The role of a cytochrome c-552-cytochrome c complex in the oxidation of sulfide in Chromatium vinosum

The sulfide:cytochrome c oxidoreductase activity of the flavocytochrome c-522 from the purple sulfur bacterium Chromatium vinosum has been investigated. The oxidized sulfur product of the sulfide:cytochrome c reductase activity has been shown to be elemental sulfur. Cytochrome c-552 has been found to form a stable complex with horse heart cytochrome c that appears to be held together by electrostatic interactions. The stability of this complex and the sulfide:cytochrome c reductase activity of cytochrome c-552 are both ionic strength dependent, with maximal rates of cytochrome c reduction and extent of complex formation occurring over the same ionic strength range. Trifluoroacetylated cytochrome c is not reduced in the presence of cytochrome c-552 and sulfide, nor does it form a complex with cytochrome c-552. These results suggest the possible involvement of cytochrome c lysine residues in complex formation. Cytochrome c-552 migrates with an anomalously high apparent molecular weight on gel filtration columns equilibrated with low ionic strength buffers, suggesting the possibility of conformational changes or dimerization of the protein. However, complexation of cytochrome c-552 with cytochrome c still occurs at low ionic strength.     

Bacillussubtilis holo-cytochrome c-550 can be synthesised in aerobic Escherichia coli

Bacillus subtilis membrane-bound holo-cytochrome c-550 was found to be expressed from the structural gene cloned on a plasmid vector in aerobically grown Escherichia coli and exhibited normal biochemical properties. This occurs despite the lack of endogenous eytochrome c and suggests that eytochrome c-heme lyase activity is also present in aerobic E. coli. The membrane topology of B. subtilis eytochrome c-550 was studied using fusions to alkaline phosphatase (PhoA). The results show that the heme domain (at least when fused to PhoA) can be translocated as apo-cytochrome and confirm that the N-terminal part of the cytochrome functions as both export signal and membrane anchor for the C-tenninal heme domain. A model for the organisation of B. subtilis cytochrome c-550 in the cytoplasmic membrane is presented.     

The reconstitution of energy transfer in membranes from a bacteriochlorophyll-less mutant of Rhodopseudomonas sphaeroides by addition of light-harvesting and reaction centre pigment-protein complexes

Antenna and reaction centre complexes purified from photosynthetically-grown cells of Rhodopseudomonas sphaeroides have been mixed with cytoplasmic membranes prepared from an aerobically-grown bacteriochlorophyll-less mutant of Rp. sphaeroides (designated 01) in the presence of 1% sodium cholate. After removal of the cholate by dialysis, the dialysate was subjected to isopycnic centrifugation. Reconstituted cytochrome c₂ photooxidation and cytochrome b photoreduction were demonstrated in a pigmented fraction recovered from the sucrose gradient, suggesting that the pigment-proteins were incorporated into the 01 membrane.

The fluorescence properties of the system were examined. The appearance of a variable component after the initial fast fluorescence rise indicated that energy transfer occurred between the antenna and reaction centre proteins in the presence of 01 membrane. The order in which the system was assembled was important. Reconstituted energy transfer with a pre-dialysed reaction centre-antenna complex was more effective than when all the components were mixed at once. Energy transfer was also reconstituted between added reaction centre protein and the endogenous antenna present in membranes from the pigmented, but aerobically-grown reaction centre-less mutant PM8dp of Rp. sphaeroides.

Preparations of 01 membranes reconstituted with reaction centre exhibited a light intensity dependent cytochrome c₂ photooxidation. At low exciting light intensities, preparations containing reconstituted antenna protein in addition to reaction centres showed greater membrane cytochrome c₂ photooxidation than preparations with the antenna omitted; this improvement was maximal when a pre-dialysed antenna-reaction centre complex was used.     

