From the Laboratoire de Biochimie des Porphyrines, Institut Jacques
Monod, Université Paris 7, 2 place Jussieu,
75251 Paris, France
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INTRODUCTION |
Hemoglobins (Hbs)1 are
very ancient hemoproteins that are widely distributed in nature; they
are found in all groups of organisms, including many invertebrates,
plants, fungi, and bacteria (1). They all bind reversibly molecular
oxygen, but their avidities vary greatly. The hemoglobin from the worm
Ascaris binds oxygen 4 orders of magnitude more tightly than
does human Hb (2). Some hemoglobins bind other molecules besides
oxygen; the Hbs from the mollusc Lucina pectinata (3) and
the deep-sea hydrothermal vent worm Riftia pachyptila (4)
bind hydrogen sulfide and transport it to the sulfur-oxidizing
bacterial symbionts which feed them. Hbs also vary greatly in size and
composition, from the single-domain monomeric myoglobin to the complex
heteromultimeric Riftia Hbs (4); but their heme-binding
domains all seem to have retained the characteristic "globin fold,"
although there are subtle variations in the amino acid residues lining
the heme pocket, and these are responsible for the differences in their
properties (Refs. 1-3 and references therein).
Hb is normally involved in oxygen transport, storage, and facilitated
diffusion, as is well established in animals. In plants, the
specialized leghemoglobins transport oxygen in the nodules during
symbiotic nitrogen fixation, while the nonsymbiotic Hbs presumably
facilitate oxygen diffusion in actively respiring cells (5). These Hbs
are expressed in several tissues with high metabolic activity (5) and,
in barley, they are induced by hypoxia and by inhibitors of respiration
and oxidative phosphorylation (6). The homodimeric Hb from the
obligately aerobic bacterium Vitreoscilla, whose synthesis
is strongly stimulated in hypoxic conditions, probably plays a role in
facilitation of oxygen diffusion or in oxygen delivery (7, 8). The
overproduction of Vitreoscilla Hb in such hosts as
Escherichia coli (9), yeast (10), and plants (11), enhanced
growth metabolism by increasing the availability of oxygen and/or the
supply of ATP in the cells.
A class of intriguing Hbs, the flavohemoglobins, has recently been
described in yeast and in several bacteria. These "two-domain" globins contain an N-terminal globin module fused to a reductase module
that is homologous to the ferredoxin-NADPH reductase family, with one
FAD and one NAD(P)H-binding domains (12-18). Their physiological function remains uncertain and the published data on them are often
confusing. The flavoHb in Erwinia chrysanthemi is essential for pathogenicity (16). The flavoHbs of the denitrifier
Alcaligenes eutrophus, Bacillus subtilis, and
E. coli are all induced by nitrate and nitrite under
anaerobic conditions (15, 17, 19); nitric oxide also induces expression
of the E. coli flavoHb (19). These findings suggest a role
of these flavoHbs in the anaerobic metabolism of nitrogen oxide
compounds, but the way in which they act is not known. In
vitro studies on purified E. coli flavoHb suggest that
it may function as an NADH oxidase (20), with the release of superoxide
anions (21), or also as a reductase (22), acting perhaps as an oxygen
sensor in vivo. E. coli flavoHb has also been
implicated in oxidative stress: it produces superoxide in vivo when overproduced (21) and its expression is induced by the
redox cyclic agent, paraquat, which generates intracellular superoxide
(23).
Hb was first identified in yeasts by Keilin (24). The flavoHb of
Candida mycoderma has a very high affinity for oxygen, similar to that of cytochrome oxidase but a thousand times higher than
that of human Hb (but with same oxygen dissociation rate constant);
there is no evidence that it facilitates oxygen diffusion in the cells
(25, 26). In Saccharomyces cerevisiae, the expression of the
gene YHB1, encoding the flavoHb in this yeast, is regulated by the growth phase and oxygen in a manner opposite to that found in
most bacteria, suggesting that it has a different function in this
yeast (27). The fact that cells deleted of YHB1 become sensitive to some conditions promoting oxidative stress, and that these
conditions also enhance YHB1 expression suggest that Yhb1p contributes to the response to oxidative stress (28). This study explores the regulation of YHB1 gene expression by
respiration and by different agents that cause oxidative stress
(H2O2, diamide, menadione, DTT,
Cu2+), and the effect of a lack or excess of Yhb1p on
sensitivity of cells to these various stresses.
