Occurrence of heme O in photoheterotrophically growing, semi-anaerobic cyanobacterium Synechocystis sp. PCC6803.

Extraction and identification of the non-covalently bound heme groups from crude membrane preparations of photoheterotrophically grown Synechocystis sp. PCC 6803 by reversed phase high performance liquid chromatography and optical spectrophotometry led to the detection of heme O in addition to hemes B and A which latter was to be expected from the known presence of aa3-type cytochrome oxidase in cyanobacteria. In fully aerated cells (245 microM dissolved O2 in the medium) besides heme B only heme A was found while in low-oxygen cells (< 10 microM dissolved O2) heme O was present at a concentration even higher than that of heme A. Given the possible role of heme O as a biosynthetic intermediate between heme B and heme A, together with generally much higher Km values of 5-50 microM O2 for oxygenase as compared to Km values of 40-70 nM O2 for typical cytochrome-c oxidase, our findings may permit the conclusion that the conversion of heme O to heme A is an obligately oxygen-requiring process catalyzed by some oxygenase directly introducing oxygen from O2 into the 8-methyl group of heme O. At the same time thus the occurrence of heme O (cytochrome o) in cyanobacteria does of course not imply the existence of an 'alternative oxidase' since according to the well-known 'promiscuity of heme groups' both hemes O and A are likely to combine with one and the same apoprotein.

Immunologically cross-reactive and redox-competent cytochrome b6/f-complexes in the chlorophyll-free plasma membrane of cyanobacteria.

Plasma and thylakoid membranes were separated and purified from cell-free extracts of the cyanobacteria Anacystis nidulans, Synechocystis 6714, Anabaena variabilis and Nostoc sp. strain Mac. Immunoblots of the membrane proteins using antisera raised against subunits I-IV of the chloroplast b6/f-complex gave evidence for the presence of a homologous complex in both plasma and thylakoid membranes from the four species of cyanobacteria investigated. Both plasma and thylakoid membranes catalyzed the electron transfer from (exogenous) plastoquinol-9 and NADH to horse heart ferricytochrome c. However, while with plasma membranes these reactions were severely inhibited by low concentrations of antimycin A and rotenone, respectively, the inhibitors were without major effect on thylakoid membranes. The results will be discussed in terms of a possible similarity (analogy and/or homology?) of cyanobacterial plasma membranes to the inner mitochondrial membrane.

Immunological and spectral characterization of partly purified cytochrome oxidase from the cyanobacterium Synechocystis 6714.

Membranes were isolated by French pressure cell extrusion of lysozyme-preincubated cells of the cyanobacterium Synechocystis 6714 after growth in the presence of 0.4 M NaCl for 4 days. These cells showed up to 6-fold respiratory activity (oxygen uptake) when compared to control cells. Separation of plasma and thylakoid membranes revealed that the major part of cytochrome c oxidase was associated with the latter. Immunoblotting of sodium dodecylsulfate polyacrylamide gel electrophorized membranes with antisera raised against subunit I, subunit II, and the holoenzyme of the aa3-type cytochrome oxidase from Paracoccus denitrificans gave specific and complementary cross-reactions at apparent molecular weights of about 25 and 17-18 kDa, respectively. Crude membranes were solubilized also with n-octyl glucoside, and the cytochrome oxidase was separated from the extract by affinity chromatography using immobilized cytochrome c from Saccharomyces cerevisiae. The enzyme was eluted with KCl/octyl glucoside. Dialysed and concentrated enzyme solution, which was free of b- and c-type cytochromes, gave reduced alpha- and gamma-peaks around 603 and 443 nm, respectively. Upon treatment of the sample with carbon monoxide the peaks were found at 593 and 433 nm, respectively. Photodissociation spectra of the CO-complexed enzyme were in full agreement with cytochrome aa3 being a functional cytochrome oxidase in Synechocystis 6714.

Acidic cytochrome c6 of unicellular cyanobacteria is an indispensable and kinetically competent electron donor to cytochrome oxidase in plasma and thylakoid membranes.

Cytochromes c6 from three cyanobacteria were tested as substrates for membranous cyt. c oxidase(aa3) of Anacystis and Synechocystis using intact spheroplasts or isolated plasma(CM) and thylakoid(ICM) membranes. Neither spheroplasts nor CM/ICM gave significant O2 uptake rates with NADH without added cyt. c. Horse cyt. c (at low ionic strength) or cyt. c6 from Anacystis, Synechocystis or Microcystis (at high ionic strength) supported substantial HCN- & CO-sensitive NADH oxidase activity, consistent with in vivo O2 uptake. Cyanobacterial respiratory electron transport involves NADH dehydrogenase(fpN), plastoquinone, cyt. b/c(f), cyt. c6 & cyt. aa3, in both CM & ICM. In ICM, fpN and cyt. aa3 are functionally replaced in the light by PS II and PS I, respectively. In both membranes, cyt. c6 is an obligatory electron donor to cyt. aa3 &/or to P700. Respiratory action of acidic cyt. c6 (in unicellular species) may be unmasked only under conditions of elevated ionic strength.

