Transient accumulation of heme O (cytochrome o) in the cytoplasmic membrane of semi-anaerobic Anacystis nidulans. Evidence for oxygenase-catalyzed heme O/A transformation.

Incubation of obligately photoautotrophic and aerobic cyanobacterium Anacystis nidulans (Synechococcus sp. PCC 6301) in the light in the presence of the photo-system II inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea and equilibrated with approximately 1% (v/v) O2 in N2 (10 microM O2 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 microM dissolved O2). 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 O2 uptake by the membranes, and pyridine hemochrome spectra pointed to either heme belonging to a functional form of the terminal oxidase. The NADH:O2 oxidoreductase reaction catalyzed by membranes from both high O2 and low O2 cells was strictly dependent on the addition of catalytic amounts of cytochrome c, fully inhibited by 1.2 microM KCN, and insensitive to 5 microM 2-n-heptyl-4-hydroxyquinoline-N-oxide. O2 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 O2-reducing center, is a cytochrome c rather than a quinol oxidase.

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-dimethylurea and equilibrated with approximately 1% (v/v) O 2 in N 2 (10 M O 2 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 M dissolved O 2 ). 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 2 uptake by the membranes, and pyridine hemochrome spectra pointed to either heme belonging to a functional form of the terminal oxidase. The NADH:O 2 oxidoreductase reaction catalyzed by membranes from both high O 2 and low O 2 cells was strictly dependent on the addition of catalytic amounts of cytochrome c, fully inhibited by 1.2 M KCN, and insensitive to 5 M 2-n-heptyl-4-hydroxyquinoline-N-oxide. O 2 uptake by the membranes was effectively catalyzed by N,N,N,N-tetramethyl-pphenylenediamine 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 2 -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 3 -type enzyme very similar to the mitochondrial or Paracoccus denitrificans enzyme (Peschek, 1981;Peschek et al., 1988Peschek et al., , 1989aPeschek et al., , 1989bAlge and Peschek, 1993;Alge et al., 1994). In most cyanobacteria the cytochrome c oxidase was localized in both (chlorophyll-free) cytoplasmic (CM) 1 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., , 1989a(Peschek et al., , 1989b(Peschek et al., , 1994aMolitor et al., 1990). The most widely investigated species Anacystis nidulans was repeatedly, but in contrast to initial reports 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(Peschek et al., , 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 . A major obstacle to successful heme extraction and HPLC identification (Svensson et al., 1993;Lü bben and Morand, 1994) from cyanobacteria has been the tremendous amount of photosynthetic pigments (viz. chlorophyll and phycobilins) in these cells. 2 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., , 1989a(Peschek et al., , 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 3 heme O 3 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 2 in the biosynthesis of chlorophyll b (formyl group) from chlorophyll a (methyl group) was recently * Financial support was received from the Austrian Research Community, the Austrian Science Foundation, and the Kulturamt der Stadt Wien. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. demonstrated (Porra et al., 1993), and a "heme O synthase gene" is being cloned and sequenced from Synechocystis sp. PCC 6803. 3