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EXPERIMENTAL PROCEDURES |
Yeast Strains and Plasmids--
The S. cerevisiae
strains used in this study are described in Table
I. The rho
derivatives were prepared by growing the cells in the presence of
ethidium bromide (35). The YHB1 gene (reading frame YGR234w of chromosome VII (36)) was disrupted by "
-transformation" (31)
using routine DNA manipulation procedures (37). Two DNA fragments
(nucleotides
679 to +7 and +990 to +1202; the A of the initiating ATG
is +1) flanking the coding region were amplified by polymerase chain
reaction using yeast genomic DNA, and cloned into the plasmid pRS403
(31) in tandem, but in reverse order, leaving an EcoRI site
between them. After linearization at this site, the construct was
transformed into yeast cells by a modified lithium acetate method (38).
Integration of the deleted/disrupted yhb1-
1::HIS3 allele at the proper locus was
confirmed by polymerase chain reaction analysis of whole cells
(39).
The plasmids pADE/YHB1 and pPGE/YHB1 for overproducing Yhb1p were
constructed as follows. The coding region of YHB1 was
polymerase chain reaction-amplified from yeast genomic DNA using
forward (5'-CGCGGATCCATTCATTatgCTAGCCG-3') and reverse
(5'-CCGGAATTCCGCctaAACTTGCACGG-3') primers containing
sequences for BamHI and EcoRI restriction sites (underlined) and the initiating ATG and stop TAG codons (in lowercase type), respectively. The resulting DNA fragment was inserted into the
polylinker of the multicopy expression vectors pYADE4 and pYPGE2 (40).
This placed the expression of YHB1 under the control of the
glucose-regulatable ADH2 or the constitutive PGK1
promoters. The entire YHB1 sequence was verified by
sequencing using the Sequenase kit (U. S. Biochemical Corp.). The
YHB1-lacZ fusion was constructed by ligating a polymerase
chain reaction-amplified DNA fragment encompassing 679 nucleotides of
the 5'-untranslated region and the first two codons of YHB1
to the lacZ gene of the episomal YEp357 plasmid (41).
Growth Conditions--
Yeast cells were grown at 30 °C with
vigorous agitation (200 rpm) on a platform shaker (Infors Multitron,
Bottmingen, Switzerland). Milder agitation (160 rpm in a Infors
Aquatron water bath) was used for the experiments on sensitivity to
oxidative stress. Rich media contained 1% yeast extract (Difco), 1%
Bacto-peptone (Difco), and 2% glucose (YPglu), or 2% galactose
(YPgal). Synthetic complete media contained 0.67% Yeast Nitrogen Base
(Difco), 0,1% casamino acids (Difco), appropriate nutrients (Ura, Ade,
Trp) at 20 mg/liter, 2% glucose (SCglu), or 2% glycerol + 2% ethanol
(SCgly). Tween 80 (0.2%) and ergosterol (30 mg/liter) were added for
culturing heme-deficient cells and for anaerobic growth carried out in
special air-tight flasks equipped with ground joints, outlets, and a
mercury valve (29); flasks were bubbled with nitrogen for 30 min after inoculating the medium. Cultures were inoculated with fresh precultures in exponential growth phase in the same medium. Growth was followed by
measuring optical density at 600 nm (OD600) in a Kontron
Uvikon 860 spectrophotometer; one OD600 equals 0.3 mg dry
weight of cells/ml, or 107 cells/ml. Cells were collected
in the logarithmic growth phase (OD600 = 0.3-1.5) and used
immediately; they were stored at
80 °C for extracting RNA.
Respiration--
The oxygen consumption of whole cells was
monitored at 30 °C with a Clark-type electrode in 67 mM
KH2PO4, using glucose or ethanol as the
respiratory substrate. KCN was used at a concentration of 2 mM final. Respiration is expressed as nanomole of
O2/min/mg dry weight of cells.
-Galactosidase Assay--
-Galactosidase activity was
measured on chloroform-permeabilized cells grown in selective minimal
medium (SCglu -ura) at OD600 = 0.3-0.5 (35). It is
expressed as Miller units (A420 nm × 1000/min/OD600). The values reported are the averages of
duplicate determinations of at least two separate cultures, each
inoculated with 10-12 independent transformants.