Characterization of the cytochrome c oxidase in isolated and purified plasma membranes from the cyanobacterium Anacystis nidulans.

Functionally intact plasma membranes were isolated from the cyanobacterium (blue-green alga) Anacystis nidulans through French pressure cell extrusion of lysozyme/EDTA-treated cells, separated from thylakoid membranes by discontinuous sucrose density gradient centrifugation, and purified by repeated recentrifugation. Origin and identity of the chlorophyll-free plasma membrane fraction were confirmed by labeling of intact cells with impermeant protein markers, [35S]diazobenzenesulfonate and fluorescamine, prior to membrane isolation. Rates of oxidation of reduced horse heart cytochrome c by purified plasma and thylakoid membranes were 90 and 2 nmol min-1 (mg of protein)-1, respectively. The cytochrome oxidase in isolated plasma membranes was identified as a copper-containing aa3-type enzyme from the properties of its redox-active and EDTA-resistant Cu2+ ESR signal, the characteristic inhibition profile, reduced minus oxidized difference spectra, carbon monoxide difference spectra, photoaction and photodissociation spectra of the CO-inhibited enzyme, and immunological cross-reaction of two subunits of the enzyme with antibodies against subunits I and II, and the holoenzyme, of Paracoccus denitrificans aa3-type cytochrome oxidase. The data presented are the first comprehensive evidence for the occurrence of aa3-type cytochrome oxidase in the plasma membrane of a cyanobacterium similar to the corresponding mitochondrial enzyme (EC 1.9.3.1).

Extended heme promiscuity in the cyanobacterial cytochrome c oxidase: characterization of native complexes containing hemes A, O, and D, respectively.

The cyanobacteria Anacystis nidulans (Synechococcus sp. PCC6301), Synechocystis sp. PCC6803, Anabaena sp. PCC 7120, and Nostoc sp. PCC8009 were grown photoautotrophically under reduced oxygen tension in a medium with sulfate replaced by thiosulfate and nitrate replaced by ammonium as the S- and N-sources, respectively. In addition, Anabaena and Nostoc were grown under dinitrogen-fixing conditions in a medium free of combined nitrogen. Membranes were isolated from late-logarithmic cells (culture density corresponding to approximately 3 microliters packed cells per milliliter); cytoplasmic and thylakoid membranes were separated and purified according to established procedures. Acid-labile hemes were extracted from the membranes and subjected to reversed-phase high-performance liquid chromatography. Separated hemes were analyzed spectroscopically and identified by comparison with authentic standards. In addition to hemes B, A, and O, the latter of which was induced under semianaerobic conditions only, substitution of thiosulfate and ammonium for the oxy-anions sulfate and nitrate led to the appearance of spectrally discernible heme D in the membranes and extracts therefrom. However, spectroscopic and kinetic investigation of the membrane-bound heme D rather disproved any reaction with oxygen or carbon monoxide. Kinetic measurements performed with the membrane-bound respiratory oxidase gave evidence for only two kinetically competent terminal oxidases, a3 and o3, both apparently associated with a single type of apoprotein, viz. subunit I of the known cyanobacterial aa3-type cytochrome c oxidase. The heme D, on the other hand, seems to form a spectrally distinguished, yet kinetically ill-defined hemoprotein complex which does not qualify as a fully functional d-type terminal oxidase on our (wild-type) cyanobacteria even after growth under semianaerobic pseudo-reducing conditions. Also growth (of Anabaena and Nostoc) under dinitrogen-fixing conditions did not change this situation. Thus, we are left with (wild-type) cyanobacteria forming an unbranched respiratory chain with only a single type of terminal oxidase protein, viz. the known aa3-type cytochrome c oxidase. This oxidase, however, may incorporate different prosthetic (heme) groups in the sense of "heme promiscuity." Biosynthesis of the different heme groups thereby seems to respond to the ambient redox environment. In particular, however, conditions for expression of the two quinol oxidases potentially and additionally coded for by the genome of, e. g., Synechocystis sp. PCC6803 (see http://www.kazusa.or.jp/cyano), have not yet been found.

