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.     

Do photosynthetic bacteria contain cytochrome c1?

A method is described for characterizing, c-type cytochromes in bacterial membrane preparations according to molecular weight on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Applied to the photosynthetic bacterium Rhodopseudomonas sphaeroides this technique is used, together with spectroscopic measurements, to demonstrate that a membrane-bound cytochrome c of mol.wt. 30000 is active in photosynthetic electron transport in addition to the well-known soluble cytochrome, cytochrome c2. The membrane cytochrome has a midpoint potential (E'0) at pH 7 of +290 mV, as compared with +360 mV for purified cytochrome c2. Its alpha-band has a peak near 552 nm, as compared with 550 nm for cytochrome c2. Evidence is presented that chromatophores contain roughly equal amounts of the two cytochromes.     

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.     

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.     

Purification and characterization of two soluble cytochromes from the alkalophile Bacillus firmus RAB

A soluble cytochrome c and soluble cytochrome b were purified from the alkalophilic Bacillus firmus RAB. The cytochrome c, with an alpha band at 552 nm, had an apparent molecular weight of 16,500 and was acidic, with a pI of 3.4. At both pH 7.0 and 8.3, the midpoint potential of c-552 was +66 mV. Above pH 8.3, the cytochrome exhibited a pH-dependent decrease in midpoint potential. This property, among others, distinguished the cytochrome c-552 from other membrane-associated c-type cytochromes. The soluble cytochrome b, with an alpha band maximum at 558 nm, had a molecular weight of approx. 15,500 and was also an acidic protein, with a pI of 3.07. It exhibited a pH-independent midpoint potential of +28 mV.     

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.     

Spectral and potentiometric analysis of cytochromes from Bacillus subtilis

Bacillus subtilis cytoplasmic membranes contain several cytochromes which are linked to the respiratory chain. At least six different cytochromes have been separated and identified by ammonium sulphate fractionation and ion-exchange chromatography. They include two terminal oxidases with CO-binding properties and cyanide sensitivity. One of these is an aa3-type cytochrome c oxidase which has characteristic absorption maxima in the reduced-oxidized difference spectrum at 601 nm in the alpha-band and at 443 nm in the Soret band regions. In the alpha-band two separate electron transitions with Em = +205 mV and Em = +335 mV can be discriminated by redox potentiometric titration. The other CO-binding cytochrome c oxidase contains two cytochrome b components with alpha-band maxima at 556 nm and 559 nm. Cytochrome b556 can be reduced by ascorbate and has an Em + +215 mV, whereas cytochrome b559 has an Em = +140 mV. Furthermore a complex consisting of a cytochrome b564 (Em = +140 mV) associated with a cytochrome c554 (Em = +250 mV) was found. This cytochrome c554, which can be reduced by ascorbate, appears to have an asymmetrical alpha-peak and stains for heme-catalyzed peroxidase activity on SDS-containing polyacrylamide gels. A protein with a molecular mass of about 30 kDa is responsible for this activity. A cytochrome b559 (Em = +65 mV) appears to be an essential part of succinate dehydrogenase. Finally a cytochrome c550 component with an apparent mid-point potential of Em = +195 mV has been detected.     

The c-type cytochromes of the gram-positive bacterium Bacillus licheniformis

The release of soluble c-type cytochromes from cells of the gram-positive bacterium Bacillus licheniformis was effected by treatment with lysolytic buffer. After further purification three different c-type cytochromes designated c-551, c-552, and c-554 were isolated. Oxidized and reduced spectra, molecular weight, isoelectric point, and amino acid compositions are reported for each of these monoheme proteins.     

Two membrane-bound b-type cytochromes in Nitrosomonas europaea

Abstract Membranes from Nitrosomonas europaea were found to contain two b -type cytochromes. One had an α-band centred at 562 nm and E _m,7=+ 155 mV; the other had an α-band maximum close to 558 nm and E _m,7=+ 40 mV. A b -type cytochrome ran at an apparent M _r of 32000 on lithium dodecyl sulphate/polyacrylamide gels at 4°C.     

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.     

Bradyrhizobium japonicum cytochrome c550 is required for nitrate respiration but not for symbiotic nitrogen fixation.