Northern Blot Analysis--
Total RNA (15 µg), isolated from
cells in exponential growth (42), was electrophoresed on
agarose-formaldehyde gels, transferred to nylon membranes, and probed
with 32P-labeled DNA fragments under standard conditions
(35, 37). The probes were a 1.2-kilobase
BamHI-EcoRI fragment from plasmid pPGE/YHB1 for
the YHB1 gene, and a 1.1-kilobase
XhoI-HindIII fragment of the actin gene
ACT1 used to monitor RNA loading. The autoradiograms were
quantified by scanning densitometry.
Total Heme and Cytochrome Content--
The whole cell cytochrome
content was estimated by low temperature (liquid nitrogen)
spectrophotometry of cells (40 mg dry weight) filtered from liquid
cultures in exponential growth phase (43), using a Leres S-66
spectrophotometer. The filtered cells were also resuspended in 5 ml of
water, bubbled with CO for 5 min, or treated with sodium dithionite (a
few grains) or KCN (5 mM final), and the treated cells
processed for spectrophotometric analysis as above. This rapid,
reliable method allowed the direct, quantitative comparison of the
cytochrome and hemoglobin contents of different cells. The total heme
contents of whole cells and mitochondrial membranes were quantified
from the reduced-minus-oxidized difference spectra of the pyridine
hemochromes (44). Membranes were prepared after mechanical disruption
of the cells (45).
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RESULTS AND DISCUSSION |
Assignment of the Absorption Band at 575 nm to Oxyhemoglobin
(HbO2)--
Low temperature spectra of whole cells of
S. cerevisiae often have an absorption band near 575-577 nm
which we attributed previously to porphyrins and Zn-porphyrins (46). We
have now shown that this band is in fact due to the oxygenated form of the flavohemoglobin Yhb1p. It disappears after cells are reduced with
sodium dithionite and is replaced by a more diffuse band at 568-570 nm
in cells treated with carbon monoxide (CO) (Fig. 1), which is characteristic of a
hemoprotein that combines reversibly with molecular oxygen. The band at
575 nm is also absent from S/
HB cells lacking the YHB1
gene (
Yhb1), and reappears when these cells are
transformed with a plasmid expressing YHB1 (pADE/YHB1) (Fig.
1). Identical results were obtained with the strains YPH499 and W303-1B
and their
Yhb1 derivatives. Control experiments with hem12 mutant cells partially deficient in uroporphyrinogen
decarboxylase (47) showed that the peak at 575 nm due to the
accumulated Zn-porphyrins does not change when cells are treated with
dithionite or CO (data not presented).

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Fig. 1.
Low temperature (liquid nitrogen) absorption
spectra of whole cells showing the peak due to oxyhemoglobin
(HbO2) at 575 nm. Strain S150-2B (WT) and
its derivative S/ HB deleted for YHB1
( yhb1), transformed or not with the plasmid pADE/YHB1,
were grown in glucose synthetic medium to OD600 = 1-1.5.
Spectra were recorded with 40 mg dry weight of rapidly filtered cells
(43) reduced by endogenous substrates. Where indicated, cells were
treated with sodium dithionite (Red) or CO. The positions of
the cytochromes c, c1, b,
and aa3 absorption peaks are shown by
arrows.
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Respiratory Activity and Expression of YHB1--
Yhb1p is found
mainly in rho
cells and in cells growing in
the presence of antimycin A. It was therefore proposed that its expression is activated by the disruption of mitochondrial respiration (13, 28, 48). We have examined the effects of respiratory deficiency on
the presence of Yhb1p, detected by the absorption at 575 nm of its
oxygenated form, in parallel with the expression of the YHB1
gene estimated by Northern blot and the activity of a
YHB1-lacZ fusion. Strains S150-2B and W303-1B were grown in a glucose medium in the presence of antimycin A, an inhibitor of the
mitochondrial electron transport chain, or chloramphenicol, an
inhibitor of mitochondrial protein synthesis (for S150-2B, cells in the
rho
state were used, as these are
phenotypically similar to cells grown with chloramphenicol). Full
derepression of the respiratory chain was achieved by growth with
glycerol + ethanol. In contrast to S150-2B cells (Fig. 1), the peak at
575 nm was very small or absent in the low temperature spectra of
W303-1B grown in glucose, unless the cells were treated with KCN (Fig.