Characterization of cytochrome c from Nitrobacter Agilis

Cytochrome c from Nitrobacter agilis was isolated and purified approx. 60-fold. Absorption spectra of both the oxidized and the reduced Nitrobacter cytochrome c and the oxidized minus reduced difference spectrum of this cytochrome were essentially identical to the corresponding spectra of horse-heart cytochrome c. The redox potential of this cytochrome was determined by spectrophotometric titration with ferrocyanide/ferricyanide and found to be +0.282 V over the pH range 6.0 to 8.7, while a potential of +0.265 V was determined in the same manner for horse-heart cytochrome c. The titration also indicated that the Nitrobacter ferrocytochrome is oxidized by a single electron transfer.     

Further studies on the NADH oxidase of the cytoplasmic membrane of Mycobacterium tuberculosis

The cytoplasmic membrane of the H₃₇Ra strain of Mycobacterium tuberculosis has been isolated free of cell wall.

These membrane preparations contain very small quantities of cytochromes c, b and cytochrome oxidase. The cytochrome c is not extracted by any method attempted. The cytochrome b is reducible only by dithionite and is believed not to be involved in the direct transfer of electrons during the oxidation of NADH by these preparations. The NADH oxidase activity of the membrane is inhibited by high concentrations of cyanide and also by 2-(n-heptyl)-4-hydroxyquinoline-N-oxide (HQNO). The cytochrome oxidase of the membrane contains both cytochromes a and a₃ and is present in low concentrations relative to cytochrome c. The cytochrome a₃ component was identified by characteristic complexes with both CO and cyanide and shows a γ-band absorption maximum at a slightly lower wavelength than the cytochrome oxidase of mammalian mitochondria (442 nm vs. 445 nm). The functional activity of the cytochrome oxidase is indicated by the inhibition of reoxidation of reduced cytochromes c and a in the presence of cyanide.     

Cytochrome c1 and b-type cytochrome with two heme centers in the photosynthetic membranes from Rhodopseudomonas palustris

The photosynthetic membranes from Rhodopseudomonas palustris contained one species of membrane-bound c-type cytochrome, presumably cytochrome c₁, and a b-type cytochrome with two heme centers. The molecular weight and midpoint potential of cytochrome c₁ were 30000 and 275 mV, respectively. The peak of the reduced-minus-oxidized difference spectrum of cytochrome c₁ was at 552 nm. Molecular weight of the b-type cytochrome was 32000 and the cytochrome had two midpoint potentials of 60 mV and −55 mV. The peaks of the reduced-minus-oxidized difference spectra of the high and low midpoint potential heme centers were at 560 and 562 nm, respectively. These results suggested that there was a cytochrome b-c₁ complex in Rps. palustris.     

Lipid specificity for membrane mediated partial unfolding of cytochrome c

In this study we investigated the lipid specificity for destabilization of the native structure of horse heart cytochrome c by model membranes. From (i) the enhanced release of deuterium from deuterium-labelled cytochrome c and (ii) the increased proteolytic digestion of the protein in the presence of anionic lipids, it was concluded that these lipids are able to destabilize the native structure of cytochrome c. Changes in the absorbance at 695 nm indicated that the destabilization was accompanied by a diminished ligation of Met-80 to the heme. Beef heart cardiolipin was found to be more effective than DOPS, DOPG or DOPA, while no protein destabilization was observed in the presence of the zwitterionic lipid DOPC or, surprisingly, in the presence of E. coli cardiolipin. Experimnts with mitoplasts showed that the protein can also be destabilized in its native structure by a biological membrane.     

A nitrite reductase with cytochrome oxidase activity from Micrococcus denitrificans

The formation of nitrite reductase and cytochrome c in Micrococcus denitrificans was repressed by O₂. The purified nitrite reductase utilized reduced forms of cytochrome c, phenazine methosulphate, benzyl viologen and methyl viologen, respectively, as electron donors. The enzyme was inhibited by KCN, NaN₃ and NH₂OH each at 1 mM, whereas CO and bathocuproin, diethyl dithiocarbamate, o-phenanthroline and ,'-dipyridyl at 1 mM concentrations were relatively ineffective. The purified enzyme contains cytochromes, probably of the c and a₂ types, in one complex. A K_m of 46 μM for NO₂⁻ and a pH optimum of 6.7 were recorded for the enzyme. The molecular weight of the enzyme was estimated to be around 130000, and its anodic mobility was 6.8·10⁻⁶ cm²·sec⁻¹·V⁻¹ at pH 4.55.