Chlorophyll precursors in the plasma membrane of a cyanobacterium, Anacystis nidulans. Characterization of protochlorophyllide and chlorophyllide by spectrophotometry, spectrofluorimetry, solvent partition, and high performance liquid chromatography.

Plasma membranes were isolated and separated from thylakoid membranes by discontinuous sucrose density gradient centrifugation of crude membranes prepared by French pressure cell extrusion of lysozyme-treated Anacystis nidulans. Two distinct populations of chlorophyll-free plasma membrane vesicles were obtained exhibiting buoyant densities of 1.087 and 1.100 g/cm3 as opposed to a uniform density of 1.192 g/cm3 for thylakoid membranes. Plasma and thylakoid membranes were characteristically different also with respect to fatty acid and protein composition, cytochrome oxidase activity, and pigment content as analyzed by spectrophotometry, spectrofluorimetry, and high performance liquid chromatography. Apart from carotenoids, chlorophyll a was the only major photosynthetic pigment detected in thylakoid membranes while plasma membranes contained virtually no chlorophyll a but (besides large amounts of carotenoids) protochlorophyllide a and chlorophyllide a as revealed by solvent partition (between n-hexane and acetone or methanol), room and low temperature fluorescence emission and excitation spectra, and analytical separation and identification by high performance liquid chromatography and comparison with authentic standards. The protochlorophyllide in the plasma membrane could be transformed into chlorophyllide in the dark in vitro by incubating the membrane preparation with NADPH; NADP+ effected the reverse transition.

Promiscuity of heme groups in the cyanobacterial cytochrome-C oxidase.

The cyanobacteria Nostoc sp. strain Mac, Anabaena 7937, Synechocystis 6803, and Anacystis nidulans (Synechococcus 6301) were grown and incubated in the light under three different oxygen regimes: Phase-A cells were harvested from photoautotrophically growing cultures at a cell density of 2.8-3.2 microliter packed cell mass/ml and an oxygen concentration of approx. 350 microM (corresponding to > 150% air saturation). Phase-B cells were harvested 24 hrs after 20 microM 3-(3,4-dichlorophyl)-1,1-dimethylurea had been added to the culture and gassing switched to 1% oxygen (< 10 microM). Phase-C cells originated from phase-B cells after 12 hrs of gassing the illuminated, yet non-growing cultures with air (21% oxygen or 200-220 microM in the medium). Cytoplasmic membranes were isolated and purified from each of the three cell types. Non-covalently bound hemes were extracted and identified by reversed-phase high performance liquid chromatography. Besides ubiquitous heme B only heme A was detected in phase-A membranes while phase-B and phase-C membranes contained both hemes A and O proportions of which depended on the oxygen status of the cells. CO/difference spectra, photo-action spectra of CO-inhibited oxygen uptake, and polarographic determination of oxygen-affinities clearly showed that both hemes A and O were part of a functional form of cytochrome-c oxidase which, however, exhibited a single subunit-I apoprotein as verified by immunoblotting. Also electron transport characteristics did not give evidence for a quinol or any other alternate oxidase functioning in cyanobacteria.

Allosteric properties of cyanobacterial cytochrome c oxidase: inhibition of the coupled enzyme by ATP and stimulation by ADP.

Thorough analysis of the cta operon of Synechocystis sp. PCC6803 (grown in high-concentration salt medium to enhance the expression of respiratory proteins) showed that, apart from ctaCDE and Fb genes potentially encoding subunits I, II, III, and a small pseudo-bacteria-like subunit-IV of unknown function, a large mitochondria-like cta-Fm gene and a pronounced terminator structure are additional components of the operon. The deduced cta Fm gene product shows approximately 50% and 20% sequence identity to the Saccharomyces cerevisiae and beef heart mitochondrial COIV proteins, respectively. It also shows amino acid regions (near the N terminus, on the cytosolic side) with conspicuous sequence similarities to adenylate-binding proteins such as ATP synthase beta subunit Walker A and B consensus regions or to adenylate kinase. We suggest that, similar to the situation with beef heart mitochondria, it is the mitochondria-like subunit-IV of the cyanobacterial aa3-type cytochrome-c oxidase that confers allosteric properties to the cyanobacterial enzyme, the H+/e- ratios of cytochrome c oxidation being significantly lowered by ATP (intravesicular or intraliposomal) but enhanced by ADP. Therefore, the antagonistic action of ATP and ADP was in a way that the redox reaction proper, was always significantly less affected than the coupled proton translocation. Evolutionary and ecological implications of the unusual allosteric regulation of a prokaryotic cytochrome-c oxidase is discussed.