Bradyrhizobium japonicum possesses three soluble c-type cytochromes, c550, c552, and c555. The genes for cytochromes c552 (cycB) and c555 (cycC) were characterized previously. Here we report the cloning, sequencing, and mutational analysis of the cytochrome c550 gene (cycA). A B. japonicum mutant with an insertion in cycA failed to synthesize a 12-kDa c-type cytochrome. This protein was detectable in the cycA mutant complemented with cloned cycA, which proves that it is the cycA gene product. The cycA mutant, a cycB-cycC double mutant, and a cycA-cycB-cycC triple mutant elicited N2-fixing root nodules on soybean (Nod+ Fix+ phenotype); hence, none of these three cytochromes c is essential for respiration supporting symbiotic N2 fixation. However, cytochrome c550, in contrast to cytochromes c552 and c555, was shown to be essential for anaerobic growth of B. japonicum, using nitrate as the terminal electron acceptor.     

N-terminal amino acid sequence of cytochrome c-552 from Nitrosomonas europaea

Nitrosomonas europaea is an ammonia-oxidizing bacterium which contains multiple c-type cytochromes. Few of these components have been assigned physiological roles, but on the basis of molecular weight and redox potential cytochrome c-552 has been considered to be an analogue of the mitochondrial cytochrome-c family of proteins. We present the N-terminal amino acid sequence (47 residues) of cytochrome c-552 and show that this protein is most closely related to the group of small cytochrome-c components from pseudomonads (cytochromes c-551) and is probably evolutionarily distant from the analagous protein (cytochrome c-550) from the nitrite-oxidizing bacterium Nitrobacter agilis.     

Two types of cytochrome cd1 in the aerobic photosynthetic bacterium, Erythrobacter sp. OCh 114

Components I and II of cytochrome cd1 which had different spectral features were purified from the aerobic photosynthetic bacterium, Erythrobacter sp. strain OCh 114. Component I showed an absorption maxima at 700 and 406 nm in the oxidized form, and at 621, 552.5, 548 and 416 nm in the reduced form. Component II showed an absorption maxima at 635 and 410 nm in the oxidized form and at 628, 552.5, 548 and 417 nm in the reduced form. The relative molecular mass, Mr, of both cytochromes was determined to be 135,000 with two identical subunits. Components I and II showed pI values of 7.6 and 6.8, respectively. The redox potential of hemes ranged from +234 mV to +242 mV, except for the heme d1 of component I (Em7 = +134 mV). Components I and II showed both cytochrome c oxidase and nitrite reductase activities. Cytochrome c oxidase activity was strongly inhibited by a low concentration of nitrite and cyanide. Erythrobacter cytochromes c-551 and c-552 were utilized as electron donors for the cytochrome c oxidase reaction. The high affinity of cytochrome c-552 to component II (Km = 1.27 microM) suggested a physiological significance for this cytochrome. Erythrobacter cytochromes cd1 are unique in their presence in cells grown under aerobic conditions as compared to other bacterial cytochromes cd1 which are formed only under denitrifying conditions.     

A partial resolution and reconstitution of the ammonia-oxidizing system of Nitrosomonas europaea: role of cytochrome c554

The cell-free ammonia-oxidizing system of Nitrosomonas europaea was resolved into three major fractions: a membrane fraction containing cytochrome a1 and c-type cytochromes, a fraction with hydroxylamine-cytochrome c reductase and a cytochrome c fraction. The ammonia-oxidizing activity was reconstituted by the combination of these three fractions. The activity was more consistently reconstituted by adding Nitrosomonas cytochrome c554 to the membrane fraction. The hydroxylamine-cytochrome c reductase activity of the membrane fraction increased with the addition of cytochrome c554, but the oxidation of hydroxylamine to nitrite required a further addition of cytochrome c552. The ammonia oxidation by the membrane plus cytochrome c554 was affected by the concentration of phosphate and the addition of bovine serum albumin, spermine, or MgCl2.     

A comparative study of the resonance Raman spectra of bacterial cytochromes

Resonance Raman spectra were obtained for two newly isolated bacterial cytochromes, Alcaligenes faecalis (ATCC 8750) c554 and Alcaligenes faecalis c556. Their spectra were compared with those of mammalian cytochrome c and two other bacterial cytochromes, Paracoccus denitrificans c550 and Pseudomonas aeruginosa c551. The positions of the Raman bands indicated that, while Al. c554 and Al. c556 were c-type cytochromes with two thioether linkages, several common features found in their Raman spectra were anomalous. These features suggest that the electronic charge density of the porphyrin macrocycle of Al. c554 and Al. c556 is more asymmetric than that of other bacterial and mammalian c-type cytochromes. The Raman evidence indicates that the electronic properties of the heme are controlled by the protein in these two Alcaligenes cytochromes.     