The most highly purified nitrite reductase still exhibited cytochrome c oxidase activity with a K_m of 27 μM for O₂. This activity was also inhibited by KCN, NaN₃ and NH₂OH and by NO₂⁻.

A constitutive cytochrome oxidase associated with membranes was also isolated from cells grown anaerobically with NO₂⁻. It was inhibited by smaller amounts of KCN, NaN₃ and NH₂OH than the cytochrome oxidase activity of the nitrite reductase enzyme and also differed in having a pH optimum of about 8 and a K_m for O₂ of less than 0.1 μM. Spectroscopically, cytochromes b and c were found to be associated with the constitutive oxidase in the particulate preparation. Its activity was also inhibited by NO₂⁻.

The physiological role of the cytochrome oxidase activity associated with the purified nitrite reductase is likely to be of secondary importance for the following reasons: (a) it accounts for less than 10% of total cytochrome c oxidase activity of cell extracts; (b) the constitutive cytochrome c oxidase has a smaller K_m for O₂ and would therefore be expected to function more efficiently especially at low concentrations of O₂.     

Arrangement of the light-harvesting chlorophyll a/b protein complex in the thylakoid membrane

The transverse orientation of the light-harvesting chlorophyll a/b protein complex of Photosystem II (LHC II) in the thylakoid membrane of pea was investigated using surface radioiodination with Iodo-Gen^TM. The labelling effects on LHC II of four different membrane preparations were compared. One preparation was oriented right-side-out (intact thylakoids); two of them had an inside-out orientation exposing the lumenal surface (inside-out vesicles; PS II particles) and one had both sides of the membrane exposed (mechanically damaged thylakoids). It was found that LHC II could be iodinated only in membrane preparations with an exposed lumenal surface. Isolated apoproteins were chemically cleaved. Fragments analysis revealed a tyrosine residue located eight amino acids from the C-terminus as the single iodination site. It is concluded that the C-terminus of LHC II points towards the lumental side of the thylakoid. Differences in the labelling behaviour of the LHC apoproteins could be assigned to a heterogeneity in the C-terminal region in which the tyrosine residue is replaced by phenylalanine.     

Mitochondrial cytochrome components of Paragonimus adult worms

The cytochrome components of adult Paragonimus miyazakii mitochondria were investigated by polyacrylamide gel electrophoresis. The mitochondria were found to contain cytochromes b, c₁, c and aa₃. Two types of mitochondria, lightweight mitochondria (LWMt) and heavyweight mitochondria (HWMt), were obtained by centrifugation from the mitochondrial fraction of the adult Paragonimus ohirai. The succinate-reduced and oxidized difference spectrum of LWMt and HWMt at −196°C revealed that both mitochondria contained at least functional levels of cytochromes b, c₁, c and a low value of aa₃. Although succinate-reduced cytochromes of LWMt reoxidized in the presence of air, those of HWMt did so only minimally.     

The adaptation of the electron transfer chain of Rhodopseudomonas capsulata to different light intensities

(1) Cells of Rhodopseudomonas capsulata (wild-type) were grown photoheterotrophically in a turbidostat under very high and very low light intensity. Membranes were isolated from cells adapted to the respective light conditions and fractionated by sucrose density centrifugation. The molar ratios of ubiquinone and cytochromes c₂, c₁, b-561 and b-566 per reaction center were 3-fold to 5-fold higher in high-light than in low-light membranes. (2) Most of the Cyt(c₁ + c₂) and Cyt b-561 detected in dark redox titrations undergoes light-induced redox changes, both in high- and in low-light membranes. (3) The fractions of the total photooxidizable reaction center and Cyt(c₁ + c₂) oxidized under continuous light in the absence of antimycin are higher in membranes from low-light- than from high-light-grown cells. (4) From these data and results of kinetic studies it is proposed that cyclic electron flow under saturating light intensities is faster in high-light-grown cells.     

