Cytochrome c-550 mediates electron transfer from inducible periplasmic c-type cytochromes to the cytoplasmic membrane of Paracoccus denitrificans

Electron transfer from periplasmic cytochromes c to the membrane-bound respiratory chain has been studied with the isolated cytochromes and membrane preparations from Paracoccus denitrificans. When reduced cytochromes were incubated with spheroplasts only the constitutive cytochrome c-550 was rapidly oxidized. The inducible cytochromes c-551i and c-553i were not oxidized at appreciable rates. Cytochrome c-550 was able to mediate the transfer of electrons from either cytochrome c-551i or cytochrome c-553i to the membrane preparation.     

Properties of oxygen-evolving photosystem-II particles from Phormidium laminosum, a thermophilic blue--green alga.

1. O2-evolving Photosystem-II particles from the thermophilic blue--green alga Phormidium laminosum contained 1 mol of Mn/13--17 mol of chlorophyll a compared with 1 mol of Mn/65--75 mol of chlorophyll a in unfractionated membranes. 2. At least two-thirds of the Mn in the Photosystem-II particles was removed by mild heating and by treatment with Tris or EDTA, with concomitant loss of O2 evolution. However, irreversible inactivation was also caused by washing in buffers without MgCl2, and this inactivation was not accompanied by a corresponding loss of Mn. 3. Bivalent cations (Mg2+ or Ca2+), Cl- or Br- ions and at least 20% (v/v) glycerol were required for maximum stability of O2 evolution. 4. The Photosystem-II particles were enriched in high-potential cytochrome b-559 (1 mol of cytochrome/50--60 mol of chlorophyll a) and in component C-550, and had a photosynthetic-unit size of 40--70 molecules of chlorophyll a. 5. The absorption spectrum at 77 K showed a preponderance of shorter-wavelength forms of chlorophyll a in the Photosystem-II particles, and in the fluorescence emission spectrum at 77 K there were major chlorophyll fluorescence bands at 684 nm and 695 nm, with almost no fluorescence in the far-red region. 6. Analysis of the lipid and protein contents showed that the Photosystem-II particles were not chemically pure (for example, all of the membrane-bound cytochromes and cytochrome c-549 were present), but their high O2-evolution activity and good optical properties make them useful for functional studies on Photosystem-II and O2 evolution.     

Oxidation-reduction potentials of respiratory chain components in Thiobacillus A2

(1) Cells of Thiobacillus A2 grown chemoautotrophically on thiosulfate or heterotrophically on succinate with oxygen contained b-, c-, o-, a- and a3-type cytochromes. The amount of cytochrome per mg of cell protein was much greater in thiosulfate-grown cells and differences in the relative concentrations of cytochromes were observed for the different growth conditions. (2) The half-reduction potentials at pH 7.0 (Em,7.0) and spectral maxima of c-, b-, a- and a3-type cytochromes were similar in cells grown aerobically with thiosulfate or with succinate as the growth substrate. (3) The half-reduction potential of the 'invisible', or high-potential copper, as determined from the potentiometric behavior of the carbon monoxide-reduced cytochrome a3 complex at pH 8.0, was 365 mV. (4) Reducing equivalents from thiosulfate appear to enter the respiratory chain at the cytochrome c level; however, studies in cell-free extracts were limited due to a loss in respiratory activity with thiosulfate as a substrate upon cell disruption.     

Soluble cytochrome composition of the purple phototrophic bacterium, Rhodopseudomonas sphaeroides ATCC 17023

A detailed study of the soluble cytochrome composition of Rhodopseudomonas sphaeroides (ATCC 17023) indicates that there are five c-type cytochromes and one b-type cytochrome present. The molecular weights, heme contents, amino acid compositions, isoelectric points, and oxidation-reduction potentials were determined and the proteins were compared with those from other bacterial sources. Cytochromes c2 and c' have previously been well characterized. Cytochrome c-551.5 is a diheme protein which has a very low redox potential, similar to certain purple bacterial and algal cytochromes. Cytochrome c-554 is an oligomer, which is spectrally similar to the low-spin isozyme of cytochrome c' found in other purple bacteria (e.g., Rhodopseudomonas palustris cytochrome c-556). An unusual high-spin c-type heme protein has also been isolated. It is spectrally distinguishable from cytochrome c' and binds a variety of heme ligands including oxygen. A large molecular-weight cytochrome b-558 is also present which appears related to a similar protein from Rhodospirillum rubrum, and the bacterioferritin from Escherichia coli. None of the soluble proteins appear to be related to the abundant membrane-bound c-type cytochrome in Rps. sphaeroides which has a larger subunit molecular weight similar to mitochondrial cytochrome c1 and chloroplast cytochrome f.     