Purification and properties of the soluble cytochromes c-550 and c-556 from the bacterium Aquaspirillum itersonii

Two c-type cytochromes were isolated from cells of the gram-negative bacterium Aquaspirillum itersonii grown under low aeration in the presence of nitrate. The major component, cytochrome c-550, was equated with the (single) c-type cytochrome previously reported to be present in this organism [Clark-Walker, G. D. & Lascelles, J. (1970) Arch. Biochem. Biophys. 136, 153-159], although a significantly higher molecular mass was apparent in the present work. The complete amino acid sequence of this cytochrome is reported in the accompanying paper. A second soluble c-type cytochrome, designated c-556, was also isolated. The molecular mass, isoelectric point, spectrum, midpoint oxidation reduction potential and amino acid composition of this monoheam cytochrome are reported. The possible relationship of this cytochrome to other cytochromes c-556 is discussed.     

Properties of a cytochrome c-enriched light particulate fraction isolated from the photosynthetic bacterium Rhodopseudomonas spheroides.

Differential centrifugation of suspensions of French-press-disrupted Rhodopseudomonas spheroides yielded a light particulate fraction that was different in many properties from the bulk membrane fraction. It was enriched in cytochrome c and had a low cytochrome b content. When prepared from photosynthetically grown cells this fraction had a very low specific bacteriochlorophyll content. The cytochrome c of the light particles differed in absorption maxima at 77K from cytochrome c2 attached to membranes; there was pronounced splitting of the alpha-band, as is found in cytochrome c2 free in solution. Potentiometric titration at A552--A540 showed the presence of two components that fitted an n = 1 titration; one component had a midpoint redox potential of +345mV, like cytochrome c2 in solution, and the second had E0' at pH 7.0 of +110 mV, and they were present in a ratio of approx. 2:3. Difference spectroscopy at 77K showed that the spectra of the two components were very similar. More of a CO-binding component was present in particles from photosynthetically grown cells. Light membranes purified by centrifugation on gradients of 5--60% (w/w) sucrose retained the two c cytochromes; they contained no detectable succinate-cytochrome c reductase or bacteriochlorophyll and very little ubiquinone, but they contained NADH-cytochrome c reductase and some phosphate. Electrophoresis on sodium dodecyl sulphate/polyacrylamide gels showed that the light membranes of aerobically and photosynthetically grown cells were very similar and differed greatly from other membrane fractions of R. spheroides.     

Thermostability of cytochrome c-552 from the thermophilic hydrogen-oxidizing bacterium Hydrogenobacter thermophilus

The denaturation of the c-type cytochrome of the thermophilic bacterium Hydrogenobacter thermophilus cytochrome c-552 by heat and guanidine hydrochloride was studied by measuring the change in circular dichroic spectra. The melting temperature (T1/2) of cytochrome c-552 in the presence of 1.5 M guanidine hydrochloride was 34 degrees C higher than that of the c-type cytochrome of Pseudomonas aeruginosa cytochrome c-551. Hydrogenobacter cytochrome c-552 is a much more stable protein than cytochrome c-551 of the mesophilic bacterium P. aeruginosa, even though their amino acid sequences are 56% identical and they have numerous other similarities. However, notwithstanding these similarities between the sequences of the cytochromes c-552 and c-551 that were compared, it is very likely that these differences in stability could be due to some heretofore undefined differences in their spatial structures. It has been suggested that alpha-helix structure and electrostatic interaction could be the source of the stable spatial structure of cytochrome c-552.     

Two membrane-bound c-type cytochromes of Thiobacillus ferrooxidans: Purification and properties

Abstract Membrane-bound cytochrome c, cytochrome c-552 (m) was purified from Thiobacillus ferrooxidans . It showed an absorption peak at 410 nm in the oxidized form, and peaks at 552, 523 and 416 nm in the reduced form. Its molecular mass, E _m,7 and isoelectric point were 22,300, +0.336 volt and 9.1, respectively. Another membrane-bound cytochrome c , cytochrome c -550 (m) was also purified. It showed an absorption peak at 408 nm in the oxidized form, and peaks at 550, 523 and 418 nm in the reduced form. Its molecular mass was estimated to be 51,000. Ferrocytochromes c -552 (m) and c -55 (m) were oxidized by cytochrome c oxidase of the bacterium. The reactivity with the oxidase of cytochrome c -550 (m) was higher than that of cytochrome c -552 (s) (soluble cytochrome) of the bacterium, while the reactivity of cytochrome c -552 (m) was greatly lower than that of cytochrome c -552 (s).     

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.