2A, curves 1). This is because
the W303-1B cells have a greater respiratory activity than S150-2B
cells, and "cyanide, by inhibiting the reoxidation of the reduced
components of cytochrome, prevents the utilization of oxygen in yeast
respiration and thus protects oxyhemoglobin from deoxygenation" (24).
KCN must also be added to observe the band at 575 nm in fully
derepressed cells grown in glycerol + ethanol (Fig. 2A, curve
4, and Fig. 3B), and the
further addition of dithionite or CO abolished or displaced it as was
shown in Fig. 1.

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Fig. 2.
Expression of YHB1 in different
respiratory conditions. A, low temperature absorption
spectra of whole cells of strain W303-1B recorded as described in the
legend to Fig. 1. Cells were grown in synthetic medium containing
glucose (curve 1), in the presence of 1 mg/liter antimycin A
(curve 2), or 4 g/liter chloramphenicol (curve
3), or glycerol + ethanol (curve 4). Where noted, the
cells were treated with KCN. B, Northern blot analysis of
the expression of YHB1 in the S150-2B and W303-1B strains.
Total RNA was prepared and probed for YHB1 and
ACT1 mRNA levels. The YHB1/ACT1 ratios were determined
by densitometry and normalized to 1 for normal growth in glucose
(lane 1). Lane HB, strain S/ HB
(yhb1- 1::HIS3) used as control for
YHB1 mRNA; lanes 1-4, same growth conditions
as those described above for curves 1-4, except that strain
S150-2B/rho was used in lane
3.
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Fig. 3.
Low temperature absorption spectra of whole
cells overexpressing Yhb1p. S150-2B (WT) and S150-2B
cells transformed with pPGE/YHB1 were grown aerobically in synthetic
medium containing glucose (panel A) or glycerol + ethanol
(panel B), or anaerobically in synthetic glucose medium
(panel C). Spectra were recorded as described in the legend
to Fig. 1, with untreated cells or cells treated with carbon monoxide
(CO), dithionite (Red), or KCN. The two spectra
for each strain shown in panel C correspond to the different
experiments described in Tables III and IV.
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Disrupting the mitochondrial respiration with antimycin A or
chloramphenicol did not appear to increase the intensity of the absorption band at 575 nm (Fig. 2A, curves 2 and
3), indicating no activation of the YHB1 gene. This is
confirmed by the lack of any significant change in the steady-state
amount of YHB1 mRNA (Fig. 2B, lanes 1-3), or
in the activity of a YHB1-lacZ fusion (Table
II). YHB1 expression was
decreased to about 60% of the control value in the
rho
derivative of strain S150-2B (Fig.
2B, lane 3 and Table II). Growing the cells in glycerol + ethanol slightly increased the amounts of YHB1 mRNA
(Fig. 2B, lane 4) and caused a 2.5-3.5-fold increase in
-galactosidase activity (Table II), confirming that glucose exerts
very little, if any, repression on YHB1 expression (27).
Heme-deficiency and anaerobiosis both reduced considerably the
expression of YHB1, but to different extents (Table II), as described previously (27, 28); this is in contrast to most bacterial Hb
genes, which are induced during growth under oxygen limitation (7, 8,
15, 17).
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Table II
Effects of different physiological conditions on the expression of a
YHB1-lacZ fusion
Strains S150-2B and W303-1B carrying the plasmid-borne
YHB1-lacZ fusion were grown overnight in synthetic medium
supplemented with glucose or glycerol + ethanol to OD600 = 0.3-0.5. Antimycin A, when added, was 1 mg/liter. For respiration
deficiency, the S150-2B/rho strain was used, and
strain W303-1B was grown with 4 g/liter chloramphenicol. For heme
deficiency, the strain S/ H1 (hem1- ) was used.
-Galactosidase activity was assayed in duplicate on cultures
inoculated with 10-12 independent transformants. Values reported
(A420nm × 1000/min/OD600 of cells) are the
mean ± S.E.
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Our results clearly show that mitochondrial respiratory activity need
not be disrupted for optimal expression of YHB1 and the
production of the holoprotein. The reported absence of Yhb1p in
respiratory-proficient cells (13) is probably due to the fact that the
cells were in late stationary phase: because the concentration of
YHB1 mRNA drops sharply upon exit from the exponential growth (27), Yhb1p is probably diluted out during the oxidative diauxic
growth, what would not occur in respiratory deficient cells.
