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INTRODUCTION |
In the years following the discovery of hemoglobin in the
Gram-negative bacterium Vitreoscilla in 1986 (1), other
hemoglobins and flavohemoglobins have been found in a variety of
microbes, indicating the widespread occurrence of Hb-like proteins in
the microbial world (2-6). However, at present Vitreoscilla
hemoglobin (VHb)1 remains the
most studied of these bacterial hemoglobins, including its potential
use in biotechnological applications. The cellular concentration of VHb
in Vitreoscilla increases roughly two orders of magnitude
(to about 50 nmol heme/g wet weight) when the oxygen concentration of
its growth medium falls to a microaerobic level (7). Enhanced
biosynthesis of VHb is mediated at the transcriptional level by an
oxygen-sensitive promoter that turns on under hypoxic conditions (below
~10% of air saturation) in both its native and recombinant host,
Escherichia coli (8-11). The presence of a relatively large
cellular concentration of VHb under oxygen-limiting conditions suggests
that its primary function is to trap molecular oxygen and facilitate
its transfer to the respiratory apparatus to enable Vitreoscilla to survive under these conditions despite its
being a strict aerobe.
It has been demonstrated through genetic engineering that the
intracellular expression of VHb in various heterologous hosts often
results in the enhancement of cell density, oxidative metabolism, engineered product formation, and bioremediation, especially under oxygen-limiting conditions. Some examples of the in vivo
effects of VHb include (i) increased cell density in recombinant
E. coli and Pseudomonads (9, 12-14), (ii)
increased production of
-amylase in E. coli (15) and
cephalosporin C in Acremonium chrysogenum (16), and (iii)
enhanced degradation of toxic wastes such as benzoic acid
degradation by Pseudomonads (17) and 2,4-dinitrotoluene degradation by Burkholderia (18). Studies conducted so
far on biosynthesis, functional characteristics, and genetic regulation of VHb suggest two possible working models to account for the mechanism
of VHb action. The first one, called the facilitated diffusion
hypothesis (19), implies that the presence of VHb enhances the oxygen
flux to one or both of the terminal oxidases (cytochromes bo
and bd) under hypoxic conditions. This is supported by the
fact that the respiratory activity and ATP production increased in an
E. coli strain that contained VHb relative to a control strain lacking VHb (20). The oxygen-binding properties of VHb are
presumed to contribute to its postulated function; it has a relatively
normal association rate constant (kon) for
oxygen binding, thus showing a relatively high "avidity" for
oxygen, but its rate constant for oxygen dissociation
(koff) is unusually large (21). The relatively
large static equilibrium dissociation constant, Kd,
which differs 10-fold from the one determined kinetically (22), is 6 µM (equivalent to P50 = 3.3 mm).
These oxygen-binding characteristics are consistent with its putative role of sequestering oxygen from the environment and feeding it to the
respiratory terminal oxidases. A possible alternative mechanism of
action of VHb may be that oxy-VHb influences the activity of some key
redox-sensitive component of the cell, which could be a sensor, a
regulator, or even an allosteric site of a respiratory enzyme. Such an
influence could in turn be transduced into an increase in the
efficiency of energy conservation. There is also the possibility that
VHb has a totally different function or more than one function. For
example, it has recently been demonstrated that the related
flavohemoglobin from E. coli is a nitric-oxide dioxygenase
that dioxygenates nitric oxide to form nitrate (23) to protect the cell
from this free radical that can be generated by oxidation-reduction
systems including the respiratory chain.