EXPERIMENTAL PROCEDURES
Materials and Growth Conditions-A. nidulans (Synechococcus sp. strain PCC 6301) was obtained from the Pasteur culture collection (courtesy of Mme. Rosi Rippka-Herdman) and grown axenically and photoautotrophically in the presence of 0.5 M NaCl at 40°C and 20 watts⅐m Ϫ2 warm white fluorescent light as measured with a YSI radiometer, model 65, at the surface of the growth vessels as described (Molitor et al., , 1990Peschek et al., 1994a). Actively growing cultures were sparged with 1.5% (v/v) CO 2 in air, steady-state concentration of O 2 in the suspensions amounting to 30 -35% (v/v) oxygen saturation (150 -175% air saturation) corresponding to a steady-state O 2 concentration in the liquid medium of between 310 and 360 M O 2 . When cell concentrations had reached approximately 3 l of packed cell mass/ml (light-limited, linearly growing cultures; see Peschek et al. (1988) and ) aliquots of the culture were harvested by centrifugation at room temperature, washed three times with distilled water, and finally suspended in the desired preparation or assay medium. A second aliquot of the culture was supplemented with 20 M DCMU and sparged with bacteriologically pure, "technical" nitrogen gas (containing approximately 1% O 2 ) under continued illumination for another 24 h until harvest. A third aliquot of the culture was derived from DCMU-poisoned 1% O 2 cells by switching to a gassing regime with normal air (21%, v/v, O 2 ) for a final 12-h period. During each condition of incubation the O 2 concentration in the cell suspensions was continuously monitored with a Clark-type oxygen electrode (YSI oxygen monitor, model 53). Cells from O 2 -oversaturated (310 -360 M O 2 ), semianaerobic (Ͻ10 M O 2 ), and normally re-aerated (210 -215 M O 2 ) cultures were designated "phase A," "phase B," and "phase C" cells, respectively (see Fig. 1).
Membrane Isolation and Respiratory Measurements-Cytoplasmic membranes were isolated and purified from harvested and washed cells after lysozyme treatment and French pressure cell extrusion as described (Peschek et al., 1989a(Peschek et al., , 1989c and resuspended in 10 mM potassium phosphate buffer, pH 7.0 or 7.6, for spectrophotometric and polarographic assays, respectively. Oxidation of horse heart ferrocytochrome c (7 M initial concentration) was measured in a Shimadzu UV-300 dual wavelength spectrophotometer , and O 2 uptake by the membranes in the presence of 6.0 mM sodium ascorbate, 0.1 mM TMPD, 3.5 mM NADH, 3 or 25 M horse heart cytochrome c, 60 M plastoquinol-1, and/or 60 M 2-methylnaphthoquinol was measured polarographically as described (Nicholls et al., 1992;Kraushaar et al., 1990). Temperature of measurements was 30°C.
Reversed-phase HPLC-Non-covalently bound hemes were extracted from a total of 2.0 ml of cytoplasmic membrane suspensions (approximately 30 mg of protein/ml), each from phase A, B, and C cells, and divided into 0.05-ml aliquots for experimental handling, with acetone/ HCl (19:1, v/v) followed by ethyl acetate/acetonitrile treatment (Svensson et al., 1993;Lü bben and Morand, 1994). The heme composition was analyzed on an ISCO HPLC System 2004i equipped with a Deltapak C18 (3.9 ϫ 150 mm, Zorbax) reversed-phase HPLC column. Hemes were eluted with acetonitrile/0.5% trifluoroacetic acid/water gradients according to Puustinen and Wikström (1991), Sone and Fujiwara (1991), Svensson et al. (1993), and Lü bben and Morand (1994) and detected spectrophotometrically at 406 nm (UVIS 205 detector, ISCO). The hemes were identified by comparison with heme A and B standards prepared by extraction of commercially available bovine cytochrome c oxidase and reductase, respectively (Sigma), by comparison with a heme BO standard prepared by extraction of membranes from a cytochrome bo 3 -overproducing Escherichia coli strain (kindly donated by Dr. M. Lü bben), by CO/reduced-minus-reduced difference spectra of cytoplasmic membranes solubilized with 1%, w/v, n-octyl glucoside (Peschek et al., 1989a), by transforming hemes that eluted after Ͼ30 min from the reversed-phase HPLC column (see Fig. 1) directly into alkaline pyridine hemochromes (Lü bben and Morand, 1994;Berry and Trumpower, 1987), and by recording photoaction spectra of CO-inhibited O 2 uptake in the presence of 25 M horse heart cytochrome c, 6.0 mM sodium ascorbate, and 0.1 mM TMPD, membranes having been sparged with a CO/O 2 mixture (9:1, v/v) for 20 min prior to polarographic measurements and illuminated with an Oriel 1000-watt xenon lamp equipped with an Oriel monochromator (half-bandwidth, 10 nm), light intensity being normalized to 20 watts⅐m Ϫ2 at each wavelength by use of Kodak Wratten neutral gray filters (Peschek et al., 1989a).
Determination of Protein and Chlorophyll-Protein and chlorophyll were determined according to Bradford (1976) and MacKinney (1941), respectively. The chlorophyll-to-protein ratios of the purified cytoplasmic membranes (fraction CM-II; see Peschek et al. (1989c) and Nicholls et al. (1992)) were Ͻ5 g/mg. 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 Ϯ3%) 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 (Lü bben and Morand, 1994; Lemberg and Barrett, 1973). 3 N. Murata, personal communication. The only other acid-labile heme detectable in these membranes is heme A (Fig. 1A) from the aa 3 -type cytochrome c oxidase, which has been characterized in cytoplasmic and/or thylakoid membranes from 27 different cyanobacterial species (Peschek et al., 1989a(Peschek et al., , 1989bWastyn et al., 1988). 4 After a 24-h incubation period in the light under low oxygen tension (Ͻ10 M final concentration of O 2 in the medium, which is still above the K m of typical cytochrome c oxidases but below the K m 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 2 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 2 only (Fig. 1C). When de-aeration and re-aeration of the cells were performed in strict darkness or in the presence of 20 g/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.)