Studies on the mechanism of resistance of Pseudomonas aeruginosa to neomycin. II. Correlation between neomycin resistance and hemoprotein concentration

The cells of a newly isolated neomycin sensitive and neomycin resistant strains of Pseudomonas aeruginosa contained similar level of catalase activity. The difference spectrum of the cells revealed the presence of cytochrome c-554 and cytochrome b-560-. The presence of both cytochromes in the same molecular ratio in the cells and subcellular fractions indicates that they are tightly bound to the cytoplasmic membrane. The neomycin resistant strain contained five times less cytochromes than the sensitive strain. The lower cytochrome level in the neomycin resistant cells was found to be independent of the presence or absence of neomycin in the medium. The difference spectra of the cells and particulate fractions did not reveal any cytochromes of alpha-type.     

The roles of c-type cytochromes in algal photosynthesis. Extraction from algae of a cytochrome similar to higher plant cytochrome f.

A membrane-bound cytochrome resembling higher plant cytochrome f in many respects has been extracted from the algae Chlamydomonas. Euglena and Anacystis, and partially purified. The spectra of the cytochromes from Chlamydomonas and Euglena are virtually identical to that of parsley cytochrome f, with alpha-band maxima near 554 nm, very asymmetrical beta-bands, and gamma-band maxima at 421 nm. The cytochrome from Anacystis had alpha and gamma-bands both shifted to slightly longer wavelengths. The redox potential of the cytochrome from Chlamydomonas was determined as +350 mV, and its minimum molecular weight in sodium dodecyl sulphate as 31 000. The cytochrome from Euglena showed a rate of reaction with higher plant plastocyanin at least 100 times that of the soluble Euglena cytochrome c-552, and was unaffected by Euglena cytochrome c-552 antiserum. A very fast rate of electron transfer occurred between this cytochrome purified from Euglena and cytochrome c-552. The roles of the membrane-bound and soluble c-type cytochromes in algal photosynthesis are discussed, and it is recommended that the name cytochrome f should be reserved for the membrane-bound cytochrome (to emphasize its affinity with higher plant cytochrome f), while the soluble one should be named by its alpha-band (c-552, c-553, etc.) to make clear its distinctness from higher plant cytochrome f and homology with mitochondrial cytochrome c.     

Components and activity of the photosynthetic electron transport system of intact heterocysts isolated from the blue-green alga Nostoc muscorum

Heterocysts of the blue-green alga Nostoc muscorum have been isolated by prolonged treatment with lysozyme. Quantitative data are presented which show the occurrence of cytochromes c-553, f-557 and b-563 in heterocysts in amounts comparable to vegetative cells. Particularly the content of the water-soluble cytochrome c-553 can be used to evaluate the intactness of a heterocyst preparation. Cytochrome f-557 has been partially purified and found to be a c-type cytochrome corresponding to cytochrome f of higher plants and other algae. Cytochrome b-559 is present in vegetative cells but not in heterocysts. The content of plastoquinone in heterocysts is reduced to 42% of the amount present in vegetative cells. These data suggest a degradation of Photosystem II during heterocyst differentiation. Measurements of photosynthetic electron transport in heterocysts proved the inability of the photosynthetic apparatus to carry out electron transport with electrons donated by water or diphenylcarbazide. In Tris-washed thylakoids from vegetative cells, however, diphenylcarbazide can act as an electron donor to Photosystem II.     