The proposed function of VHb as an oxygen carrier suggests
that close proximity to the respiratory membrane would enable it to
perform its cellular function most efficiently. It was reported previously that ~40% of the VHb expressed in E. coli is
found in the periplasmic space (24). This result was based on the isolation of periplasmic VHb protein from the recombinant E. coli by lysozyme osmotic shock treatment. Further, a
phoA-VHb fusion study indicated that the N terminus of VHb
may have a transport function. These observations supported the
function of VHb as a facilitator of oxygen transfer by placing it
closest to the environmental source of oxygen. However, the N-terminal
sequence of VHb is not a typical export signal sequence, and it is
known that recombinant protein that is overexpressed in E. coli often ends up in the periplasm or inclusion bodies or is even
secreted from the cell (25). On the other hand, the localization of
oxy-VHb near the respiratory apparatus would be preferable if VHb
facilitates oxygen delivery by generating sufficient oxygen flux and
directly interacting with the respiratory apparatus of the cell. In the homologous mitochondrial protein, cytochrome-c oxidase, the
oxygen-reactive sites are oriented in the membrane on the cytoplasmic
side (26). Thus, a specific cellular location for VHb may be required
for the optimal performance of its cellular function. Because the periplasmic localization of VHb was determined through indirect experimental observations and could be an artifact, we reexamined the
question of the cellular localization of VHb by determining the
location of VHb inside the cell through electron microscopy. This led
to a derivative study of the membrane binding properties of VHb. This
paper shows that the location of VHb in Vitreoscilla and
recombinant E. coli is cytoplasmic and concentrated adjacent to the cell membrane, and evidence is provided that because of its
membrane association properties, it may perform its cellular function
by interacting directly with the respiratory apparatus of the cell.
 |
MATERIALS AND METHODS |
Bacterial Strains, Plasmids, and Growth
Conditions--
Vitreoscilla sp. strain C1, E. coli JM109, and E. coli BL21DE3 carrying various
recombinant plasmid constructs were used for the experimental work.
Recombinant plasmid pUC8:16 carrying vgb has been described
previously (27). Vitreoscilla was routinely grown in PYA
medium (1% peptone, 1% yeast extract, and 0.02% sodium acetate, pH
7.8) at 25 °C. E. coli strains were grown in Luria Broth
(1% tryptone, 0.5% yeast extract, and 1% NaCl, pH 7.5) at 37 °C,
and the oxygen level was adjusted according to the method adopted by
Narro et al. (28) and as described previously (29). The
plasmid, Bluescript KS(+), was obtained from Stratagene and the
expression-secretion vector, pTMNO (30), was used for the generation of
the ompA-vgb gene fusion. E. coli BL21DE3
carrying the ompA-vgb fusion was induced with 0.1 mM isopropyl-1-thio-
-D-galactopyranoside to
induce VHb. Ampicillin was added when needed at 50 µg/ml.
Generation of Antibodies--
The antibodies against VHb were
generated as described (29) and saturated against a crude extract of
E. coli. The specificity of the antibodies was determined by
Western blotting using standard protocol.
Immunogold Electron Microscopy of E. coli Containing
VHb--
Cells were harvested in late log phase for both the high
aeration studies (grown at 200 rpm in 500-ml baffled flasks containing 100 ml of medium) and the low aeration studies (grown at 75 rpm in
500-ml flasks containing 300 ml of medium) by centrifugation (5,000 × g at 4 °C), washed twice with Dulbecco's
phosphate-buffered saline (PBS), and resuspended in 0.2%
glutaraldehyde plus 4% paraformaldehyde for 20 min. Cells were then
dehydrated with a graded series of ethanol and embedded in LR White
resin (polymerization at 55 °C for 24 h). Ultrathin sections
cut with a Reichert Ultracut Ultramicrotome (Leica Reichart Jung,
Austria) were picked up on 200-mesh nickel grids. Nonspecific binding
sites were blocked with 2% skim milk/0.001% Tween 20 in PBS (blocking
buffer). The grids carrying the ultrathin sections were then washed in
0.05% Tween 20 in PBS (washing buffer) and incubated overnight with
rabbit anti-VHb antibody (diluted 1:500 in 1:10 diluted blocking
buffer) at 4 °C. The grids were then washed in the washing buffer
and incubated for 2 h at room temperature with goat anti-rabbit
antibody conjugated to 10-nm colloidal gold spheres (diluted 1:20 in
1:10 diluted blocking buffer). This was followed by washing the grids
in washing buffer and subsequently in distilled water. The sections
were then stained in 2% aqueous uranyl acetate for 40 min in the dark
followed by a final washing with double-distilled water. The grids thus
prepared were examined in a JEOL 1200 EXII transmission electron
microscope (TEM, operating voltage 60-80 kV), and random fields were
photographed. The prints of the micrographs were then made at the
desired magnification for further analysis. Controls included the
labeling of each set of samples with the preimmune serum
(i.e. normal rabbit serum) instead of anti-VHb serum.