Effects of Overexpressing YHB1--
The effects of deleting the
flavohemoglobin gene on cell growth and sensitivity to oxidative stress
have been described (28). But the effects of Yhb1p overproduction have
not previously been reported. Cells overexpressing Yhb1p from the
plasmid pPGE/YHB1 exhibited a red-brown tint when collected. They grew
a little slower than control cells carrying the vector pYPGE2 (Table
III), but reached the same final cell
density. For instance, the increase in doubling time was 14% for the
strain S150-2B and 7% for the strain W303-1B growing in glucose
synthetic medium. Overproducing Yhb1p might be detrimental to the
cells, and this is consistent with plasmid loss experiments showing
that 80-85% of the cells contained the plasmid pYPGE2, but only
32-36% contained pPGE/YHB1 at the end of the culture on glucose rich
medium.
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Table III
Effect of Yhb1p overexpression on heme and cytochrome synthesis
Strain S150-2B carrying plasmid pYPGE2 (control) or pPGE/YHB1 (Yhb1p
overproduction), was grown aerobically (O2) in SC medium
containing glucose (Glu) or glycerol + ethanol (Gly), and
anaerobically (N2) in SC glucose medium. Cells were collected
in exponential growth phase at OD600 = 1-1.2. The respiration
of whole cells and the total heme content of whole cells and membranes
were measured. Results are the averages of duplicate assays done in two
separate experiments. Standard errors were ±7% for respiration and
±5-15% for heme content. The values reported for anaerobic cells are
the ranges of values determined in the four separate experiments
described in Table IV.
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Low temperature spectra of whole cells overexpressing YHB1
showed the presence of large amounts of hemoglobin absorbing at 575 and
541 nm in its oxygenated form, and at 568 and 538 nm in its CO-liganded
form (Fig. 3, A and B). The amount of Yhb1p,
which was comparable to that of the mitochondrial cytochromes in the control cells, was estimated to be 1-2% of the total cellular proteins, similar to that reported for the production of various human
Hbs in S. cerevisiae (49, 50). This represented a doubling of the heme synthesis capacity (Table III), which was not increased further by adding the heme precursor 5-aminolevulinic acid to the
culture. It is interesting that the synthesis of large amounts of
holohemoglobin occurred at the expense of mitochondrial cytochromes. The decrease in their concentrations, which can be seen in the low
temperature spectra (Fig. 3, A and B), was
estimated to be 2-4-fold by spectrophotometric analysis and measuring
the total heme contents of mitochondrial membranes (Table III).
Glucose-grown cells also showed a concomitant decrease in cell
respiration (Table III). Cyanide-insensitive respiration was very low
in glucose-grown cells (0.5 nmol of O2/min/mg dry wt), and
zero in cells grown in glycerol + ethanol. These data indicate that the
accumulated flavohemoglobin does not contribute significantly to the
oxygen consumption of whole cells, and, consequently that Yhb1p
probably has no oxidase activity in vivo. This is consistent
with the fact that the flavoHb purified from the yeast C. mycoderma is relatively stable in its oxygenated form (26).
Thus, the cells appear to produce more heme in response to an increased
demand made by the apohemoglobin, and this heme is used by the
cytosolic globin rather than by the mitochondrial cytochromes. This
implies that the heme newly synthesized in the mitochondria is first
used in the cytosol before being incorporated into the mitochondrial
cytochromes. Alternatively, overproduction of hemoglobin may reduce the
transcription of the genes encoding the apocytochromes, by disturbing
heme traffic or by some other unknown means. Our results (Table
IV) suggest that this might be the
case.
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Table IV
Effect of Yhb1 overproduction on the expression of HEM13-lacZ,
CYC1-lacZ, and SOD2-lacZ fusions
Strains S/H13-Z and S/SOD2-Z carried the HEM13-lacZ and
SOD2-lacZ fusions integrated at the HEM13 or
SOD2 loci. Strain S150-2B carried the CYC1-lacZ
fusion on the episomal plasmid pLG669-Z (34). Cells contained plasmid
pYPGE2 (control) or plasmid pPGE/YHB1 (Yhb1p overexpression). Cells
were grown overnight under aerobic (O2) or anaerobic
(N2) conditions in glucose synthetic medium, until
OD600 = 0.5-0.7. Values (A420 × 1000/min/OD600 of cells) are the averages (±S.E.) of duplicate
assays on 4 separate cultures, run on different days, each inoculated
with a different pool of 10-12 individual transformants.