Bacillus subtilis 13-kilodalton cytochrome c-550 encoded by cccA consists of a membrane-anchor and a heme domain

Little is known about c-type cytochromes in Gram-positive bacteria in contrast to the wealth of information available on this type of cytochrome in Gram-negative bacteria and in eucaryotes. In the present work, the strictly aerobic bacterium Bacillus subtilis was analyzed for subcellular localization and number of different cytochromes c. In vivo labeling with radioactive 5-aminolevulinic acid, a precursor to heme, showed that the proteins containing covalently bound heme are predominantly found in the membrane fraction. One major membrane-bound cytochrome c of about 15 kDa and with an alpha-band absorption peak in the reduced state at 550 nm was analyzed in more detail. Cytochrome c-550 has the properties of an integral membrane protein. The physiological function of this relatively high redox potential cytochrome is not known. Its structural gene, cccA, was cloned, sequenced, and overexpressed in B. subtilis. The gene maps adjacent to rpoD (sigA) at 223 degrees on the chromosome. The amino acid sequence of cytochrome c-550 as deduced from the DNA sequence consists of 120 residues and contains one heme c binding site (Cys-Ile-Ala-Cys-His) located approximately in the middle of the polypeptide. From the hydropathy distribution and from comparisons to soluble c-type cytochromes of known three-dimensional structure, cytochrome c-550 seemingly consists of two domains; an N-terminal membrane-anchor domain and a C-terminal heme domain. A model for the topography of the cytochrome in the cytoplasmic membrane is suggested in which the N-terminal part spans the membrane in the form of a single segment in an alpha-helical conformation and the C-terminal heme domain is exposed on the extracytoplasmic side of the membrane. Deletion of cccA from the chromosome revealed another membrane-bound cytochrome with absorption maximum at 550 nm in the reduced state. Analysis of cccA deletion mutants demonstrated that the cytochrome c-550 encoded by cccA is not essential for growth of B. subtilis on rich or minimal media.     

Isolation and properties of the soluble c-type cytochromes of the dinoflagellate Peridinium cinctum

Four soluble cytochromes of the c type were isolated from the freshwater dinoflagellate Peridinium cinctum collected from Lake Kinneret, Israel. Cytochrome c with alpha-band maximum at 550 nm in the reduced state had a molecular mass of 10,200 Da, pI 7.4, and Em of 278 m V. This cytochrome was active in the respiratory chain of beef heart Keilin-Hartree particles. Cytochrome c-553 had a molecular mass of 13,200 Da, pI 4.9, and Em of 384 m V, and was active in light induced electron transport of Euglena gracilis chloroplast fragments. Cytochrome c-554 had a molecular mass of 13,500 Da, pI 4.4, and Em of 326 m V. This cytochrome was inactive in light induced electron transport but competed with cytochrome c-552 of Euglena in the assay. The acidic cytochrome c-557 was present in very small quantities. The properties of the soluble c-type cytochromes of P. cinctum are compatible with the classification of dinoflagellates as primitive eucaryotes.     

Expression of plastocyanin and cytochrome f of the cyanobacterium Phormidium laminosum in Escherichia coli and Paracoccus denitrificans and the role of leader peptides.

The gene for plastocyanin from the cyanobacterium Phormidium laminosum was successfully expressed in Escherichia coli. Expression of the gene for cytochrome f resulted in the production of holocytochrome f in the periplasmic space of E. coli, but the yield was low. Expression in Paracoccus denitrificans yielded no holoprotein. When the region encoding the cytochrome f leader sequence was replaced with more typical bacterial leader sequences (those from the P. laminosum plastocyanin gene and the Paracoccus versutus cytochrome c-550 gene), much higher yields were consistently obtained in both species. Overexpressed proteins were compared to those isolated from P. laminosum and found to be identical in mass, isoelectric point, redox midpoint potential and (for plastocyanin) 1H-NMR spectrum.     