Electron transfer components of wild-type and photosynthetic mutant strains of Scenedesmus obliquus D3

To investigate the possible alteration of various components of the photosynthetic electron transport system of certain mutants of Scenedesmus techniques were developed for their extraction and purification from whole cells of this alga. The components identified in the normal alga were cytochrome c 549, cytochrome b 562, a cytochrome c 551, flavoprotein-ferredoxin reductase, plastocyanin, cytochrome c 552, and ferredoxin. Lamellar-bound cytochrome c 552 and cytochromes b were also detected. Application of the extraction and purification techniques to two photosynthetic mutants revealed that Mutants 26 and 50 lacked cytochrome f in both the bound and soluble forms (Mutant 50) or in only the bound form (Mutant 26). Chloroplasts prepared from either of these mutants lacked Hill reaction activity with a variety of oxidants with water as the electron donor but photoreduced NADP⁺ with 2,6-dichlorophenolindophenol and ascorbate as the electron donor system. No photophosphorylation in vivo was detected with either mutant, but isolated chloroplasts performed a cyclic photophosphorylation with phenazine methosulphate as cofactor. Fluorescence analysis revealed that both mutants possess a measurable Photosystem II activity.

It was concluded that the loss of cytochrome f prevents the normal flow of electrons from Photosystem II to NADP and also to a variety of other Hill reaction oxidants. Furthermore, cytochrome f is not required for the reduction of NADP with electron donor systems other than water nor is it an essential component of the mechanism of cyclic photophosphorylation with phenazine methosulphate as cofactor.     

Biochemical and biophysical studies on cytochrome c oxidase XIV. The reaction with cytochrome c as studied by pulse radiolysis

1. The reduction of cytochrome c oxidase by hydrated electrons was studied in the absence and presence of cytochrome c.

2. Hydrated electrons do not readily reduce the heme of cytochrome c oxidase. This observation supports our previous conclusion that heme a is not directly exposed to the solvent.

3. In a mixture of cytochrome c and cytochrome c oxidase, cytochrome c is first reduced by hydrated electrons (k = 4 · 10¹⁰ M⁻¹ · s⁻¹ at 22 °C and pH 7.2) after which it transfers electrons to cytochrome c oxidase with a rate constant of 6 · 10⁷ M⁻¹ · s⁻¹ at 22 °C and pH 7.2.

4. It was found that two equivalents of cytochrome c are oxidized initially per equivalent of heme a reduced, showing that one electron is accepted by a second electron acceptor, probably one of the copper atoms of cytochrome c oxidase.

5. After the initial reduction, redistribution of electrons takes place until an equilibrium is reached similar to that found in redox experiments of Tiesjema, R. H., Muijsers, A. O. and Van Gelder, B. F. (1973) Biochim. Biophys. Acta 305, 19–28.     

Cytochrome c interaction with the mitochondrial membrane: A spin label study

Horse-heart ferrocytochrome c has been labeled with N-(2,2,5,5-tetramethyl-3-pyrrolidinyl-1-oxyl) iodoacetamide at methionine-65. The paramagnetic resonance spectrum of labeled ferricytochrome c indicates a weak immobilization of the radical (τ_c = 9.3·10⁻¹⁰ sec) which becomes stronger upon binding of labeled cytochrome c to cytochrome c-depleted mitochondrial membranes (τ_c = 3.3·10⁻⁹ sec). The hyperfine coupling constant remains, however, unchanged (16.7 ± 0.1 gauss) indicating that the cytochrome c binding site is highly polar. The region where cytochrome c is bound to the membrane is insensitive to large variations of medium viscosity.     