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We then asked whether overproduction of Yhb1p occurs in anaerobically
grown cells. Although both coproporphyrinogen and protoporphyrinogen oxidases (sixth and seventh enzymes in the heme biosynthetic pathway) require molecular oxygen for their activity in vitro, a
limited amount of heme is synthesized in vivo by cells
growing in anaerobiosis, probably at the expense of traces of
contaminating air that are virtually impossible to avoid (46). This
heme is associated with the microsomal cytochromes
b5 and P450 and with Yhb1p. Yhb1p is
overproduced in the anaerobic cells containing pPGE/YHB1, but its
concentration varied from one culture to another (Fig. 3C and Table III). This most likely reflects the differences in the amount
of air that leaked into the flasks. The possibility of significantly
increasing the amount of heme made by anaerobic cells led us to examine
the transcriptional activity of the HEM13 and
CYC1 genes in these cells, as their expression is controlled by oxygen and heme in an opposite fashion, negative for
HEM13 and positive for CYC1 (see Ref. 51 for
review). Since there is less heme synthesized in anaerobic cells, heme
is believed to mediate oxygen regulation (51). Overproduction of Yhb1p, and hence of endogenous heme, should thus reverse the effects of oxygen
deficiency and lead to aerobic levels of expression. This is obviously
not the case (Table IV). The activities of the HEM13-lacZ
and CYC1-lacZ fusions were unchanged in the anaerobic cells,
whatever the amount of heme made, indicating that oxygen regulation is
not simply mediated by the heme concentration. In contrast, a 2-3-fold
difference in the expressions of the two fusions was consistently found
in aerobic cells overproducing Yhb1p, suggesting that Yhb1p
overproduction influenced the aerobic transcription of HEM13
and CYC1. The expression of SOD2, encoding the
mitochondrial manganese-superoxide dismutase, was not induced by Yhb1p
overexpression (Table IV), as would have been expected (30) if Yhb1p
had generated superoxide anions during NAD(P)H oxidation, as has been
described for E. coli flavoHb (21).
Expression of YHB1 in Response to Agents Causing Oxidative
Stress--
The expression of YHB1 was not significantly
altered in cells grown in the presence of antimycin A, or in
rho
cells (Fig. 2), conditions that enhance
(antimycin A (see Ref. 52 and references therein and also, Ref. 53)) or
decrease (rho
state (54, 55)) the mitochondrial
production of superoxide anions and other reactive oxygen species.
These results differ from those of a recent report (28) indicating that
YHB1 is induced in cells exposed to reagents that promote
oxidative stress, including antimycin A. We therefore analyzed the
expression of YHB1 in response to oxidative stress generated
by antimycin A, H2O2, the thiol-oxidizing agent
diamide (which decreases the cell glutathione content), the
redox-cycling agent menadione bisulfite (that generates superoxide anions and H2O2 extracellularly (56)), DTT,
which is a reductant in a metal-catalyzed oxidation system (Ref. 57 and
references therein), and Cu2+, a transition metal ion that
can initiate Fenton chemistry. Cultures of the strains S150-2B and
W303-1B, with or without the plasmid-borne YHB1-lacZ fusion,
were grown in glucose minimal medium to exponential phase
(OD600 = 0.3-0.4), the drugs were added, and the cells
were incubated for an additional 1 h. The
-galactosidase
activity was then assayed and RNA extracted for Northern blot analysis. OD600 measurements indicated that the growth of the cells
treated with H2O2, diamide, DTT, and
Cu2+ was slowed. As shown in Fig.