E. coli cells not expressing VHb were also included as a
control for the recombinant E. coli.
Estimation of the Distribution of VHb in the Cytosol of E. coli--
Prints of the negatives were made and overlaid with
transparency sheets. Cell outlines clearly delineating the outer and
inner membranes were traced, cut out, and weighed. The gold particles in the cytoplasm and periplasm of each section were counted with the
help of an ×5 magnifying glass. The area of each section was computed
by comparison with the weight of an area corresponding to 100 square
microns cut out from the same transparency sheet. To calculate the
probe density in the two cellular compartments, at least 35 sections
were analyzed per sample, and the results were subjected to statistical
analysis using Sigma Plot (version 3.0) and t tests to test
the level of significance. A more detailed description of the
immunogold labeling procedure and analytical methods are described by
Ramandeep et al. (31).
Construction of ompA-vgb Gene Fusion--
An NcoI
site was incorporated at the N terminus of vgb by PCR using
the oligomer HB-P (below). The C-terminal oligomer HB-3 used for
amplification incorporated a BamHI site. The
sequences of these primers are HB-P
(5'-GCCATGGCAGACCAGCAAACCATTAAC-3') and HB-3
(5'-GGGATCCGTTTTGGCCAACAGCCAAACTGCTGCTGTG-3'). The
restriction sites are underlined.
The amplified product was cloned in pBluescript KS(+), and its complete
nucleotide sequence was checked to validate the authenticity of the
clone. The NcoI-BamHI fragment was then ligated
in frame with the OmpA signal peptide-coding sequences carried on the
expression-secretion vector pTMN (30). The fusion junction of
ompA and vgb was ascertained through nucleotide sequencing.
Preparation of Membranes and Proteoliposomes--
Membranes of
both E. coli and Vitreoscilla were obtained by
lysozyme treatment of the cells as described previously (32). These
were sonicated for 5 min at 25% duty cycle at full power with a
Branson Sonifier Cell Disruptor 35 fitted with a microtip to produce
the inverted membrane vesicles. Phospholipids purified from
Vitreoscilla (33) were used to make liposomes that were produced by suspending 62 mg of the phospholipids in 2.0 ml of 25 mM Tris-Cl, pH 7.2, and sonicating for 1 min at 0 °C.
Cytochrome bo proteoliposomes were prepared using a
modification of a procedure described previously (33) by adding
purified cytochrome bo to the liposomes at a protein/lipid
ratio of 1:150, adding octyl glucoside at a final concentration of
1.25% and slowly stirring at 0 °C for 30 min. The suspension was
diluted with 20 volumes of the Tris buffer and incubated at room
temperature for 20 min. The proteoliposomes were then collected by
centrifugation at 120,000 × g for 2 h at 4 °C
and stored at
70 °C.
Purification of VHb and Cytochrome bo--
The VHb was purified
by a modification of the procedure described previously (34): lysozyme
lysis of Vitreoscilla cells, 45-70% ammonium sulfate
fractionation, DEAE-cellulose chromatography, and Sephadex G-100
chromatography. Cytochrome bo from Vitreoscilla was also purified by the modification of a published method (35). The
membranes obtained by lysozyme treatment were extracted with deoxycholate followed by chromatography first on Bio-Gel A (0.5 M) and then on DEAE-Sepharose CL-6B.