Characterization of the c-type cytochromes of Nitrosomonas europaea with the aid of fluorescent gels

When a total soluble extract of Nitrosomonas europaea was denatured with dodecyl sulphate, subjected to dodecyl sulphate/polyacrylamide-gel electrophoresis and illuminated with near-u.v. light, eight bands of protein fluorescence were observed. All but one of these bands were red in colour, a property characteristic of c-type cytochromes. Standard techniques were used to purify soluble c-type cytochromes from this organism, and it was then possible to assign all but two very minor bands to specific c-type cytochromes, namely hydroxylamine oxidase, cytochrome c-554, cytochrome c-552 and a cytochrome c-550 not previously described. The eight band had fluorescence peaking in the green region of the spectrum, probably caused by covalently bound flavin, and co-purified with hydroxylamine oxidase. The following physical properties were determined for these components: isoelectric point, molecular weights according to gel filtration and mobility on dodecyl sulphate/polyacrylamide gels, and alpha-band spectra at room temperature and 77K. Redox potentials were measured as follows: cytochrome c-554, E(m,7) = +20mV; cytochrome c-552, E(m,7) = +230mV; cytochrome c-550, E(m,7) = +140mV. When washed membranes were applied to dodecyl sulphate/polyacrylamide gels in the same way, a number of fluorescent bands were observed that could be matched by soluble proteins. In addition, there was one band that could not be detected in supernatants, migrating with an apparent molecular weight of 24000. This species is probably coincident with a c-type cytochrome having E(m,7) = +170mV found in redox titration of these membranes. In future studies, gel fluorescence should form a useful complement to spectroscopy for analysis of cytochrome composition in active cell-free preparations or semi-purified material.     

Comparison of cytochromes from anaerobically and aerobically grown cells of Pseudomonas perfectomarinus.

Pseudomonas perfectomarinus (ATCC 14405) is a facultative anaerobe capable of either oxygen respiration or anaerobic nitrate respiration, i.e., denitrification. A comparative study of the electron transfer components of cells revealed five c-type cytochromes and cytochrome cd in the soluble fraction from anaerobically grown cells and four c-type cytochromes in the soluble fraction from aerobically grown cells. Purification procedures yielded three c-type cytochromes (designated c-551, c-554, and acidic c-type) from both kinds of cells as indicated by similarities in absorption spectra, molecular weight, and electrophoretic mobility. Cytochrome cd, a diheme c-type cytochrome (cytochrome c-552), and a split-alpha c-type cytochrome were recovered only from anaerobically grown cells. A c-type cytochrome with a low ratio of alpha to beta absorption peak heights was uniquely present in the aerobically grown cells. Liquid N2 temperature absorption spectroscopy on the membrane fraction from anaerobically grown cells revealed residual cytochrome cd as well as differences in the relative amounts of c-type and b-type cytochromes in membranes prepared from cells grown under the two different conditions.     

Effect of NADP on light-induced cytochrome changes in membrane fragments from a blue-green alga.

The effect of NADP+ on light-induced steady-state redox changes of membrane-bound cytochromes was investigated in membrane fragements prepared from the blue-green algae Nostoc muscorum (Strain 7119) that had high rates of electron transport from water to NADP+ and from an artificial electron donor, reduced dichlorophenolindophenol (DCIPH2) to NDAP+. The membrane fragments contained very little phycocyanin and had excellent optical properties for spectrophotometric assays. With DCIPH2 as the electron donor, NADP+ had no effect on the light-induced redox changes of cytochromes: with or without NADP+, 715- or 664-nm illumination resulted mainly in the oxidation of cytochrome f and of other component(s) which may include a c-type cytochrome with an alpha peak at 549nm. With 664 nm illumination and water as the electron donor, NADP+ had a pronounced effect on the redox state of cytochromes, causing a shift toward oxidation of a component with a peak at 549 nm (possibly a c-type cytochrome), cytochrome f, and particularly cytochrome b559. Cytochrome b559 appeared to be a component of the main noncyclic electron transport chain and was photooxidized at physiological temperatures by Photosystem II. This photooxidation was apparent only in the presence of a terminal acceptor (NADP+) for the electron flow from water.     

