 10 mM Tris-HCl buffer,
pH 7.5 (0.0141 s1); in 50 mM sodium phosphate
buffer, pH 7.5 (0.0133 s
1); and in 50 mM
CAPS-NaOH buffer, pH 10.5 (0.0140 s
1). The same rate was
observed by trapping dissociated oxygen with dithionite alone. These
data show that dithionite does not react with the oxygen adduct, but
does remove dissociated oxygen. The rates measured in the presence of
dithionite reflect oxygen dissociation per se.
Carbon Monoxide Dissociation--
Inhomogeneous apparent rate
constants could be resolved into a major rate (about 90% of the
total), loff = 2.14 × 103
s
1, and a faster minority rate. The major rate is similar
to those of soybean leghemoglobin and sperm whale myoglobin (Table
II).
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Oxygen, Carbon Monoxide, and Nitric Oxide Combination--
Single
rates following apparent first order kinetics, independent of
wavelength, were observed at pH 9.5 at high oxygen concentrations (>200 µM, Fig. 11) and
were also observed in the combination of nitric oxide and carbon
monoxide with ferrous Chlamydomonas hemoglobin at all
concentrations examined. Oxygen binding measurements at low
pO2 were complicated by competing oxidation of
the ferrous protein to the ferric form. As shown in Fig. 11, the rates
of combination of oxygen, carbon monoxide, and nitric oxide with
alkaline ferrous Chlamydomonas hemoglobin tend toward a
limiting value, ~160 s1, at high ligand concentration.
We propose that the rate of conversion of the 6C alkaline species to a
5C species prior to ligand binding limits the rate of combination with
ligands (see "Discussion").
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The rates of combination with oxygen and carbon monoxide at pH 7.5 were
too fast to measure by stopped-flow spectrophotometry (>107 M1 s
1) in
the range of ligand concentration where pseudo-first order kinetics
could be achieved.
Partition of Ferrous Chlamydomonas Hemoglobin between Oxygen and Carbon Monoxide-- The partition coefficient expresses the relative affinity of the protein for carbon monoxide and oxygen. A plot of the ratio [HbCO]/[HbO2] against the ratio pCO/pO2 was linear over the range examined (Fig. 12). The partition coefficient, M, given by the slope of this relation, is M = 3.70. This value is expressed in terms of the gaseous pressures. A value, M', corrected for the solubility of the gases and expressed in molar terms, is related to M by M' = 1.34 × M = 5.0 (Table II).
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Chlamydomonas Hemoglobin Mutants--
In order to identify the
sixth ligand of the alkaline form of ferrous Chlamydomonas
hemoglobin, four single amino acid substitutions were studied:
Tyr-63 Leu (B10), Met-81
Ala (E4), Gln-84
Gly (E7),
and Lys-87
Ala (E10). A multiple sequence alignment, Fig. 1,
indicates how the positions of these residues were assigned. Optical
and resonance Raman spectra were used to investigate the role of these
residues. Although none of these substitutions allowed us to identify
the sixth ligand, they nevertheless revealed distinctive features of
the heme pocket structure (Table
III).
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First, the 4-coordinate form observed at acidic pH depends on the
presence of these four residues. Indeed, in contrast to the wild type
recombinant protein, none of the mutants showed the 4-coordinate form.
Second, the sixth ligand of the alkaline species is stabilized by both
tyrosine 63 and lysine 87. As shown in Table III, mutagenesis of these
residues has a major disruptive effect on the heme pocket. The Tyr-63
Leu mutant remains 6-coordinate, low spin from pH 5.0 to 9.5, whereas the Lys-87
Ala mutant is always a mixture of a majority
6-coordinate low spin species and a minority 5-coordinate, high spin
species over the whole pH range studied (pH 5.0-9.5). In contrast,
mutation of the glutamine 84 or methionine 81 residues has only minor
effects on the pKa and ligand coordination.
Observations of 6-coordinate species in all of the mutants at alkaline
pH indicate that the sixth ligand in the alkaline wild type form is not
necessarily the same as that present in the various mutants.
