Marnik Wastyn

[45] Characterization of cytochrome-c oxidase in isolated and purified plasma and thylakoid membranes from cyanobacteria

Publisher Summary Recently methods have been devised for the successful separation of physiologically active plasma (CM) and thylakoid (ICM) membranes from cyanobacteria. The results, however, with respect to the presence or absence of aa 3 -type cytochrome oxidase in the CM at first were inconsistent. This chapter focuses on membrane isolation, purification, and identification, growth conditions that permit optimum recovery of aa 3 -type cytochrome oxidase activity in the CM of certain cyanobacteria. Identification of CM can be achieved by external labeling of intact cells prior to disruption and membrane separation using an impermeant covalent protein marker, diazobenzene [ 35 S]sulfonate (DABS), or fluorescamine which binds covalently to primary amino groups to give a fluorescent conjugate with maximum emission at 475 nm (excitation at 390 nm) but which itself is nonfluorescent and decomposes very rapidly in aqueous media to give nonfluorescent products. Identification of the oxidase is carried out by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE), followed by Western blotting, and cross-reaction of transferred polypeptide bands with (polyclonal) antibodies raised against subunits I and II, and the holoenzyme, of Paracoccus denitrificans aa 3 -type cytochrome oxidase.

Respiratory activities and aa3-type cytochrome oxidase in plasma and thylakoid membranes from vegetative cells and heterocysts of the cyanobacterium Anabaena ATCC 29413

Abstract Plasma and thylakoid membranes were separated by discontinuous sucrose density gradient centrifugation from crude membrane preparations of vegetative cells and heterocysts of the nitrogen-fixing cyanobacterium Anabaena ATCC 29413. Isolated and purified plasma membranes were devoid of spectroscopically detectable chlorophyll. Intact heterocysts were separated from vegetative cells by selective lysozyme degradation of the latter, followed by low-pressure French-press extrusion and differential centrifugation. Each of the four types of membrane preparation oxidized horse heart ferrocytochrome c , rates ranging from 35 nmol/min per mg protein (plasma membrane from vegetative cells) to 2400 nmol/min per mg protein (thylakoid membranes from heterocysts). The reactions were stimulated up to 6-fold by 0.05% (w/v) n -octyl glucoside; they were completely inhibited by 1.2 μM KCN and severely inhibited by CO. The cytochrome oxidase present in n -octylglucoside-solubilized membranes could be enriched about 10-fold by affinity chromatography on immobilized yeast cytochrome c . Extraction with acidic butanone-2 or acetone, followed by preparation of the alkaline pyridine ferrohemochrome derivatives, proved the presence of a -type cytochrome. Sodium dodecylsulfate polyacrylamide gel electropherograms of the four membrane types were characteristically different from each other. Immunoblotting of the membrane polypeptides with antisera against the aa 3 -type cytochrome of Paracoccus denitrificans (subunits I and II and holoenzyme) and rat liver mitochondria (subunit II) gave specific and complementary cross-reaction at about 48–49 and 36 kDa (subunits I and II, respectively) irrespective of the membranes used. In addition to oxidizing ferrocytochrome c , the isolated membranes were also found to reduce horse heart ferricytochrome c to variable extents with NAD(P)H.

‘Respiratory protection’ of the nitrogenase in dinitrogen-fixing cyanobacteria

Several filamentous and unicellular cyanobacteria were grown photoautotrophically with nitrate or dinitrogen as N-sources, and some respiratory properties of the cells or isolated plasma (CM) and thylakoid (ICM) membranes were compared. Specific cytochrome c oxidase activities in membranes from dinitrogen-fixing cells were between 10- and 50-times higher than those in membranes from nitrate-grown cells, ICM of heterocysts but CM of unicells being mainly responsible for the stimulation. Whole cell respiration (oxygen uptake) of diazotrophic unicells paralleled increased cytochrome oxidase activities of the isolated membranes. Mass spectrometric measurements of the uptake of isotopically labeled oxygen revealed that (low) light inhibited respiration of diazotrophic unicells to a much lesser degree than that of nitrate-grown cells which indicates the prevailing (respiratory) role of CM in the former. Normalized growth yields of diazotrophic unicells grown in continuous light were significantly higher than those of cells grown in a 12/12 hrs light/dark cycle. Mass spectrometry showed that overall nitrogen uptake by the former was higher than by the latter; in particular, and in marked contrast to the time course of nitrogenase activity (acetylene reduction) there was no appreciable nitrogen uptake or protein synthesis during dark periods; likewise, there was no 14-CO2 fixation, nor chloropholl synthesis, nor cell division in the dark. By contrast, growth in continuous light gave sustained rates of nitrogen and carbon dioxide incorporation over the whole time range. Our results will be discussed in terms of ‘respiratory protection’ as an essential strategy of keeping apart nitrogenase and oxygen, either atmospheric or photosynthetically produced within the same cell.