The role of soluble cytochrome c-551 in cyclic electron flow-driven active transport in Chromatium vinosum

Spheroplasts have been prepared from the photosynthetic purple sulfur bacterium Chromatium vinosum by lysozyme plus ethylenediaminetetraacetic acid treatment. These spheroplasts are able to take up alanine in the light, but light-dependent alanine uptake is lost upon subsequent washing of the spheroplasts. The observations that alanine uptake driven by a potassium plus valinomycin-induced membrane potential (outside positive) is not affected by washing and that light-dependent alanine uptake can be restored by addition of the supernatant from washing suggest that a soluble electron carrier is lost during washing. Light-dependent alanine uptake in washed spheroplasts could be restored by addition of C. vinosum cytochrome c-551. Other soluble electron carriers from C. vinosum (high-potential iron protein, cytochrome ‘f’, cytochrome c′ and the flavocytochrome c-552) did not restore alanine uptake nor did a variety of other soluble electron carrier proteins from other organisms. These results suggest that cytochrome c-551 functions as an electron carrier in the cyclic electron transfer chain of C. vinosum. Mitochondrial cytochrome c (equine heart) and cytochrome c-551 from Pseudomonas aeruginosa were highly effective in restoring light-dependent alanine uptake in washed spheroplasts, making it likely that C. vinosum cytochrome c-551 is related by evolution to the same cytochrome c family as these other two c cytochromes.     

The participation of cytochromes in the reduction of N20 to N2 by a denitryfying bacterium.

The oxidation of cytochromes during the reduction of N2O to N2 by a denitrifying bacterium was studied spectrophotometrically. The reduced b- and c-type cytochromes are partially oxidized when N2O is added to intact cells reduced with lactate under anaerobic conditions. The oxidation of cytochromes is inhibited non-competitively by azide, cyanide, 2,4-dinitrophenol and CuSO4, which inhibit the reduction of N2O to N2. In the presence of each inhibitor at a high concentration, at which the reduction of N2O to N2 is perfectly inhibited, cytochromes are not oxidized by N2O, while when an adequate, low concentration of inhibitor is added, b-type cytochrome is partially oxidized but c-type cytochrome is apparently not oxidized. In cell-free extracts, prepared by the sonic disruption of cells, that have entirely lost their activity in the reduction of N2O to N2, cytochromes are not oxidized by N2O. From the above results, it was concluded that b-type and c-type cytochromes should participate in the electron transport mechanism of the reduction of N2O to N2.     

Soluble cytochromes and ferredoxins from the marine purple phototrophic bacterium, Rhodopseudomonas marina

Four soluble c-type cytochromes, the high redox potential 4-Fe-S ferredoxin known as HiPIP, a large molecular weight 2-Fe-S ferredoxin and a 4-Fe-S 'bacterial' ferredoxin, were isolated from extracts of two strains of Rps. marina. Cytochrome c-550, cytochrome c' and cytochrome c-549 were previously described, and we have extended their characterization. Cytochrome c-558, which has not previously been observed in Rps. marina, appears to be a low-spin isozyme of the more commonly observed high-spin cytochrome c'. HiPIP, which was not observed in previous work, was found to be abundant in Rps. marina. The 2-Fe-S ferredoxin, which has previously been observed only in Rps. palustris, has a native size greater than 100 kDa and a subunit size of 17 kDa. The 'bacterial' ferredoxin appears to have only a single four-iron-sulfur cluster. We examined photosynthetic membranes by difference spectroscopy and found abundant c-type cytochromes. Approximately one-quarter of the heme can be reduced by ascorbate and the remainder by dithionite. There is 2 nm difference between the high-potential heme (554 nm) and the low (552 nm). These characteristics resemble those of the tetraheme reaction center cytochrome of Rps. viridis. In addition to the electron transfer components, we found small amounts of a fluorescent yellow protein which has spectral resemblance to a photoactive yellow protein from Ec. halophila.     

The role of cytochrome c4 in bacterial respiration. Cellular location and selective removal from membranes.