Assay for Ubiquinol-1 Oxidase Activity--
The assay medium
contained 60 mM Tris-Cl, pH 7.7, 400-500 µg of membrane
protein or 0.16 µg of cytochrome bo in the
proteoliposomes, and 10 mM dithiothreitol in a final volume
of 3.65 ml. After a 2-min preincubation at 30 °C, 15 µM ubiquinol-1 was added to start the reaction, which was
monitored polarographically for 4 min at 30 °C using a YSI Model 53 oxygen monitor. The O2 uptake in the absence of membranes
was measured similarly to correct for the autoxidation of ubiquinol-1.
This assay was performed at normal conditions (100% atmospheric
O2 saturation) and hypoxic conditions (18-22% of
atmospheric O2 saturation). Hypoxic conditions were produced by bubbling the reaction mixture (minus the ubiquinol-1) in a
special test tube containing a long needle connected to a nitrogen gas
supply, a tube connected to a vacuum pump, and sealed with cork and
parafilm. Nitrogen gas was bubbled for 5 min, and then the gas phase
inside the test tube was evacuated for 5 min. These steps were repeated
four times, and then the reaction buffer was transferred using another
long needle back to the oxygen monitor to determine the oxygen
concentration and start the reaction with ubiquinol-1.
Determination of Binding of Hbs to Membranes--
This is an
adaptation of a procedure that was originally developed for studying
the binding of ligands to proteins (36). The Sephadex G-50 used in the
original method was replaced by Sephadex G-100, which with its larger
pore size excludes membrane fragments but not Hb from the gel. After
mixing the Sephadex, the Hb, and membrane fragments and allowing the
suspension to settle, the Hb concentration in the supernatant was
determined. If the Hb has a binding affinity for the membrane, the Hb
concentration in the external gel space will be higher in the presence
of the membranes than in their absence. The following equations
summarize the procedure used.
|
(Eq. 1)
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|
(Eq. 2)
|
Hb is the amount of hemoglobin added, Hbo and
Hbi are the amounts outside and inside the gel,
respectively,
is the ratio of the concentration of membranes (in
terms of protein content) outside the gel to that of the membrane
solution added to the dried gel, and
is defined as the ratio of the
hemoglobin concentration outside the gel to that of the solution of
hemoglobin added.
is determined by plotting the amount of membrane
fragments outside the gel versus the amount of membrane
fragments added, as illustrated in Fig. 1
for Vitreoscilla membranes, and
is determined as shown in Fig. 2 for control horse
hemoglobin. Because
|
(Eq. 3)
|
it can be shown that the concentrations of the bound and free
hemoglobin outside the gel are
|
(Eq. 4)
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|
(Eq. 5)
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where
' is defined as
in the absence of the
membranes.

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Fig. 1.
Determination of for Vitreoscilla membranes. See
"Materials and Methods" for experimental details.
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Fig. 2.
Determination of for control horse Hb. See "Materials and Methods" for
experimental details. a, no membranes added; b,
0.7 mg of protein/ml of Vitreoscilla membranes added;
c, 1.4 mg of protein/ml of Vitreoscilla membranes
added.
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The final operational equation is a modified Scatchard equation,
|
(Eq. 6)
|
where R = Hbo{bound}/mg
membrane, Kd = dissociation constant for the
hemoglobin binding to the membranes, Hbo{free} = concentration of unbound hemoglobin, and N = the
maximum number of binding sites per mg membrane.
Experimentally, 50 mg of the dried Sephadex G-100 was suspended in 1.5 ml of 0.1 M potassium phosphate, pH 7.2, and allowed to
swell for 4 h at room temperature, and then 0.5 ml of the buffer solution containing the membranes and/or globins was added. This suspension was incubated for 10 min at room temperature with continuous stirring, the resin was allowed to settle for 10 min, and a 100-µl aliquot of the supernatant was removed. The globin concentration was
determined by using heme absorption at A410
(179, 188, and 214 mM
1 cm
1 for
equine Hb, Mb, and VHb, respectively), membrane protein using A280 (E = 0.675 ml
mg
1 cm
1, corrected for globin protein when
present), and liposome concentration by measuring the inorganic
phosphate released after total hydrolysis using the Fiske-Subbarow
colorimetric method (37).
 |
RESULTS |
Immunolocalization of VHb in Vitreoscilla--
A previous study of
VHb indicated that this bacterial hemoglobin is partially transported
into the periplasmic space both in its native host,
Vitreoscilla, and recombinant host, E. coli (24).