A chlorophyll b-less mutant of Chlamydomonas reinhardii lacking in the light-harvesting chlorophyll a/b-protein complex but not in its apoproteins

The mutant pg 113, derived from Chlamydomonas reinhardii, arg₂⁻ mt⁺ (parent strain), completely lacks chlorophyll (Chl) b but is still able to grow under autotrophic conditions. The light-harvesting Chl a/b-protein complex (LHCP) is absent. This is shown (a) by the lack of the corresponding signal in the CD spectrum of thylakoids and (b) by the absence of the band of the LHCP after electrophoresis of partially solubilized thylakoid membranes on lithium dodecyl sulfate polyacrylamide gels. All the other chlorophyll-protein complexes are present. In spite of the absence of the LHCP, all the polypeptide components of this complex are present in the mutant in the same ratios as in the parent strain, although in slightly reduced amounts. The LHC apoproteins are synthesized, processed and transported into the thylakoid membrane of the mutant. Moreover, the phosphorylation of thylakoid membrane polypeptides, which is related to the regulation of the energy distribution between Photosystem I and II, is the same in the mutant and in the parent strain, indicating that phosphorylation is not dependent on the presence of Chl b. Electron micrographs of thin sections of whole cells show that there are stacked regions of thylakoids in both the mutant and the parent strain chloroplasts. However, in the mutant, stacks are located near the chloroplast envelope, while long stretches or sometimes circles of unstacked membranes are found in the interior, mostly around the pyrenoid.     

Cytochrome c involved in the reductive decomposition of organic mercurials. Purification of cytochrome c-I from mercury-resistant Pseudomonas and reactivity of cytochromes c from various kinds of bacteria

Cytochrome c-I which was involved in the decomposition of organic mercurials as an electron carrier was purified from the cell-free extract of the mercury-resistant strain, Pseudomonas K62, by means of (NH₄)₂SO₄ precipitation and column chromatography on Sephadex G-150, DEAE-Sephadex and Sephadex G-75. The cytochrome was crystallized in a needle-like form. It showed absorption maxima at 550, 521, and 416.5 nm in the reduced form, and the pyridine ferrohemochrome had absorption maxima at 549, 520, and 413 nm, suggesting it to be a c-type cytochrome.

Cytochromes c prepared from type cultures of bacteria belonging to the genera Aeromonas, Micrococcus, Bacillus, Corynebacterium, Staphylococcus, Aerobacter, and Pseudomonas were all inactive with respect to the decomposition of phenylmercuric acetate. However, cytochrome c prepared from Pseudomonas CF, which was isolated from the activated sludge acclimatized with HgCl₂ and phenylmercuric acetate, as well as the cytochrome c-I of Pseudomonas K62, were active in this respect.     

The P-700-chlorophyl a-protein complex and two major light-harvesting complexes of Acrocarpia paniculata and other brown seaweeds

Acrocarpia paniculata thylakoids were fragmented with Triton X-100 and the pigment-protein complexes so released were isolated by sucrose density gradient centrifugation. Three main chlorophyll-carotenoid-protein complexes with distinct pigment compositions were isolated.

1. (1) A P-700-chlorophyll a-protein complex, with a ratio of 1 P-700: 38 chlorophyll a: 4 ta-carotene molecules, had similar absorption and fluorescence characteristics to the chlorophyll-protein complex 1 isolated with Triton X-100 from higher plants, green algae and Ecklonia radiata.

2. (2) An orange-brown complex had a chlorophyll a : c₂ : fucoxanthin molar ratio of 2 : 1 : 2. This complex had no chlorophyll c₁ and contained most of the fucoxanthin present in the chloroplasts. This pigment complex is postulated to be the main light-harvesting complex of brown seaweeds.

3. (3) A green complex had a chlorophyll a : c₁ : c₂ : violaxanthin molar ratio of 8 : 1 : 1 : 1. This also is a light-harvesting complex.