Oxygen Dissociation Rates and Autoxidation Rates of Chlamydomonas
Hb Mutants--
The oxygen dissociation rates measured for the
putative distal glutamine mutant, Gln-84 Gly, and the B10 tyrosine
mutant, Tyr-63
Leu, increased by 30- and 70-fold (Table
IV), and the autoxidation rates increased
by 90- and 25-fold, respectively, as compared with the wild type
protein (Table IV). In contrast, another heme pocket mutant, Lys-87
Ala, shows little difference in the oxygen dissociation and the
autoxidation rates compared with those of the wild type protein (Table
IV).
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DISCUSSION |
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C. eugametos chloroplast hemoglobin (encoded by the gene LI637) is expressed in response to light and requires photosynthesis for its full expression (1, 2). Within the chloroplast of the wild type cells, the hemoglobin concentration does not exceed 130 nM, far too little to store a metabolically significant amount of oxygen or to facilitate oxygen movement, except possibly within very limited structural domains. Within the cell, delivery of oxygen to possible oxygen-consuming enzymes will be limited by the rate of oxygen dissociation. It would appear that oxygen dissociation from Chlamydomonas hemoglobin, among the slowest known, t1/2 = 49 s, is far too slow to support a metabolic function requiring dissociation of hemoglobin-bound oxygen. The function of Chlamydomonas hemoglobin remains to be determined.
Sequence alignment, and comparison with myoglobin, suggests that the
proximal heme residue of Chlamydomonas hemoglobin is histidine; the distal residue is glutamine; and tyrosine occupies position B10 (1) (Fig. 1). EPR and resonance Raman spectra reported
here confirm the assignment of histidine as the proximal residue.
Site-directed mutagenesis of the putative proximal histidine residue,
His-111 Gly, leads to expression of an apoprotein, which does not
bind heme (data not shown). Several hemoglobins, many characterized by
very high oxygen affinity, share with Chlamydomonas hemoglobin the simultaneous presence of a distal glutamine and a
tyrosine residue in position B10. Among these are the hemoglobins of
numerous nematodes (25) including Caenorhabditis elegans (25-27) and Ascaris (28-31) and, in addition, a hemoglobin
of the clam Lucina, HbII (31-33). Trematode hemoglobins
which have very high oxygen affinities have a tyrosine residue in
position B10, in addition to tyrosine in the distal E7 position (34,
35). Tyrosine B10 alone, in the absence of a distal glutamine residue, as in the symbiotic and nonsymbiotic plant hemoglobins (36, 37), plays
a poorly understood role in defining oxygen affinity.
Oxy Chlamydomonas Hemoglobin--
Several residues may interact
with the bound oxygen and stabilize it. Site-directed mutagenesis
implicates two distal pocket residues in stabilizing the bound oxygen.
Mutation of the distal glutamine residue, Gln-84 Gly, or of
tyrosine B10, Tyr-63
Leu, increases the rate of oxygen dissociation
30- and 70-fold and increases the rate of autoxidation 90- and 20-fold,
respectively, as summarized in Table IV. We suggest that these two
residues may form hydrogen bonds stabilizing the bound oxygen molecule. The rate of oxygen dissociation from wild type oxy
Chlamydomonas hemoglobin is the same from pH 7.5 to pH 10.5. This suggests that the structure of the oxygen complex does not depend
on protonatable groups titrating within this range.
Instances in which the distal residue forms stabilizing hydrogen bonds to the hemoglobin-ligated oxygen molecule are many (38, 39). Imidazole from histidine plays this role in the majority of vertebrate hemoglobins and myoglobins but may be replaced by glutamine, as in elephant myoglobin (40) or in mutant versions of sperm whale myoglobin (41). When glutamine replaces histidine, hydrogen bond formation from the distal glutamine to the bound oxygen molecule becomes a common theme among invertebrate hemoglobins and myoglobins (38). Among these, Ascaris hemoglobin offers a strong analogy to Chlamydomonas hemoglobin. In Ascaris hemoglobin, tyrosine B10, together with the distal glutamine residue, contributes to a network of hydrogen bonds believed to stabilize the bound oxygen molecule, see Table II (28-31). A still more elaborate network of hydrogen bonds involving the distal glutamine, tyrosine B10, and, in addition, a water molecule, accounts for the very slow dissociation of oxygen from Lucina HbII (31-33), see Table II.
