Occurrence of cytochrome aa3 in Anacystis nidulans.

The cytochrome content of membrane fragments prepared from the blue-green alga (cyanobacterium) Anacystis nidulans was examined by difference spectrophotometry. Two beta-type cytochromes and hitherto unknown cytochrome alpha could be characterized. In the reduced-minus-oxidised difference spectra the alpha-type cytochrome showed an alpha-band at 605 nm and a gamma-band at 445 nm. These bands shifted to 590 and 430 nm, respectively, in CO difference spectra, NADPH, NADH and ascorbate reduced the cytochrome through added horse heart cytochrome c as electron mediator. In presence of KCN the reduced-minus-oxidised spectrum showed a peak at 600 nm and a trough at 604 nm. Photoaction spectra of O2 uptake and of horse heart cytochrome c oxidation by CO-inhibited membranes showed peaks at 590 and 430 nm. These findings are consistent with cytochrome aa3 being the predominant respiratory cytochrome c oxidase in Anacystis nidulans.

Anaerobic hydrogenase activity in Anacystis nidulans. H2-dependent photoreduction and related reactions.

1. Anaerobic hydrogenase activity in whole cells and cell-free preparations of H2-induced Anacystis was studied both manometrically and spectrophotometrically in presence of physiological and artificial electron acceptors. 2. Up to 90% of the activity measured in crude extracts were recovered in the chlorophyll-containing membrane fraction after centrifugation (144 000 X g, 3 h). 3. Reduction of methyl viologen, diquat, ferredoxin, nitrite and NADP by the membranes was light dependent while oxidants of more positive redox potential were reduced also in the dark. 4. Evolution of H2 by the membranes was obtained with dithionite and with reduced methyl viologen; the reaction was stimulated by detergents. 5. Both uptake and evolution of H2 were sensitive to O2, CO, and thiolblocking agents. The H2-dependent reductions were inhibited also by the plastoquinone antagonist dibromothymoquinone, while the ferredoxin inhibitor disalicylidenepropanediamine affected the photoreduction of nitrite and NADP only. 3-(3,4-Dichlorophenyl)-1,1-dimethylurea did not inhibit any one of the H2-dependent reactions. 6. The results present evidence for a membrane-bound 'photoreduction' hydrogenase in H2-induced Anacystis. The enzyme apparently initiates a light-driven electron flow from H2 to various low-potential acceptors including endogenous ferredoxin.

Aerobic hydrogenase activity in Anacystis nidulans. The oxyhydrogen reaction.

1. The oxyhydrogen reaction of Anacystis nidulans was studied manometrically and polarographically in whole cells and in cell-free preparations; the activity was found to be associated with the particulate fraction. 2. Besides O2, the isolated membranes reduced artificial electron acceptors of positive redox potential; the reactions were unaffected by O2 levels less than 10--15%; aerobically the artificial acceptors were reduced simultaneously with O2. 3. H2-supported O2 uptake was inhibited by CO, KCN and 2-n-heptyl-8-hydroxyquinoline-N-oxide. Inhibition by CO was partly reversed by strong light. Uncouplers stimulated the oxyhydrogen reaction. 4. The kinetic properties of O2 uptake by isolated membranes were the same in presence of H2 and of other respiratory substrates. 5. Low rates of H2 evolution by the membrane preparations were found in presence of dithionite; methyl viologen stimulated the reaction. 6. The results indicate that under certain growth conditions Anacystis synthesizes a membrane-bound hydrogenase which appears to be involved in phosphorylative electron flow from H2 to O2 through the respiratory chain.

The gene encoding cytochrome-c oxidase subunit I from Synechocystis PCC6803.

The gene (coxI or CoxA) encoding subunit I (COI) of cytochrome-c oxidase (cytochrome aa3) of Synechocystis PCC6803, Synechococcus PCC7942 (Anacystis nidulans R2) and Nostoc PCC8002 (Nostoc Mac), was identified by heterologous hybridization of chromosomal digests with a 17-bp oligodeoxyribonucleotide (probe C) derived from the coxI of Paracoccus denitrificans. A single genomic fragment was found to bind to probe C in all chromosomal digests. Due to its favorable signal-to-noise ratio, the genome of Synechocystis was chosen for the isolation and sequencing of this gene. A genomic DNA library in pUC18 was screened with probe C. The two probe C-positive plasmids, pDAUV1 and pDAUV2, contained a 1-kb overlapping region, with the conserved 17-bp sequence encoding the CuB-binding region of the COI polypeptide. These plasmids were subcloned into competent Escherichia coli DH5 alpha cells, and the nucleotide sequences were determined. The deduced amino acid (aa) sequences of Synechocystis COI and homologous proteins from a variety of prokaryotic and eukaryotic organisms showed an overall similarity of between 38.6 and 45.8%. Hydropathy plots revealed 12 potential transmembrane helices. All of the six histidines needed for the binding of heme a and the heme a3/CuB bimetallic center are present in the expected positions of the Synechocystis COI protein (533 aa, M(r) 59,390). A monospecific antibody raised against P. denitrificans COI gave an unequivocal immunological cross-reaction on Western blots of membrane preparations from Synechocystis, Anacystis and Nostoc, showing that the product of gene coxI is indeed synthesized and incorporated into cyanobacterial membranes.