The absorption and fluorescence spectral characteristics and other physical properties were consistent with the pigments of these three major complexes being bound to protein. Differential extraction of brown algal thylakoids with Triton X-100 showed that a chlorophyll c₂-fucoxanthin-protein complex was a minor pigment complex of these thylakoids.     

Characterization of a respiratory mutant of Escherichia coli with reduced uptake of aminoglycoside antibiotics

A strain of Escherichia coli (NSW77) which is partially resistant to streptomycin was isolated by selecting for growth on plates supplemented with 12.5 μg/ml streptomycin, a concentration which completely inhibits growth of wild-type strains. The low-level resistance of the mutant appears to result from a reduced ability to accumulate streptomycin intracellularly. In addition, the mutant strain is unable to use succinate for growth because of a defective respiratory chain. Thus, membranes of the mutant strain were found to have approximately half the NADH and D-lactate oxidase activity of the parent strain. Succinate oxidase activity was reduced more drastically, to a level of 7% that of the parent strain. Moreover, membranes of the mutant were found to contain demethyl-menaquinone and, in place of ubiquinone, a structural analogue, 2-octaprenyl-3-methyl-6-methoxy-1,4 benzoquinone. The mutation responsible for both the Suc⁻ phenotype and partial resistance to streptomycin was found to be located near minute 15 on the bacterial chromosome. Both the biochemical and genetic evidence suggests that the mutation in strain NSW77 resides in the ubi F gene. Another previously characterized ubi F strain was also found to have a reduced capacity to take up an aminoglycoside antibiotic (gentamicin). These results suggest that the respiratory defects in ubi F strains are responsible for the reduced capacity of such strains to accumulate aminoglycosides.     

Properties of a nitrate reductase of Chlorella

1. An NADH-nitrate oxidoreductase (EC 1.6.6.1) of Chlorella has the unusual property of existing in cell-free extracts mainly in the form of an inactive precursor which can be activated by a variety of procedures. This enzyme is associated with a cytochrome of the b type.

2. The inhibitors, azide, cyanate, thiocyanate and nitrite, react rapidly with the enzyme, with kinetics which show that they are competitive with nitrate.

3. The inhibitors, cyanide and hydroxylamine, react slowly with the reduced form of the enzyme to give an inactive product which can slowly be reactivated in the presence of nitrate. There is at least a superficial similarity between the reactivation of the inhibited enzyme and the activation of the enzyme precursor in fresh extracts.

4. Mammalian cytochrome c, dichlorophenolindophenol and ferricyanide can substitute for nitrate as oxidants for NADH in the presence of the enzyme. This “diaphorase” reaction does not require activation, but is fully active in fresh extracts. It is not inhibited by cyanide, hydroxylamine, azide, cyanate, thiocyanate, or by the substrate, nitrate. Oxidized cytochrome c, on the other hand, inhibits the reduction of nitrate by NADH in the presence of the enzyme.

5. Pyridoxal phosphate inhibits both nitrate reductase and cytochrome c reductase to about the same extent.     

Autocatalytic peroxidation of ferrocytochrome c

1. Ferricytochrome c acts as a catalyst in the peroxidation of ferrocytochrome c thereby giving rise to an autocatalytic reaction.

2. The rate of the peroxidation reaction is proportional to the concentration of H₂O₂ and ferricytochrome c but is independent of the concentration of ferrocytochrome c in the concentration ranges studied.

3. Integration of the rate equation, d[c³⁺]/dt = k[c³⁺][H₂O₂], gives a theoretical expression which fits the experimental time courses for the ferrocytochrome c peroxidation reaction.

4. No direct spectral evidence was found for the formation of a catalytically active ferricytochrome c-H₂O₂ derivative. Kinetic evidence is presented, however, which indicates the existence of such an intermediate.

5. Ferricytochrome c was more susceptible than ferrocytochrome c to an apparent degradation reaction caused by excess H₂O₂, thus supporting the idea that the cytochrome c heme iron is more accessible in the oxidized form.