Acidic Ferrous Chlamydomonas Hemoglobin-- The optical spectra of ferrous Chlamydomonas hemoglobin are pH-dependent (Figs. 2, 4, and 5a). This dependence is best fit by a model involving two pKa values (8.5 and 6.4) and three different optical entities. In the following sections we discuss the properties of these species.
At pH 5.0, ferrous Chlamydomonas hemoglobin exists as a stable 4-coordinate species (Figs. 2, 3, and 5a and Table I). Observation of a 4-coordinate heme in Chlamydomonas hemoglobin at acidic pH indicates that the proximal histidine-iron bond is broken. This is unusual because mammalian ferrous myoglobins and hemoglobins retain their proximal ligand in a similar pH range. Hence, the heme pocket structure and strain on the proximal ligand bond must be very different from other hemoglobins and myoglobins. Breaking of the proximal histidine ligation in Chlamydomonas hemoglobin is not caused by protein unfolding, indicated by the absence of any significant increase in the tryptophan fluorescence in the pH range 5-9.5. In addition, the heme does not fall out of the heme pocket, which would also be expected to result in a large increase in fluorescence. The optical absorption spectrum is also consistent with the above suggestion as the Soret band of the 4-coordinate species of Chlamydomonas hemoglobin is located at 421 nm in comparison to highly blue-shifted Soret band (~380 nm) in acid-denatured deoxymyoglobin (42) and model heme complexes in aqueous solutions.4 Thus, in Chlamydomonas hemoglobin in the absence of its axial ligation, the heme is stabilized in the pocket by non-bonded interactions. This is consistent with reports of stable 5-coordinate, hydroxide-bound heme groups in mutants of myoglobin and cytochrome c peroxidase in which the proximal histidine has been replaced by glycine (43, 44). The protein structures of these mutants do not unfold. On the other hand, in sperm whale deoxymyoglobin at low pH (pH < 4), unfolding has been proposed to be a prerequisite for cleavage of the proximal histidine bond (42). Reversible formation of a 4-coordinate species at acid pH has been observed only in wild-type recombinant Chlamydomonas hemoglobin. None of the mutants examined formed this species, suggesting that each of the mutated heme pocket residues contributes toward stabilizing this unique structure.
Neutral Ferrous Chlamydomonas Hemoglobin--
The optical spectrum
of neutral ferrous Chlamydomonas hemoglobin (Fig. 5 and
Table I) is consistent with a high spin 5- coordinate heme as found in
other globins at neutral pH. Furthermore, the resonance Raman spectra
supply strong evidence that the fifth ligand is histidine. A line at
232 cm1 (Fig. 7) is assigned to the Fe-His stretching
mode of this 5-coordinate species (45). Our assignment of this mode is
based on the following lines of evidence. First, the resonance Raman
spectrum in the high frequency region demonstrates that the ferrous
protein is 5-coordinate. It is only under such conditions that the
Fe-His mode appears in the resonance Raman spectrum. Second, EPR of the CN complex and of the NO complex establish that histidine is
coordinated to the proximal position of both the ferric and ferrous
hemes, respectively. Third, on photodissociation of the CO-bound
species, the 232-cm
1 line, which was absent in the
presence of CO, reappeared in the spectrum. The frequency of 232 cm
1 is very high for a hemoglobin under equilibrium
conditions (46). It is the same as in photodissociated hemoglobins
prior to structural relaxation in which there is no strain on the
proximal histidine to iron bond (47), and it is the same in peroxidases
when the proton on the proximal histidine is either absent or strongly hydrogen-bonded to amino acid residues (48). To distinguish between
these two possibilities, we turn to the EPR spectra. The EPR spectrum
of the ferric cyanide complex is consistent with a neutral imidazole
such as that found in myoglobin and is not consistent with the
deprotonated imidazole group. We note that the (first derivative) EPR
spectrum of the FeNO protein (Fig. 10, upper) has poorly
resolved hyperfine splittings commonly observed for NO-bound globins,
whereas for the Fe-NO complexes of peroxidases, it is possible to
resolve hyperfine splittings for both axial nitrogens without the use
of second derivative analysis such as that shown in the lower
part of Fig. 10 (23). Based on the comparison of these two
spectroscopic studies, we conclude that the histidine adopts a
perpendicular orientation to the heme, and there is no strain induced
on the proximal histidine to iron bond in ferrous Chlamydomonas hemoglobin at neutral pH. This should
contribute to a high ligand affinity since the iron can be pulled into
heme plane without the restraints imposed to it by the protein as
occurs in most other hemoglobins.