The question of the subcellular location of VHb was addressed further
by tracing it in Vitreoscilla using immunogold electron microscopy. Vitreoscilla cells grown under high and low
aeration were probed with an anti-VHb antibody and examined with an
electron microscope. Vitreoscilla cells cultivated at low
aeration had a significantly higher level of labeled VHb (as compared
with aerobically grown cells), which was predominantly cytoplasmic (Fig. 3). The standardized results
indicated that more than 90% of the VHb was confined to the cytoplasm
(Table I), much of it adjacent to the
cytoplasmic membranes; of the total signal in the cytoplasm, 57% of
the VHb-bound gold particles were found localized within 0.1 µm of
the inner membrane (Table II).

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Fig. 3.
Immunolocalization of VHb in
Vitreoscilla cells grown under low aeration probed
with anti-VHb antibody (a) or preimmune serum
(b). Arrow heads, gold
particles; cy, cytoplasm; p, periplasm;
bar, 0.5 µm.
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Immunolocalization of VHb in Recombinant E. coli--
VHb
expressed in E. coli through multicopy plasmids usually
amounts to 5-10% of the total cellular protein. Because an osmotic shock method used to release periplasmic protein in recombinant E. coli indicated the partial transport of VHb into the
periplasm, we reexamined the location of VHb in recombinant E. coli using immunogold electron microscopy. Cells grown under high
and low aeration were probed with gold-labeled anti-VHb. The expression of VHb in E. coli led to an almost exclusive cytoplasmic
labeling without any significant signal for the presence of VHb in the periplasm (Fig. 4 and Table
III). These results coincided with the
observations made on Vitreoscilla as did the localization of
the VHb adjacent to the cytoplasmic membrane: 54% within 0.1 µm of
the inner membrane (Table II). Because VHb is so highly expressed in
E. coli, the immunogold signals were much stronger than in
Vitreoscilla (Figs. 4 and 3, respectively). Although the primary goal of the immunogold labeling procedure was localization and
not quantitation, the efficiency of the labeling can be roughly estimated from the section thickness, 70 nm as indicated by the interference color of the sections, and the gold particle density, 27/µm2. Assuming a concentration of 25 nmol of
dimeric VHb/g wet weight of cells, the efficiency was only ~3%. This
probably underestimates the efficiency, however, because a large
fraction of the slices (~40%) inexplicably had few or no particles,
whereas some had over 10 times the number of particles per
µm2.

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Fig. 4.
Immunolocalization of VHb in E. coli carrying vgb. a, under
high aeration probed with anti-VHb antibody; b, under low
aeration probed with an anti-VHb antibody; c, cells probed
with preimmune serum. Gold particles are indicated by arrow
heads, especially in the section marked with a.
cy, cytoplasm; p, periplasm; bar, 0.5 µm.
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OmpA-VHb Fusion and Periplasmic Transport--
To
establish that VHb can be transported via a full-fledged transport
signal and to monitor the effect of accumulation of periplasmic VHb on
the physiology of its host, an OmpA-VHb fusion was created, and
localization of VHb was monitored through immunogold electron
microscopy. In contrast to native VHb, a high level of periplasmic VHb
accumulated via OmpA-VHb fusion in E. coli (Fig. 5). Growth characteristics and oxygen
uptake of E. coli carrying the OmpA-VHb fusion were found to
be similar to E. coli carrying native VHb, indicating that
accumulation of VHb in the periplasm does not confer an advantage over
cytoplasmic VHb.

