(Received for publication, July 6, 1995; and in revised form, September 1, 1995)
From the
Incubation of obligately photoautotrophic and aerobic
cyanobacterium Anacystis nidulans (Synechococcus sp.
PCC 6301) in the light in the presence of the photosystem II inhibitor
3-(3,4-dichlorophenyl)-1,1-dimethyl-urea and equilibrated with
approximately 1% (v/v) O in N
(10
µM O
in solution) led to a decrease of the
heme A content of isolated cytoplasmic membranes and to the appearance
of heme O. The latter was not seen in membranes from fully aerated
cells (>210 µM dissolved O
). Non-covalently
bound hemes extracted from the membranes were identified by reversed
phase high performance liquid chromatography. Heme A and O contents of
the membranes changed in a reversible fashion solely depending on the
ambient oxygen regime. Both hemes A and O combine with the same
apoprotein as suggested by immunoblotting. CO/reduced-minus-reduced
optical difference spectra, photoaction spectra of CO-inhibited O
uptake by the membranes, and pyridine hemochrome spectra pointed
to either heme belonging to a functional form of the terminal oxidase.
The NADH:O
oxidoreductase reaction catalyzed by membranes
from both high O
and low O
cells was strictly
dependent on the addition of catalytic amounts of cytochrome c, fully inhibited by 1.2 µM KCN, and insensitive
to 5 µM
2-n-heptyl-4-hydroxyquinoline-N-oxide. O
uptake by the membranes was effectively catalyzed by N,N,N`,N`-tetramethyl-p-phenylenediamine but not
2-methylnaphthoquinol or plastoquinol-1 as artificial substrates.
Therefore we conclude that the cyanobacterial respiratory oxidase,
irrespective of the type of heme in its O
-reducing center,
is a cytochrome c rather than a quinol oxidase.
Cyanobacteria are phototrophic prokaryotes uniquely capable of
oxygenic (plant type) photosynthesis and aerobic (cytochrome oxidase
based) respiration (Fay and Van Baalen, 1987; Bryant, 1994). Their
immediate ancestors are generally thought to be responsible for the
first bulk amounts of oxygen gas in a previously anaerobic biosphere
and atmosphere (Barghoorn and Schopf, 1966; Broda, 1975). Consequently,
they must have been (among) the first to cope with poisonous free
oxygen (Morris, 1975; Babcock and Wikström, 1992;
Peschek, 1992). Most efficiently and significantly from the viewpoints
of ecology and evolution in the long run this happened by transforming
pre-existing photosynthetic mechanisms into those of aerobic
respiration, finally reducing oxygen back to water (``conversion
hypothesis''; see Broda (1975) and Broda and Peschek(1979)),
involving a heme-type respiratory oxidase as the crucial component.