The spectra of the CO-bound form provide additional evidence in support
of the assignment of a neutral imidazole as the proximal ligand. It is
widely accepted that the Fe-CO and C---O stretching modes display an
inverse correlation because of -electron back-donation from the
d-
(dxz and dyz)
orbitals of iron to the
* orbitals of CO (12, 49). This
results in an increase of the Fe-CO bond order and a concomitant
decrease in the C---O bond order. The correlation between these two
frequencies depends on the nature of the proximal ligand, as the
electron density in the Fe-proximal ligand bond affects the Fe-CO bond
order. The Fe-CO stretching mode, detected at 491 cm
1
(Fig. 9), and the C---O stretching line at 1957 cm
1, are
characteristic of a hemoglobin or myoglobin with an open heme pocket
with no positive groups interacting directly with the bound CO. Most
myoglobin mutants lacking a distal histidine display similar frequency
of the Fe-CO stretching mode (50). The Fe---CO and C---O frequencies
of Chlamydomonas hemoglobin fall on the correlation line
(data not shown) which is characteristic for hemeproteins that contain
histidine as the proximal ligand. The part of the Fe---CO
versus C---O frequency correlation line with the higher
Fe---CO frequencies and the lower C---O frequencies is generally
occupied by peroxidases that have a proximal imidazolate, and the part
of the line with the lower Fe---CO frequencies and the higher C---O
frequencies is occupied by hemeproteins that have a neutral proximal
imidazole (51). The fact that Chlamydomonas hemoglobin falls
on the part of the correlation line with the lower Fe---CO frequencies
and the higher C-O frequencies is consistent with a neutral imidazole
character of its proximal ligand.
In marked contrast to the 6-coordinate alkaline ferrous form which does
not react directly with oxygen, the 5-coordinate ferrous Chlamydomonas hemoglobin observed at neutral pH combines
very rapidly, >107
µM1 s
1, with oxygen and
carbon monoxide. These combination rates are comparable to those of
sperm whale myoglobin, see Table II. The behavior of
Chlamydomonas hemoglobin is analogous to that of rice hemoglobin, where a 5-coordinate mutant species, His(E7)
Leu, reacts more rapidly with oxygen than the native 6-coordinate ferrous species (52). Our finding bolsters the suggestion, made below, that
5-coordinate and 6-coordinate forms are in equilibrium, and only the
5-coordinate form is able to combine directly with ligands.
Alkaline Ferrous Chlamydomonas Hemoglobin-- The clearly resolved optical spectrum of alkaline ferrous Chlamydomonas hemoglobin is similar to those of a number of 6-coordinate low spin ferrous derivatives of hemeproteins and model compounds with oxygen, nitrogen, or sulfur atoms of the distal ligand coordinated to the heme iron. These include ferrous hemochromogens, ferrous cytochromes b and c, barley5 (53) and rice (52) hemoglobins, ferrous myoglobin or horseradish peroxidase cyanides (54), ferrous myoglobin or leghemoglobin nicotinates (55, 56), and aquo ferrous myoglobin (57). The resonance Raman spectra are also consistent with 6-coordinate low spin heme in the alkaline form of ferrous Chlamydomonas hemoglobin.