FIG. 1. Reversed-phase HPLC chromatograms of non-covalently bound hemes from cytoplasmic membranes of A. nidulans. A, phase A cells from photosynthetic
Both hemes A and O represented functional cytochromes (viz. cytochromes a 3 and o 3 , 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 2 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 Lü bben 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. Ͼ30 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 (Lü bben 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)  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 m value of 0.85 M O 2 for the a 3 -type oxidase from phase A cells (Fig. 4A) and of 0.35 M O 2 for the o 3 -type oxidase from semi-anaerobic cells (Fig. 4B). The latter two distinctly different K m values were reproducible within Ϯ10% for altogether six independent membrane preparations, each from phase A and phase B cells, respectively. Though both K m values are still about 1 order of magnitude higher than those of typical mitochondrial cytochrome c oxidase the function of a lower K m form of the oxidase under semi-anaerobic conditions is in qualitative agreement with bioenergetic expectations.
Immunoblots of membrane proteins from semi-anaerobic and re-aerated cells with monospecific antibodies raised against subunits I of aa 3 -type cytochrome c oxidase of P. denitrificans and bo 3 -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 3 -type cytochrome c oxidase of Anacystis (Peschek et al., , 1989aMolitor 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 (Lü bben 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 3 -type and an o 3 -type enzyme (Figs. 2-4).
Similar promiscuity of the heme groups appears to obtain for both high spin, O 2 -reducing hemes and low spin hemes as was found for the "conjugated" cao 3 /caa 3 -type, the bo 3 /ba 3 -type, and the oo 3 /bo 3 -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 I gives 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. FIG. 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 3type (lanes 1 and 2) and E. coli bo 3 -type (lanes 3 and 4) cytochrome c and quinol oxidases, respectively. Lane 5 represents marker proteins.

TABLE I
Oxygen uptake by the cytoplasmic membranes isolated from phase B type Anacystis (see Fig. 1

) as supported by various electron donors
Rates are given as nmol of O 2 /min/mg of protein and corrected for non-enzymatic O 2 uptake measured in the absence of membranes. Polarographic measurements were performed at 30°C in 10 mM potassium phosphate buffer, pH 7.6. Steady-state rates were calculated from linear recorder traces. In order to minimize autoxidations the oxygen concentration at the beginning of measurements was adjusted to 10% air saturation (0.117 mM O 2 in solution). After recording the nonenzymatic oxygen uptake (which was always less than 30% of the membrane-catalyzed rate) enzymatic oxygen uptake was started by addition of approximately 50 g of cytoplasmic membranes to the 2.5 ml of assay suspension in the oxygraph chamber. Inhibitors were added to the desired final concentration after a linear rate of uninhibited oxygen uptake had been established. h 25 M horse heart cytochrome c (in a parallel spectrophotometric assay the initial rate of oxidation of 7 M horse heart ferrocytochrome c was determined to be 120 nmol/min/mg of protein and also fully inhibited by 2.0 M KCN; not shown).