Interestingly, it has not been until fairly recently that the
cyanobacterial respiratory oxidase was established to be an aa-type enzyme very similar to the mitochondrial
or Paracoccus denitrificans enzyme (Peschek, 1981; Peschek et al., 1988, 1989a, 1989b; Alge and Peschek, 1993; Alge et al., 1994). In most cyanobacteria the cytochrome c oxidase was localized in both (chlorophyll-free) cytoplasmic (CM) (
)and green thylakoid membranes, relative shares of the
enzyme (and of the other respiratory electron transport components
altogether) in either membrane critically depending on growth
conditions (Peschek et al., 1988, 1989a, 1989b, 1994a; Molitor et al., 1990). The most widely investigated species Anacystis nidulans was repeatedly, but in contrast to initial
reports (Omata and Murata, 1984, 1985), shown to contain much more
cytochrome c oxidase (per mg of membrane protein) in
cytoplasmic than in thylakoid membranes (Peschek et al.,
1994a, 1994b). It was also shown that growth in salt-stressed
conditions can still markedly enhance the content of the cytochrome c oxidase in the cytoplasmic membrane (particularly in the
CM-II fraction; see Molitor et al. (1990), Nicholls et
al.(1992), and Peschek et al. (1994a)). Direct analytical
determination of cytochrome a (heme A) in cyanobacterial
membranes was previously achieved with membranes isolated from Anabaena variabilis using the laborious classical techniques
of column chromatography and alkaline pyridine ferrohemochrome
derivation (Wastyn et al., 1988). A major obstacle to
successful heme extraction and HPLC identification (Svensson et
al., 1993; Lübben and Morand, 1994) from
cyanobacteria has been the tremendous amount of photosynthetic pigments (viz. chlorophyll and phycobilins) in these cells. (
)In the present work we overcame this problem by using
chlorophyll-free cytoplasmic membranes (CM-II fraction; see Nicholls et al.(1992), Peschek et al. (1989c), and
Hinterstoisser et al. (1993)) from A. nidulans, which
contain high levels of respiratory pigments, in particular of
cytochrome c oxidase (Peschek et al., 1988, 1989a,
1989b). A-, B-, and O-type hemes could be clearly identified in these
membranes, proportions of hemes A and O reversibly depending on the
oxygen concentration present in the incubation medium. Our results are
in agreement with a biosynthetic sequence of heme B
heme O
heme A
(2-vinyl-8-methyl-/2-hydroxyethyl-farnesyl-8-methyl-/2-hydroxyethyl-farnesyl-8-formyl/iron
porphyrin) whereby some oxygenase enzyme appears to participate in the
last step. Direct involvement of O
in the biosynthesis of
chlorophyll b (formyl group) from chlorophyll a (methyl group) was recently demonstrated (Porra et al.,
1993), and a ``heme O synthase gene'' is being cloned and
sequenced from Synechocystis sp. PCC 6803. (
)
Figure 1:
Reversed-phase HPLC chromatograms of
non-covalently bound hemes from cytoplasmic membranes of A.
nidulans. A, phase A cells from photosynthetic,
O-supersaturated (310-360 µM O
in the medium) cultures grown at 40 °C. Besides heme B only
heme A is detectable. B, phase B cells from de-aerated
``semi-anaerobic'' cultures after flushing with 1% O
in N
for 24 h (approximately 10 µM O
in the medium). Heme O appears in the membranes
besides heme B and residual heme A. C, phase C cells from
re-aerated cultures equilibrated with air (21% O
in
N
) for 12 h after the semi-anaerobic phase (210-215
µM O
in the medium). The decrease of heme O in
the membranes contrasts with an increase of heme A (see panel
B). D, standards of hemes B, A, and O extracted from
authentic material as described under ``Experimental
Procedures.'' Incubation of non-growing cells was in growth medium
containing 20 µM DCMU and illuminated with 20
watts
m
warm white fluorescent light. When the
semi-anaerobic phase was extended to 72 h no heme A was detectable any
more, but the viability of the (resting) cells also had decreased by
about 20% (not shown). Re-aeration of 24-h phase B cells for 48 instead
of only 12 h led to an almost complete recovery of the original heme A
content combined with complete loss of all heme O from the membranes
(not shown).