Although we cannot make a definitive determination of the nature of the sixth ligand in alkaline ferrous Chlamydomonas hemoglobin, we can rule out several possibilities. Occupancy of the sixth coordination position by a component of the buffer is ruled out by the insensitivity of the optical and resonance Raman spectra to the nature of the buffers. Imidazole from histidine is an unlikely candidate, as there are no histidines in the distal pocket based on the amino acid sequence alignment (see Fig. 1).6 Resonance Raman data make hydroxide or water unlikely potential sixth ligands, because spectra taken in H2O (Fig. 8) or H218O were identical. Site-directed mutagenesis of heme pocket residues suggests that the Tyr-63 and Lys-87 groups may either be implicated in formation of the distal ligand of the alkaline ferrous form or may interact with that ligand, perhaps by formation of hydrogen bonds.
The 335 cm1 line in the resonance Raman spectrum of
Chlamydomonas hemoglobin at high pH changes significantly in
D2O (Fig. 8) indicating that an exchangeable proton is
close to or associated with the heme moiety. Such a change has not been
reported in other hemoglobins or myoglobins. The involvement of the
heme propionates is ruled out because the bending mode of the
propionates at 380 cm
1 (13) remains unchanged in both
solvents. We propose that the protonatable group may originate from a
chemical species adjacent to the heme, such as the distal ligand, but
not restricted to it.
Combination of Alkaline Ferrous Chlamydomonas Hemoglobin with Ligands-- Optical and resonance Raman spectra indicate that a 6-coordinate low spin species is strongly favored in an equilibrium between the 6- and 5-coordinate forms at alkaline pH. A priori, an attacking ligand would be expected to react only with a 5-coordinate form, generated by prior dissociation of the sixth ligand from the heme iron atom, as has been observed in ferrous barley and other plant hemoglobins (37, 52, 53). In general, any ligand occupying the heme-binding site must leave before it can be replaced by an incoming ligand (58).
As shown in Fig. 11, the rates of combination of oxygen, carbon monoxide, and nitric oxide with alkaline ferrous Chlamydomonas hemoglobin tend toward a common limiting value at high ligand concentration. A simple interpretation is that, at high ligand concentration, combination of ligand with the minority 5-coordinate species is very rapid, and conversion of the dominant 6-coordinate form to the 5-coordinate form is rate-limiting.
A kinetic model describing this sequence of reactions is presented below in Scheme I (Ref. 59 and references therein).
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(Eq. 1) |
At low ligand concentrations, the combination rate,
k2[L], is small relative to
k1 and is limiting. In principle, this would make it possible to calculate the apparent bimolecular rate constant at
low concentration of ligand. Under this condition, Equation 1 becomes
kobs = (k2k1/k1)[L].
The apparent bimolecular rate constant at low ligand concentration is
therefore
k2k1/k
1. However, in the case of Chlamydomonas hemoglobin, when the
concentration of ligand is sufficiently small to be considered
negligible, smaller than that at the lowest points in Fig. 11,
pseudo-first order conditions are no longer attained, and this
simplified equation cannot be applied. In addition, the apparent
bimolecular rate constant at low concentration of ligand cannot be
obtained from the initial slope of the relation shown in Fig. 11 (see
"Appendix"). Therefore, kinetics of combination with ligands for
Chlamydomonas hemoglobin and other proteins, for which the
overall ligand binding scheme contains a rate-limiting step, are best
described by k1 and the ratio of
k
1/k2.
Distinct, phylogenetically related gene families encode
Chlamydomonas hemoglobin (1) and the nonsymbiotic higher
plant hemoglobins (37, 60). Nevertheless, the two hemoglobin groups share many common properties. These include extraordinarily slow dissociation of oxygen bound to Chlamydomonas hemoglobin and
to barley (53), rice (52), and Arabidopsis (37) nonsymbiotic hemoglobins; 6-coordinate, low spin ferric forms (52, 53); 6-coordinate, low spin ferrous forms (37, 52, 53) in which the sixth
ligand to the heme iron must dissociate prior to combination of the
hemoglobin with oxygen or CO (53); and very rapid autoxidation at low
ligand pressures where the heme iron is not fully
occupied.7 These common
properties are achieved very differently in the two groups. The distal
ligand to the heme iron atom of 6-coordinate ferrous
barley5 and rice (52) hemoglobins is the distal histidine
residue, while in Chlamydomonas, it must be entirely
different, since there is no distal histidine. Likewise, stabilization
of the bound oxygen molecule, with consequent slow dissociation of
oxygen from oxyhemoglobins, is achieved very differently in the two
groups. In nonsymbiotic hemoglobins, e.g. barley
(53),6 rice (52), and possibly Arabidopsis (37)
hemoglobins, the distal histidine residue probably forms a hydrogen
bond to the bound oxygen. In contrast, stabilization of the bound
oxygen of Chlamydomonas hemoglobin requires both the distal
glutamine and tyrosine B10, each contributing to a network of
stabilizing hydrogen bonds. This similarity of behavior suggests that,
under selection pressure, proteins with different heme pocket
structures adapt to achieve similar functional properties.