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Fig. 5.
Immunolocalization of the
ompA-vgb fusion in E. coli. a, probed with anti-VHb antibody
1.5 h after induction; b, probed with anti-VHb antibody
3 h after induction; c, probed with preimmune serum.
Arrow heads, gold particles; cy, cytoplasm;
p, periplasm; bar, 0.5 µm.
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Binding of VHb and Control Equine Hb and Mb to Bacterial
Membranes--
A plot of 1/R versus
1/Hb{free} is illustrated in Fig. 6
for VHb binding to Vitreoscilla membranes. The
Kd is obtained from the slope, and N (the
maximum number of VHb molecules bound/mg of membrane protein) is
obtained from the intercept. Table IV summarizes the data for the binding of three different Hbs to three
different membrane preparations. From these data, VHb has ~4-8 times
higher affinity for Vitreoscilla membranes than the control
equine Hbs have for these membranes. Surprisingly, VHb also has about
the same high affinity for E. coli membranes. To test
whether there are binding sites accessible only on the cytosolic side
of the plasma membrane, membrane vesicles were prepared from the
membranes by sonication, which effectively reverses their topology.
However, there was essentially no difference in binding of the VHb
to these inside-out membranes compared with the periplasmic side-out
membranes (Table IV). Neither of the horse globins showed any
significant difference in binding to these two Vitreoscilla membrane preparations.

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Fig. 6.
Plot of 1/R versus
1/VHb{free} for VHb binding to Vitreoscilla
membranes. The y intercept is 1/N, and
the slope is Kd/N.
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|
Although the horse globins have a lower binding affinity than VHb to
all three membrane preparations, there seem to be more binding sites
for them with N being around 32 nmol/mg of membrane protein
for each of them versus only 7 nmol/mg of membrane protein for VHb. This indicates that horse Mb and Hb bind to a number of
nonspecific sites with relatively low affinity, whereas VHb interacts
with a limited number of higher affinity sites. Likely sites of
interaction would be components of the respiratory chain. This
possibility was examined using proteoliposomes containing purified
Vitreoscilla cytochrome bo, which had been
purified previously in our laboratory.
Binding of VHb and Control Equine Hb and Mb to Cytochrome bo
Proteoliposomes--
For this experiment, horse Mb and Hb were again
used as control Hbs, and liposomes without cytochrome bo
incorporated were used as control for the proteoliposomes. The results
(Table V) indicate that this terminal
oxidase may be one of the sites in the membrane for VHb binding.
Compared with the control equine globins, VHb had 5-11 times the
binding affinity for the cytochrome bo proteoliposomes.
VHb also had 2.4 times the affinity for the proteoliposomes than
for the control liposomes; but more significantly, N, the
number of binding sites, averaged 0.21 nmol/mM phospholipid phosphate for the proteoliposomes and 0.034 nmol/mM
phospholipid phosphate for the control liposomes.
Effect of VHb on the Respiratory Activity of
Membranes--
Because the above results indicated that VHb interacts
with bacterial membranes, a logical next step was to test the effect of
VHb on the respiration of these membranes. For this, we used their
ubiquinol-1 oxidase activity and looked at the effect of VHb at both
normal and hypoxic conditions (Figs. 7
and 8, respectively). VHb stimulated this respiratory activity in all
cases tested, the largest effect being for inside-out
Vitreoscilla membranes, especially under hypoxic
conditions.

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Fig. 7.
Effect of VHb on the ubiquinol-1 oxidase
activity of Vitreoscilla membranes,
Vitreoscilla membrane vesicles (sonicated membranes),
and E. coli membranes under saturating aerobic
conditions (236 µM
O2). VHb (0.7 µM) increased the oxygen
uptake of Vitreoscilla membranes by 11%,
Vitreoscilla membrane vesicles (sonicated membranes) by
42%, and E. coli membranes by 12%. Values are averages of
three individual measurements. Error bars, standard error of
the mean.
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Fig. 8.