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 b 6/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.

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 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 unusualallosteric regulation ofa prokaryotic cytochrome‐c oxidase is discussed.

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 μM dissolved O2 in the medium) besides heme B only heme A was found while in low‐oxygen cells (<10 μM 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 K m values of 5–50 μM O2 for oxygenase as compared to K m 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.

Extended Heme Promiscuity in the Cyanobacterial Cytochrome c Oxidase

Though there is good evidence that primordial cyanobacteria, having introduced the first substantial quantities of molecular oxygen into an essentially anoxic biosphere by way of their unique oxygenic, plant-type photosynthesis, consequently were the first aerobic respirers as well (1-3), cyanobacterial respiration has not been receiving too much attention so far (4). Only quite recently, after preliminary spectroscopic and kinetic results (5,6), was the terminal respiratory oxidase from Anacystis nidulans(Synechococcussp. PCC6301) biochemically, immunologically and genetically characterized as an aa3-type enzyme (7—9). Immunoscreening of almost thirty different strains and species of cyanobacteria has given evidence that the same type of aa3-type cytochrome-c oxidase is present in both cytoplasmic and thylakoid membranes (CM and ICM) of all species investigated (10, 11). Careful kinetic investigation on the NAD(P)H-oxidizing activity in these membranes clearly demonstrated that the terminal respiratory oxidase in wild-type cyanobacteria is a cytochrome-c oxidase, not a quinol oxidase (12,13), although the addition of external cytochrome c to the isolated membranes was not always necessary to catalyse electron transport between NADH and O2(14).

Adenylate Regulation of the Cyanobacterial Cytochrome c Oxidase

According to common reasoning primordial cyanobacteria were the first to introduce molecular oxygen into a previously near-anoxic biosphere (1—3). Thereby they basically initiated the whole evolutionary succession from aerobic prokaryotes to “primitive” eukaryotes, metaphyta, metazoa and up to the very Homo sapiensall of which essentially depend on aerobic respiration and oxidative phosphorylation for energy supply (4—6).Having identified the terminal respiratory oxidase in almost thirty different strains and species of cyanobacteria as a mitochondria-like aa3-type cytochrome c oxidase by means of conventional biochemical and immunological techniques (7—10) we succeeded in cloning and sequencing a cta operon-like genomic structure from Synechocystis sp. PCC6803with a very high degree of homology to other bacterial cta operons, particularly as far as the ctaCDE genes are concerned which, in eukaryotes, encode the mitochondrial subunits I-III (11—16). A very similar cta operon was recently published for the taxonomically more uncertain cyanobacterium Synechococcus vulcanus(17,18). When the entire genomic sequence of Synechocystis 6803was published (Ref. 19; available also on the internet under http://www.kazusa.or.jp/cyano) it turned out that this genome contained potential coding regions for an aa3-type cytochrome c oxidase, and an aa3-type and a d-type quinol oxidase. Yet, while Sugiura’s cyt-c oxidase displayed >95% deduced amino acid identity to the biochemically characterized cyanobacterial cyt-c oxidase (11—13) and >50% identity to the deduced S. vulcanusproteins (17,18) no indications of a functional quinol oxidase were so far detected in any wild-type cyanobacterium (20—23). Therefore, considering the fact that primordial cyanobacteria most probably not only were the first oxygenic photosythesizers but also the first aerobic respirers we have suggested that, of the two known types of aerobic terminal oxidases, viz. cytochrome c and quinol oxidases (24), the cytochrome c oxidase preceded the quinol oxidase in evolution (25).