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ACKNOWLEDGEMENTS |
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We are grateful to Drs. Lindsay Eltis, Robert W. Noble, and Syun-Ru Yeh for helpful discussions.
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FOOTNOTES |
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* This work was supported by Natural Sciences and Engineering Research Council of Canada Grant 06P0046306 and Fonds pour la Formation de Chercheurs et l'Aide à la Recherche Grant 96ER0350 (to M. G.) and by National Institutes of Health Grants GM54806, GM54812 (to D. L. R.), GM40168, and RR02538 (to J. P.).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: Dept. of Biochemistry, Faculty of Sciences and Engineering, Laval University, Quebec G1K 7P4, Canada. Tel.: 418-656-2131 (ext. 4530); Fax: 418-656-7176; E-mail: mguertin{at}bcm.ulaval.ca.
1 M. Guertin and M. Couture, unpublished alignment of Synechocystis hemoglobin-deduced amino acid sequence of open reading frame slr2097 with those of several hemoglobins.
3
The weak line at 228 cm1 in the
high pH spectrum (Fig. 8) is assigned as a porphyrin mode rather than a
weak Fe-His stretching mode based on the high frequency spectrum (Fig.
6b) which indicates a 6-coordinate low spin heme.
4 T. K. Das, unpublished results.
5 H. C. Lee, R. D. Hill, J. B. Wittenberg, and J. Peisach, unpublished observations.
6 If the structure of Chlamydomonas hemoglobin is very different from that of myoglobin and other hemoglobins, then it is possible that one of the two histidine residues of the G helix (see Fig. 1) may be the distal ligand to the heme iron.
7 J. B. Wittenberg and M. Couture, unpublished observations of barley and Chlamydomonas hemoglobins.
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ABBREVIATIONS |
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The abbreviations used are: HbO2, oxyhemoglobin; Hb, hemoglobin; HbCO, carbonmonoxy hemoglobin; HbNO, nitric oxide hemoglobin; MES, 2-morpholinoethanesulfonic acid; MOPS, 3-morpholinopropanesulfonic acid; TAPS, N-[tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid; CHES, 2-(cyclohexylamino)ethanesulfonic acid; CAPS, 3-(cyclohexylamino)-1-propanesulfonic acid; mW, milliwatt.
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APPENDIX |
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For most hemoglobins and myoglobins, combination of the ferrous form with ligands follows the simple Scheme II.
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(Eq. 2) |
For hemoglobins displaying a rate-limiting step in combination with
ligands, such as Chlamydomonas hemoglobin (Fig. 11), an approximation of the apparent bimolecular combination rate constant can
be obtained from the initial slope of the relation only if the apparent
rates measured at relatively low concentration of ligands, where
kobs is not rate-limited by the conversion of
the 6C species to a 5C species, follow a linear relationship. This was
the case for barley hemoglobin (53). For Chlamydomonas
hemoglobin, if a line is drawn over the first four points of the CO
combination data, the apparent straight line obtained crosses the
y axis at a value of ~40 s1, a value too
high to reconcile with the CO dissociation rate of 0.0022 s
1 measured by replacement with oxygen or nitric oxide.
To extract quantitative information from the data, we have curve fit
data with Equation 1 (see "Discussion"), the results are shown in
Fig. 13. Inspection of the curve fit of
the carbon monoxide combination data reveals that the data points at
low ligand concentration do not follow a linear relationship.
Quantitative information obtained from the mathematical treatment of
the data is reported in Table II.
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REFERENCES |
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