Fig. 1D shows the reversed-phase HPLC
chromatogram of heme B, A, and O standards prepared as described under
``Experimental Procedures.'' Retention times of the standards
were close to 26, 35, and 37 min, respectively, for hemes B, A, and O
in the order of increasing lipophilicity, reasonably reproducible
(within ±3%) from one extraction to the other, and independent
of the source of the heme. This clear-cut identification of the heme
standards permitted a reliable analysis of non-covalently bound heme
groups in cytoplasmic membranes isolated from phase A (overoxygenated,
photosynthetically growing), phase B (semi-anaerobic), and phase C
(re-aerated) Anacystis (Fig. 1, A-C,
respectively). The most abundant heme is heme B corresponding to the
ubiquitous occurrence of cytochrome b at high levels in almost
any type of biological membrane (Lübben and Morand,
1994; Lemberg and Barrett, 1973). The only other acid-labile heme
detectable in these membranes is heme A (Fig. 1A) from
the aa-type cytochrome c oxidase, which
has been characterized in cytoplasmic and/or thylakoid membranes from
27 different cyanobacterial species (Peschek et al., 1989a,
1989b; Wastyn et al., 1988). (
)
After a 24-h
incubation period in the light under low oxygen tension (<10
µM final concentration of O in the medium,
which is still above the K
of typical
cytochrome c oxidases but below the K
of most oxygenases (Jones, 1981)) heme A concentration in the
membranes was diminished, and a new peak of heme O was seen (Fig. 1B). The non-growing cells, equilibrated with 1%
(v/v) O
in the light, exhibited stable energy charge values
of up to 0.8 (Nitschmann, 1985; Nitschmann and Peschek, 1986), which
means that they went on being sufficiently energized to meet all of
their maintenance requirements. Re-aeration of the semi-anaerobic and
illuminated yet non-growing cell suspensions finally produced an
increase of the membranes' heme A content at the expense of heme
O. This shows that the transitions between hemes A and O were fully
reversible and most probably dependent on the availability of
sufficient O
only (Fig. 1C). When
de-aeration and re-aeration of the cells were performed in strict
darkness or in the presence of 20 µg/ml chloramphenicol the
reversible changes in heme A and O concentrations in the membranes were
no longer observed. Thus efficient synthesis of ATP (by
photophosphorylation) and of proteins is a prerequisite also for the
maintenance metabolism (steady-state protein turnover). (Control
experiments not shown.)
Both hemes A and O represented functional
cytochromes (viz. cytochromes a and o
, respectively) in the semi-anaerobic and
re-aerated phase B and C cells as can be seen from
CO/reduced-minus-reduced optical difference spectra of n-octyl
glucoside-solubilized membranes (Fig. 2A), photoaction
spectra of the CO-inhibited O
uptake by native membranes in
the presence of ascorbate and cytochrome c (Fig. 3),
and by polarographic measurement of different oxygen affinities of the
two different cell types (Fig. 4). Semi-anaerobic cells contain
two different CO-reactive cytochromes, one of a-type with
peaks at 430 and 590 nm and another one with peaks at 415 and 555 nm,
which probably stem from the ``o-type oxidase'' (Fig. 2A; note that spectral features of b-type and o-type cytochromes cannot be easily
distinguished due to an almost identical electronic situation in the
heme core of either cytochrome; see Lübben and
Morand(1994)).The same problem of spectrally indistinguishable hemes B
and O would also apply to the alkaline pyridine hemochrome spectra
shown in Fig. 2B. However, in this case the hemochromes
were prepared from a fraction eluting from the reversed-phase HPLC
column well after heme B (i.e. >30 min; see Fig. 1).
Thus the ``b-type'' spectral features in Fig. 2B (hemochrome difference spectrum with peaks at
415 and 555 nm) must be attributed to heme O, while 433- and 588-nm
peaks clearly belong to heme A (Lübben and Morand,
1994; Peschek, 1981). The final proof that in semi-anaerobic Anacystis both hemes A and O are part of a functional
respiratory oxidase rests on the following data. (i) Photoaction
spectra of CO-inhibited oxygen uptake (Fig. 3B) showed
peaks at 415 and 555 nm (cytochrome o
) while the
same experiments performed on membranes from photosynthetically growing
(overoxygenated) phase A cells displayed a-type peaks at 430
and 590 nm only (Fig. 3A). (ii) Direct polarographic
measurements yielded a K
value of 0.85
µM O
for the a
-type
oxidase from phase A cells (Fig. 4A) and of 0.35
µM O
for the o
-type
oxidase from semi-anaerobic cells (Fig. 4B). The latter
two distinctly different K
values were
reproducible within ±10% for altogether six independent membrane
preparations, each from phase A and phase B cells, respectively. Though
both K
values are still about 1 order of magnitude
higher than those of typical mitochondrial cytochrome c oxidase the function of a lower K
form of the
oxidase under semi-anaerobic conditions is in qualitative agreement
with bioenergetic expectations.