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.

Nucleotide sequence analysis, overexpression in Escherichia coli and kinetic characterization of Anacystis nidulans catalase-peroxidase.

Bifunctional catalase-peroxidases are the least understood type of peroxidases. A high-level expression in Escherichia coli of a fully active recombinant form of a catalase-peroxidase (KatG) from the cyanobacterium Anacystis nidulans (Synechococcus PCC 6301) is reported. Since both physical and kinetic characterization revealed its identity with the wild-type protein, the large quantities of recombinant KatG allowed the examination of both the spectral characteristics and the reactivity of its redox intermediates by using the multi-mixing stopped-flow technique. The homodimeric acidic protein (pI = 4.6) contained high catalase activity (apparent K(m) = 4.8 mM and apparent k(cat) = 8850 s(-1)). Cyanide is shown to be an effective inhibitor of the catalase reaction. The second-order rate constant for cyanide binding to the ferric protein is (6.9 +/- 0.2) x 10(5) M(-1 )s(-1) at pH 7.0 and 15 degrees C and the dissociation constant of the cyanide complex is 17 microM. Because of the overwhelming catalase activity, peroxoacetic acid has been used for compound I formation. The apparent second-order rate constant for formation of compound I from the ferric enzyme and peroxoacetic acid is (1.3 +/- 0.3) x 10(4 )M(-1 )s(-1) at pH 7.0 and 15 degrees C. The spectrum of compound I is characterized by about 40% hypochromicity, a Soret region at 406 nm, and isosbestic points between the native enzyme and compound I at 355 and 428 nm. Rate constants for reduction of KatG compound I by o-dianisidine, pyrogallol, aniline and isoniazid are shown to be (7.3 +/- 0.4) x 10(6) M(-1 )s(-1), (5.4 +/- 0.3) x 10(5) M(-1 )s(-1), (1.6 +/- 0.3) x 10(5) M(-1 )s(-1) and (4.3 +/- 0.2) x 10(4) M(-1 )s(-1), respectively. The redox intermediate formed upon reduction of compound I did not exhibit the classical red-shifted peroxidase compound II spectrum which characterizes the presence of a ferryl oxygen species. Its spectral features indicate that the single oxidizing equivalent in KatG compound II is contained on an amino acid which is not electronically coupled to the heme.

Purification and characterization of a hydroperoxidase from the cyanobacterium Synechocystis PCC 6803: identification of its gene by peptide mass mapping using matrix assisted laser desorption ionization time-of-flight mass spectrometry.

A cytosolic catalase-peroxidase from the cyanobacterium Synechocystis PCC 6803 was purified to homogeneity by a six-step purification procedure. It is a homodimeric enzyme with a subunit molecular mass of 85 kDa. The isoelectric point of the protein is at pH 5.5; Michaelis constant, turnover number, and catalytic efficiency of the catalase activity for H2O2 were measured to be 4.8 mM, 3450 s-1, and 7.2 x 10(5) M-1 s-1, respectively. Preparation and spectroscopy of the pyridine ferrohemochrome identified an iron protoporphyrin IX as the prosthetic group. The enzyme was shown to exhibit both catalase and peroxidase activities, both of which were inhibited by cyanide, leading to a high-spin to low-spin transition of the heme iron center as detected by a shift of the Soret peak from 405 to 421 nm. The catalase-specific inhibitor 3-amino-1,2,4-triazole proved ineffective. o-Dianisidine, pyrogallol and guaiacol functioned as a peroxidatic substrate, but no reaction was detected with NADH, NADPH, glutathione, and ascorbate. Peptide mass mapping using matrix assisted laser desorption ionization time-of-flight mass spectrometry showed the identity between the purified protein and a putative katG gene derived from the genome of Synechocystis PCC 6803. A comparison of amino acid sequences of the catalase-peroxidase from Synechocystis PCC 6803 and those from other bacteria showed a high homology around the assumed distal and proximal histidine residues, suggesting a highly conserved histidine as the fifth ligand of the heme iron.

Catalase-peroxidases in cyanobacteria--similarities and differences to ascorbate peroxidases.

