Susanne Fromwald

Functional and immunological characterization of both “mitochondria-like” and “chloroplast-like” electron/proton transport proteins in isolated and purified cyanobacterial membranes

Abstract Plasma membranes (CM) and thylakoid membranes (ICM) were isolated and purified from Anacystis nidulans (Synechococcus PCC6301), Synechocystis PCC6803 and Anabaena PCC7120 after growth in salt stressed and control conditions, harvested from mid-logarithmic (light saturated) and linearly growing (light limited) cultures. Immunoblotting of membrane proteins with monospecific antibodies raised against authentic cytochrome-c oxidases (subunits I and II), cytochromes f and c1, subunit IV of the chloroplast cytochrome b6f complex, NDHJ protein (a subunit of the mitochondrial NADH dehydrogenase complex), the β-subunit of the (mitochondrial or chloroplast) F2 coupling factor ATPase, and the manganese-stabilizing 33-kD-protein of photosystem II gave specific and consistent cross-reactions with both plasma and thylakoid membranes but relative intensities of the immuno-reactions on CM and ICM depended strongly on the growth conditions of the organisms. Immunological cross-reactions were in agreement with inhibition profiles of the reactions catalysed by the respective proteins. From these two lines of evidence it was concluded that, while the cytochrome-c oxidase, the cyt b6f complex, and the F-type ATPase are present in both CM and ICM (relative shares depending on growth conditions), the NDHJ protein and cytochrome c1 (cyt bc1 complex) are almost exclusively situated in the plasma membrane, as is the P-type ATPase, while the 33-kD-protein is preferentially located in the thylakoid membranes. Stress conditions lead to increased synthesis and incorporation of “mitochondria-like” electron/proton transport proteins into the plasma membrane and to elevated concentrations of the soluble electron transport protein cytochrome c6 in the periplasmic space while intrathylakoidal concentrations remain unaffected. Results are also discussed with respect to the occurrence of immunologically and functionally identical electron/proton transport proteins in two different compartments of a single (“uncompartmentalized”) prokaryotic cell, viz. a cyanobacterium.

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).