The cellular location of cytochrome c4 in Pseudomonas stutzeri and Azotobacter vinelandii was investigated by the production of spheroplasts. Soluble cytochrome c4 was found to be located in the periplasm in both organisms. The remaining cytochrome c4 was membrane-bound. The orientation of this membrane-bound cytochrome c4 fraction was investigated by proteolysis of the cytochrome on intact spheroplasts. In P. stutzeri, 78% of the membrane-bound cytochrome c4 could be proteolysed, whilst 82% of the spheroplasts remained intact, suggesting that the membrane-bound cytochrome c4 is on the periplasmic face of the membrane in this organism. Cytochrome c4 was not susceptible to proteolysis on A. vinelandii spheroplasts, in spite of being digestible in the purified state. Cytochrome c5 was shown to have a similar cellular distribution to cytochrome c4. Selective removal of cytochrome c4 from membranes of P. stutzeri was accomplished by the use of sodium iodide and propan-2-ol, with the retention of most of the ascorbate-TMPD (NNN'N'-tetramethylbenzene-1,4-diamine) oxidase activity associated with the membrane. Sodium iodide removed most of the cytochrome c4 from A. vinelandii membranes with retention of 62% of the ascorbate-TMPD oxidase activity. Cytochrome c4 could be returned to the washed membranes, but with no recovery of this enzyme activity. We conclude that cytochrome c4 is not involved in the ascorbate-TMPD oxidase activity associated with the membranes of these two organisms.     

Light inhibition of respiration is due to a dual function of the cytochrome b6-f complex and the plastocyanin/cytochrome c-553 pool in Aphanocapsa

Studies of the respiratory electron transport pathway in the blue-green alga, Aphanocapsa, demonstrated the presence of cytochrome oxidase and a cytochrome complex. The use of antimycin A showed only the occurrence of a plastidal type of cytochrome complex (the cytochrome b6-f complex), which is insensitive to this inhibitor. Determination of the extent of photooxidation of cytochromes c-553 and f-556 under conditions of high and low cytochrome oxidase activities indicated an electron flow through both cytochromes to cytochrome oxidase. Direct evidence for a common segment of photosynthetic and respiratory electron transport from plastoquinone via the cytochrome b6-f complex to the soluble plastocyanin/cytochrome c-553 pool, as well as a competition between cytochrome oxidase and Photosystem I for reductants in this pool in the light, was obtained by measurements of electron transport with suitable electron donors in this alga.     

Soluble cytochromes from the marine methanotroph Methylomonas sp. strain A4.

Soluble c-type cytochromes are central to metabolism of C1 compounds in methylotrophic bacteria. In order to characterize the role of c-type cytochromes in methane-utilizing bacteria (methanotrophs), we have purified four different cytochromes, cytochromes c-554, c-553, c-552, and c-551, from the marine methanotroph Methylomonas sp. strain A4. The two major species, cytochromes c-554 and c-552, were monoheme cytochromes and accounted for 57 and 26%, respectively, of the soluble c-heme. The approximate molecular masses were 8,500 daltons (Da) (cytochrome c-554) and 14,000 Da (cytochrome c-552), and the isoelectric points were pH 6.4 and 4.7, respectively. Two possible diheme c-type cytochromes were also isolated in lesser amounts from Methylomonas sp. strain A4, cytochromes c-551 and c-553. These were 16,500 and 34,000 Da, respectively, and had isoelectric points at pH 4.75 and 4.8, respectively. Cytochrome c-551 accounted for 9% of the soluble c-heme, and cytochrome c-553 accounted for 8%. All four cytochromes differed in their oxidized versus reduced absorption maxima and their extinction coefficients. In addition, cytochromes c-554, c-552, and c-551 were shown to have different electron paramagnetic spectra and N-terminal amino acid sequences. None of the cytochromes showed significant activity with purified methanol dehydrogenase in vitro, but our data suggested that cytochrome c-552 is probably the in vivo electron acceptor for the methanol dehydrogenase.     

The interrelation of the two c-type cytochromes in Rhodopseudomonas sphaeroides photosynthesis.

Photosynthetic electron flow in the bacterium Rhodopseudomonas sphaeroides involves two c-type cytochromes, one membrane-bound and the other a soluble protein, cytochrome c2. Membranes deficient in cytochrome c2 were used for photo-oxidation studies, with and without the addition of purified cytochrome c2. The results favour a series interrelation, membrane cytochrome c-cytochrome c2-reaction centre.