Cyanobacteria (blue-green algae) are oxygenic phototrophic bacteria carrying out plant-type photosynthesis. The only hydrogen peroxide scavenging enzymes in at least two unicellular species have been demonstrated to be bifunctional cytosolic catalase-peroxidases (CatPXs) having considerable homology at the active site with plant ascorbate peroxidases (APXs). In this paper we examined optical and kinetic properties of CatPXs from the cyanobacteria Anacystis nidulans and Synechocystis PCC 6803 and discuss similarities and differences to plant APXs. Both CatPXs and APX showed similar spectra of the ferric enzyme, the redox intermediate Compound I and the cyanide complex, whereas the spectrum of CatPX Compound II had characteristics reminiscent of the spectrum of the native enzyme. Both steady-state and multi-mixing transient-state kinetic studies were performed in order to characterize the kinetic behaviour of CatPXs. Bimolecular rate constants of both formation and reduction of a CatPX Compound I are presented. Because of its intrinsic high catalase activity (which cannot be found in APXs), the rate constants for Compound I formation were measured with peroxoacetic acid and are shown to be 5.9 x 10(4) M(-1) s(-1) for CatPX from A. nidulans and 8.7 x 10(3) M(-1) s(-1) for the Synechocystis enzyme. Compared with o-dianisidine (2.7-6.7 x 10(6) M(-1) s(-1)) and pyrogallol (8.6 x 10(4)-1.6 x 10(5) M(-1) s(-1)), the rate constant for Compound I reduction by ascorbate was extremely low (5.4 x 10(3) M(-1) s(-1) at pH 7.0 and 15 degrees C), in marked contrast to the behaviour of APXs.

The respiratory chain of blue-green algae (cyanobacteria).

Electron transport components on the way from reduced substrates to the terminal respiratory oxidase(s) are discussed in relation to analogous and/or homologous enzymes and electron carriers in the generally much better known bacteria, mitochondria and chloroplasts. The kinetic behaviour of the components, their localization within the cell and their evolutionary position are given special attention. Pertinent results from molecular genetics are also mentioned. The unprecedented role of cyanobacteria for our biosphere and our whole planet earth appears to deserve a more extended introductory chapter.

Evolutionary considerations on the thermodynamics of nitrogen fixation.

An evolutionary explanation is sought for the fact that ATP is needed for N2 fixation in spite of the exergonicity of the process. After a survey of the state of knowledge about the thermodynamics of N2 fixation in fermenters, photosynthesizers and respirers it is suggested that nitrogenase, which still shows ATP-dependent hydrogenase activity, evolved from an ATP-requiring hydrogenase that lacked nitrogenase activity. The hydrogenase action in the Archaean, reducing, biosphere may have needed ATP to ensure expulsion of H2. Extant non-nitrogenase hydrogenases have lost the dependence on ATP. Because of its complexity, nitrogenase could not rid itself of the ATP dependence or of hydrogenase activity, both wasteful. Presumably all hydrogenases evoled from ferredoxin-like Fe-S proteins.

Nitrogen fixation as evidence for the reducing nature of the early biosphere.

Probably the first nitrogen fixers were anaerobic, non-photosynthetic, bacteria, i.e. fermenters. During the evolution of N2 fixation they still needed nitrogen on the oxidation level of ammonia. Because of the complexities in structure and function of nitrogenase this evolution must have required a long time. The photosynthetic and later the respiring bacteria inherited the capacity for N2 fixation from the fermenters, but the process did not change a great deal when it was taken over. Because of the long need for NH3, which is unstable in a redoxneutral atmosphere, a long-persisting reducing atmosphere was needed. The transition to a redoxneutral atmosphere, dominated by CO2, H2O and N2, cannot have been rapid, and the NH3 in it was recycled. Probably the atmosphere contained for a long time, as was suggested by Urey but is often denied now, a great deal of methane as a reductant. The recycling occurred with participation of intermediates like cyanide, through energy input as UV radiation or as electric discharges. A stationary state was set up. The hypothesis is recalled that coloured, photosynthetic, NH3 bacteria, analogous to coloured sulphur bacteria, may have existed, or may still exist, in reducing conditions. A few remarks are made about the origin of nitrification in the later, oxidizing atmosphere.

Restoration of respiratory electron-transport reactions in quinone-depleted particle preparations from Anacystis nidulans.