4A for the strain S150-2B, the
-galactosidase activity in cells treated with antimycin A or
menadione was similar to that of untreated cells. Enzyme activity was
slightly decreased after treatment with H2O2,
and this was more pronounced (30-35%) in cells exposed to diamide,
DTT, or Cu2+. Identical results were obtained with strain
W303-1B (not shown). Thus none of the stresses tested substantially
increased the expression of YHB1, which was confirmed by the
analysis of the steady state levels of YHB1 mRNA in both
S150-2B and W303-1B cells (Fig. 4B). The amounts of
YHB1 mRNA were not significantly altered by exposure to
antimycin A or menadione. H2O2 caused a slight
decrease, while diamide and DTT led to dramatic falls in
YHB1 mRNA content. The responses of the two strains to
Cu2+ appeared to be different; this probably reflects the
difference(s) in their sensitivity (and Cu2+ metabolism?),
S150-2B being much more sensitive than W303-1B. The fact that there was
a much greater drop in mRNA than the reduction in
-galactosidase
activity is probably due to the great stability of the
-galactosidase fusion protein (t1/2 > 20 h
(58)). We also analyzed the response of YHB1 to oxidants
using the experimental procedure described by Zhao et al.
(28). Again, there was no induction with either the W303-1B strain
(Fig. 4B, lower part), or the S150-2B strain (not shown),
indicating that our results are not peculiar to one strain. Last, we
explored the possibility that YHB1 was under the control of
Yap1p, a transcriptional activator that plays a central role in the
oxidative stress response of cells by mediating the induction of the
genes for a number of antioxidant enzymes (Ref. 33, reviewed in Refs.
59 and 60). The
-galactosidase activity of the YHB1-lacZ
fusion in cells deleted of YAP1 (153 ± 5 units) or
overexpressing it (144 ± 12 units) was similar to that in
isogenic wild type cells (144 ± 16 units), indicating that Yap1p
had no effect on YHB1 expression.

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Fig. 4.
Effects of oxidants on YHB1
expression. A, expression of the fusion
YHB1-lacZ in response to oxidative stress. S150-2B cells
transformed with pHB-Z were grown overnight in glucose synthetic medium
(SCglu) to OD600 = 0.4. Aliquots of the culture
were then incubated for 1 h with 1 mg/liter antimycin A, 0.4 mM H2O2, 2 mM diamide,
2 mM menadione sodium bisulfite, 5 mM DTT, or 1 mM CuSO4, and -galactosidase activity
(A420 nm × 1000/min/OD600 of cells)
was assayed. B, Northern blot analysis of YHB1
mRNA levels in S150-2B and W303-1B cells growing exponentially in
SCglu and exposed to the different drugs for 1 h as described in
panel A. W303-1B cells were also grown in rich galactose
medium (YPgal) and treated as described in Ref. 28. At
OD600 = 1, the cells were collected by centrifugation,
suspended in an equal volume of fresh YPgal containing 2.5 mg/liter
antimycin A, 1 mM H2O2, 1.5 mM diamide, 5 mM DTT, or 1 mM
CuSO4, and incubated for 1 h at 30 °C. Total RNA
were analyzed as described in the legend to Fig. 2. The YHB1/ACT1
ratios were normalized to 1 for expression in untreated cells.
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Thus our results indicate that Yhb1p does not participate in the
genetic response to oxidative stress. Not only was the expression of
YHB1 not induced by any of the oxidants tested, but it was greatly reduced by some of them, like diamide and DTT. Whether the
discrepancies between our results and those reported recently (28) are
due to differences in strain, experimental procedures, or other factors
is not clear at present.
Sensitivity to Oxidants and the Production of Yhb1p--
The drop
in YHB1 mRNA in cells challenged with
H2O2, diamide, DTT, and Cu2+
suggested that Yhb1p was harmful for cells enduring the stresses caused
by these compounds. To test the possibility that Yhb1p plays a role in
the antioxidant enzyme system, we analyzed the effects of various
oxidants on the growth of cells containing different amounts of Yhb1p.
The strains S150-2B or W303-1B and their derivatives deleted of
YHB1 or overexpressing it from the plasmid pPGE/YHB1 were
used. The results obtained in the context of strain W303-1B are
presented in Fig. 5. Yhb1p seems to
protect the cells from damage(s) caused by Cu2+ and DTT.
The YHB1-deleted cells were sensitive to lower
concentrations of Cu2+ and DTT than were wild type cells.