Effect of VHb on the ubiquinol-1 oxidase
activity of Vitreoscilla membranes,
Vitreoscilla membrane vesicles (sonicated membranes),
and E. coli membranes under hypoxic conditions
(47 µM O2). VHb
(0.7 µM) increased oxygen uptake by 26% in
Vitreoscilla membranes, by 68% in Vitreoscilla
membrane vesicles, and by 59% in E. coli membranes. Values
are averages of three individual measurements. Error bars,
standard error of the mean.
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|
 |
DISCUSSION |
A model for studying oxygen transfer to living cells is the
Vitreoscilla-VHb system, the VHb being synthesized when
environmental oxygen becomes limiting. How the presence of VHb enables
the bacterium to grow better under oxygen limitation is not
conclusively known, but knowledge of its subcellular localization is
vital to understanding its function. The ultrastructural studies
described here have established the cytoplasmic location of hemoglobin,
both in Vitreoscilla and recombinant E. coli
overexpressing VHb. Although cells grown under low oxygen synthesize
large amounts of VHb in E. coli, little VHb-specific signal
was detected in the periplasm, a result in contrast to a previous
study, which indicated that ~40% of the VHb in E. coli
was located in the periplasmic space (24). It might seem that a
periplasmic location would have the advantage of locating the VHb
closest to the source of environmental oxygen. However, the
accumulation of large amounts of oxy-Hb in the relatively small volume
of the periplasm may change the redox environment of the periplasm,
which may not be favorable to the cell. On the other hand, the presence
of VHb in the cytosol may provide an oxygen buffer, and its subcellular
localization in close proximity to the cell membrane could facilitate
oxygen transfer to the oxygen-binding sites of the terminal oxidases
that are oriented toward the cytoplasm (26, 38). Thus, a cytoplasmic
localization of VHb in the proximity of respiratory membranes, as
demonstrated in the present study, is consistent with its proposed
function to feed molecular oxygen to the respiring membranes under
oxygen limitation.
Although the detection of VHb in the periplasmic fraction in the 1989 study (24) could have been an experimental artifact of VHb leakage
through the membranes during osmotic shock, it is more likely the
result of overproduction and extrusion of the VHb, because the
extrusion of overproduced cytoplasmic recombinant proteins into the
periplasmic space has often been observed (25). Generally, true
periplasmic proteins are compartmentalized in the periplasmic space and
not divided between there and the cytoplasm. In the work reported here,
virtually no VHb was detected in the periplasmic space, but VHb leakage
from recombinant E. coli has occasionally been observed
while washing harvested cells with buffer.2 The extrusion of VHb
could be a function of growth medium, growth phase, physiological state
of the cells, the strain of E. coli bearing the
vgb, other genetic determinants, etc.
Based on the finding of VHb in the periplasmic space, it was proposed
(24) that the first 16 N-terminal residues of VHb may be an export
signal, but this N-terminal sequence does not conform to the usual
signal-peptide characteristics, and it was not excised during
transport; further, unlike most transport signals, it is important for
the protein function. The first 16 residues of VHb are an integral part
of the A-helix structure (39), and a VHb mutant lacking the first 14 N-terminal residues is incapable of binding
heme.3 In the present study,
VHb was determined to have a cytosolic location, raising the question
of whether VHb can be transported into the periplasmic space and
whether a periplasmic location is in any way superior to a cytoplasmic
localization. The OmpA-VHb fusion study clearly suggested that there is
no hindrance to the transport of VHb across the membrane by the
structure or folding of the protein per se, because ~50%
of the peptide-VHb fusion protein was localized in the periplasm. This
enhanced periplasmic localization did not provide any distinct
advantage over the cytoplasmic accumulation of VHb under oxygen-limited
growth conditions. However, the lack of an effect could be caused by
the accumulation of nonfunctional apoprotein in the periplasmic space,
which has been observed for E. coli flavohemoglobin (40).