Figure 2: Cytochromes in the cytoplasmic membranes of phase B A. nidulans. A, CO/reduced-minus-reduced spectrum of 3 mg/ml cytoplasmic membranes in 1% (w/v) n-octyl glucoside. B, pyridine hemochrome spectra of hemes O and A directly prepared from the HPLC fraction eluting after 30 min (see Fig. 1).
Figure 3:
Photoaction spectra of CO-inhibited
O uptake by the cytoplasmic membranes of A.
nidulans. A, membranes from phase A cells; B,
membranes from phase B cells. O
uptake in the presence of
25 µM horse heart cytochrome c was measured
polarographically. Oxygen uptake by maximally inhibited membranes in
the dark was 3-5 nmol O
/min per mg of protein (see
the ordinate scale).
Figure 4: Kinetics of the oxygen uptake by the cytoplasmic membranes of A. nidulans. A, membranes from phase A cells; B, membranes from phase B cells. Lineweaver-Burk diagrams were drawn after polarographic data determined at 35 °C.
Immunoblots of membrane proteins
from semi-anaerobic and re-aerated cells with monospecific antibodies
raised against subunits I of aa-type cytochrome c oxidase of P. denitrificans and bo
-type quinol oxidase of E. coli are
shown in Fig. 5. Independent of the antibody used and of the
oxygen status of the cells, always only one single band was obtained,
which, according to its apparent molecular mass of about 55 kDa,
corresponds to subunit I of the well known aa
-type
cytochrome c oxidase of Anacystis (Peschek et
al., 1988, 1989a; Molitor et al., 1990). Therefore it
seems most likely that both hemes A and O, which are synthesized in Anacystis from heme B as in other living cells
(Lübben and Morand, 1994; Hansson and von
Wachenfeldt, 1993) and whose relative proportions just vary with oxygen
concentrations available (Fig. 1), can combine with one and the
same apoprotein. This would give rise to the spectral and functional
attitude of both an a
-type and an o
-type enzyme (Fig. 2Fig. 3Fig. 4).
Figure 5:
Immunoblotting of cytoplasmic membrane
proteins from A. nidulans. Membrane proteins from phase B
cells (lanes 1 and 3) and phase C cells (lanes 2 and 4) were immunoblotted with monospecific antibodies
raised against the subunit I of P. denitrificans
aa-type (lanes 1 and 2) and E.
coli bo
-type (lanes 3 and 4)
cytochrome c and quinol oxidases, respectively. Lane 5 represents marker proteins.
Similar promiscuity of
the heme groups appears to obtain for both high spin,
O-reducing hemes and low spin hemes as was found for the
``conjugated'' cao
/caa
-type, the bo
/ba
-type, and the oo
/bo
-type cytochrome oxidase
pairs in Bacillus PS3 (Sone and Fujiwara, 1991), Acetobacter aceti (Matsushita et al., 1992), and E. coli (Puustinen and Wikström, 1991;
Puustinen et al., 1992), respectively. Yet, this does not
imply the various promiscuous types of oxidases in cyanobacteria
representing true alternative oxidases in the classical sense; Table 1gives the rates of oxygen uptake catalyzed by the
cytoplasmic membranes from Anacystis phase B cells (see Fig. 1) incubated in the presence of different artificial and
``physiological'' electron donors and inhibitors. The results
can best be reconciled with the occurrence of a functionally (with
respect to the acceptor side) uniform cytochrome c (but not
quinol) oxidase in the non-branched respiratory electron transport
system of Anacystis and most probably of cyanobacteria in
general.