Conversely, overexpressing YHB1 increased the resistance of
cells to these compounds; in particular, the cells became much less
sensitive to killing by high concentrations of Cu2+. In
contrast, Yhb1p appears to be detrimental to cells exposed to
H2O2: YHB1-deleted cells were more
resistant, while YHB1 overexpression sensitized cells to
H2O2. The response to diamide is less clear, although it was checked repeatedly in replicate experiments. Yhb1p appears to have both beneficial (at low concentrations of diamide) and
detrimental (at high concentrations of diamide) effects on the
resistance of the cells to this compound. Essentially the same
correlations between the resistance of cells to these drugs and the
cell Yhb1p contents were obtained with strain S150-2B (not shown).
Increased sensitivity to diamide and diethylmaleate (a thiol oxidant),
but not to H2O2, upon deletion of
YHB1 has been reported (28). One possible explanation for
this difference from our results is that cells in the
rho
state were used, and these cells are much
more sensitive that rho+ cells to oxidative
stress (Ref. 61 and references therein), thus precluding the detection
of small, specific effects.

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Fig. 5.
Effect of hemoglobin deletion and
overexpression on the sensitivity of cells to oxidative stress.
SCglu medium was inoculated at 2 × 103 cells/ml from
fresh exponential precultures and distributed as 10-ml aliquots in
50-ml flasks containing the indicated drugs at the concentrations
shown. Following incubation at 30 °C for 15-17 h (10-12
generations), growth was monitored by OD600 measurements
for 6 h. Values were averaged and normalized to the OD of control
cultures without oxidative agents. The whole experiment was repeated 3 times for confirmation. Data from a representative experiment are
shown. , control strain W303-1B transformed with pYPGE2; , strain
W/ HB deleted of YHB1 and transformed with pYPGE2; ,
strain W303-1B transformed with pPGE/YHB1 overexpressing
YHB1.
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Last, we examined the effects of varying the amounts of Yhb1p on the
growth of strains made more sensitive to oxidants by deleting the genes
YAP1 (33, 59, 60) or ZWF1/MET19
encoding glucose-6-phosphate dehydrogenase (32, 62). This enzyme
catalyzes the first rate-limiting NADPH-producing step of the pentose
phosphate pathway, which is considered to be an important source of
reducing power for cells. The strains wild-type for YAP1,
overexpressing it (from the plasmid YEp351/YAP1 (33)), or
YAP1-deleted, all grew about 10% more slowly (1.8 h) when
overproducing Yhb1p from the plasmid pPGE/YHB1, compared with 1.5 h for controls. This is within the range (7-14%) mentioned for
strains S150-2B and W303-1B. But it was significantly larger for the
mutant
zwf1(met19), as its doubling time in
glucose minimal medium (1.9 h) was 50% longer when overexpressing
Yhb1p from pPGE/YHB1 (2.9 h); the increase was smaller (25%) for cells
growing in glycerol + ethanol medium, and dropped to ~10% in cells
grown under anaerobic conditions. However, we can hardly conclude from
this experiment that oxygen is required for the deleterious effect of
large production of Yhb1p in this mutant, since less holoYhb1p is made
under anaerobiosis.
Although our results clearly show a relationship between the cell Yhb1p
content and their resistance to various stresses, they are complex, for
the most part unexpected, and often contradictory. For example, one
would not have expected Yhb1p to protect cells against damage caused by
DTT, given that YHB1 expression drops in cells exposed to
it. Also surprising are the contrasting effects of Yhb1p on the
resistance of cells to H2O2 and
Cu2+. The relatively complicated situation, compared with
that described previously (28), the paucity of information on the
effects of DTT in cells (Ref. 57 and ref. therein), the variety of
reactions implicated in Cu2+ trafficking and detoxification
(see Ref. 63 and references therein), and the possible difference in
the ways cells respond to extra- and intracellular oxidative stress (by
H2O2, for instance (53)), all make it difficult
to imagine just how Yhb1p can participate in the defense of cells
against oxidative stress. This is all the more difficult because
hemoproteins in general and hemoglobins in particular are known for
their inherent ability to produce reactive oxygen species and to induce
peroxide-mediated damage in various biological systems (52, 64-67).
Clearly, better knowledge of the properties of Yhb1p itself, and
further phenotypic studies of YHB1-deleted and
overexpressing strains are required to clarify the function(s) of this
enigmatic ancient form of hemoglobin.
We thank Drs D. Thomas and W. Moye-Rowley for
the gifts of mutant strains, and O. Parkes for editorial
assistance.