Electron transport from H2, NADPH, NADH and succinate to O2 or ferricytochrome c in respiratory particles isolated from Anacystis nidulans in which hydrogenase had been induced was abolished after extraction of the membranes with n-pentane; oxidation of ascorbate plus NNN'N'-tetramethyl-p-phenylenediamine remained unaffected. Incorporation of authentic ubiquinone-10, plastoquinone-9, menaquinone-7 and phylloquinone (in order of increasing efficiency) restored the electron-transport reactions. ATP-dependent reversed electron flow from NNN'N'-tetramethyl-p-phenylenediamine to NADP+ or, via the membrane-bound hydrogenase, to H+ was likewise abolished by pentane extraction and restored by incorporation of phylloquinone. Participation of the incorporated quinones in the respiratory electron-transport reactions of reconstituted particles was confirmed by measuring the degree of steady-state reduction of the quinones. Isolation and identification of the quinones present in native Anacystis membranes yielded mainly plastoquinone-9 and phylloquinone; neither menaquinone nor alpha-tocopherolquinone could be detected. Together with the results from reconstitution experiments this suggests that phylloquinone might function as the main respiratory quinone in Anacystis nidulans.

The cytochrome f-b electron-transport complex. A common link between photosynthesis and respiration in the cyanobacterium Anacystis nidulans.

Reduced-minus-oxidized difference spectra were recorded on particle preparations of the cyanobacterium Anacystis nidulans. Physiological oxidation of anaerobic membranes was effected either by O2 or by light. In both cases the spectral changes observed in the 550-570nm region were essentially the same. The results were confirmed by dual-wavelength spectrophotometry. It is concluded that a membrane-bound cytochrome f-b complex participates in both respiratory and photosynthetic elevtron transport.

Proton pump coupled to cytochrome c oxidase in the cyanobacterium Anacystis nidulans.

Intact spheroplasts of the cyanobacterium Anacystis nidulans were found to oxidize various exogenous c-type cytochromes with concomitant proton extrusion. In the coupled state, H+/e stoichiometries close to 1 were measured, regardless of absolute reaction rates. It is concluded that the proton translocation observed is an intrinsic property of the cytoplasmic membrane-bound cytochrome c oxidase of A. nidulans.

Characterization of the Proton-Translocating Cytochrome c Oxidase Activity in the Plasma Membrane of Intact Anacystis nidulans Spheroplasts.

Intact spheroplasts of the cyanobacterium (blue-green alga) Anacystis nidulans oxidized various exogenous c-type cytochromes with concomitant outward proton translocation while exogenous ferricytochrome c was not reduced. The H(+)/e(-) stoichiometry was close to 1 with each of the cytochromes and did not depend on the actual rate of the oxidase reaction. Observed proton ejections were abolished by the uncoupler carbonyl cyanide m-chlorophenylhydrazone. Cyanide, azide, and carbon monoxide inhibited cytochrome c oxidation and proton extrusion in parallel while dicyclohexylcarbodiimide affected proton translocation more strongly than cytochrome c oxidation. The cytoplasmic membrane of A. nidulans appears to contain a proton-translocating cytochrome c oxidase similar to the one described for mitochondria.

Identification and characterization of the ctaC (coxB) gene as part of an operon encoding subunits I, II, and III of the cytochrome c oxidase (cytochrome aa3) in the cyanobacterium Synechocystis PCC 6803.

The gene (coxII = coxB = ctaC) encoding subunit II of Synechocystis PCC 6803 cytochrome c oxidase has been isolated by screening a genomic DNA library in pUC18 with a 17-bp oligonucleotide probe (probe C) derived from coxI of Paracoccus denitrificans after Southern blots with a 19-kb oligonucleotide (probe A) derived from coxII of P. denitrificans had given equivocal results. A 2.2 kb PstI-KpnI restriction fragment was subcloned into pUC 18 and the resulting plasmid pDAUV26, which contained the probe C-binding site near the downstream end was found also to contain the whole coxII gene upstream of this site. The novel plasmid pDAUV 26 was used to transform competent E. coli cells, propagated therein, and the sequence determined. The 2.2 kb insert contained the entire coding region for the coxII gene together with a GAG start codon, a TAA stop codon, and a putative Shine-Dalgarno sequence. The deduced COII polypeptide is composed of 319 aa (calculated molecular mass of 32,800) plus a N-terminal leader sequence of 20 aa. The hydropathy plot suggests two lipophilic transmembrane domains near the N-terminus connected with an extremely hydrophilic aa stretch on the cytosolic side, while an unusually long (> 50 aa) aa stretch on the periplasmic (= intrathylakoidal) side leads to a typical cyanobacterial threonine in place of the first conserved glutamate of the cytochrome c-binding region in all other COII proteins. Together with a considerably shortened and interrupted aromatic aa stretch in this region, these differences are discussed in terms of the peculiar affinity of cyanobacterial cytochrome oxidases for acidic c-type cytochromes. Other invariant features such as the strictly conserved CuA-binding aa, however, are found in correct positions.