If the primary function of VHb is to trap and feed oxygen to the
membrane-bound terminal oxidases, it may actually associate with the
cellular membrane to facilitate this process. Initial evidence for this
association was the observation that the respiratory membranes of lysed
Vitreoscilla cells retained significant amounts of VHb, and
extensive washing of the membrane fragments was required to remove it
(35). The study of VHb binding to bacterial membranes in the present
work found Kd values that were in the micromolar
range, 5.4-6.5 µM (Table IV), which were 4.2-8.3 and 4.1-8.6 times greater than the affinities of the control equine globins for Vitreoscilla and E. coli membranes,
respectively. Assuming a value of 50 nmol of VHb heme/g wet weight of
bacterial cells (7-9), it can be estimated that there will be about
15,000 molecules of dimeric VHb per bacterial cell. From the
N value of 7 nmol of binding sites/mg of membrane protein,
it can be estimated that there are roughly four times as many VHb
binding sites (69,000) in the membrane of a single bacterial cell as
there are VHb molecules in the cell. For a Kd of 6 µM, this would indicate that most of the VHb would be
bound (94%). For comparison, the Kd for human Hb
binding to human red blood cell membranes is 1.0 × 10
4 M, which was considered physiologically
significant (41). Thus, the affinity of VHb for bacterial membranes is
likely to be important physiologically and is very probably the reason
for the subcellular localization of VHb adjacent to the cellular membrane.
The binding of VHb could involve interactions of residues with membrane
lipids and/or specific membrane proteins. In the globin domains of
flavohemoglobins, which are structurally similar to VHb, Lys-11 is
generally conserved (39). Thus, a hydrophobic N terminus carrying a
positively charged residue (Lys-11 in VHb) could play a role in the
interaction of VHb with the membrane. Lys-11 is an obvious target for
site-directed mutagenesis to test whether it plays any role in the
membrane interactions of VHb. Whatever the mechanism by which VHb
associates with membranes, binding studies with proteoliposomes
implicated an interaction with a specific protein, the
cytochrome-bo terminal oxidase. When this protein was
incorporated into liposomes, the affinity for VHb increased 2.4 times
relative to the control liposomes without the cytochrome, and the
number of binding sites for VHb increased 6.1-fold to 0.21 nmol/µmol
of liposomal phospholipid phosphate. Using this latter value and
assuming a molecular mass of 700 daltons for a phospholipid
molecule, there are 4,760 molecules of phospholipid for each binding
site. Because the proteoliposomes were made using a cytochrome
bo/phospholipid ratio of 1:150 (weight/weight), there will
be ~1 cytochrome bo incorporated for every 2,240 phospholipid molecules, about twice as many protein molecules as
binding sites. Although these estimates may be fortuitous, previous
work has indicated that the cytochrome bo in these
proteoliposomes is randomly oriented, i.e. 50% in the
inside-out and 50% in the outside-in orientation (33). Thus, there may
be preferential binding to one side of the cytochrome. Although the
binding experiment with inverted membrane vesicles (Table IV) was
inconclusive, VHb did stimulate the ubiquinol-1 respiratory activity of
inverted membrane vesicles significantly more than that of periplasmic
side-out membranes (Figs. 7 and 8), suggesting a preference for
cytosolic side binding.
Although the results of this report are consistent with the proposed
role of VHb being to facilitate oxygen transfer to the terminal
respiratory apparatus under hypoxic conditions and increase respiratory
efficiency, they do not exclude other possible functions for this
protein. For example, there is evidence that it can function as a
terminal oxidase under some conditions (42). The related flavohemoglobin from E. coli has been demonstrated to be a
nitric-oxide dioxygenase that dioxygenates this free radical to form
nitrate (23). This enzyme is inducible under oxygen-limiting conditions and protects the cell from the nitric oxide, which can be generated by
the respiratory chain and other oxidation-reduction systems, probably
more so under the more reducing conditions of hypoxia. Whether VHb (in
conjunction with its flavoprotein metVHb reductase) has a similar role
remains to be tested.