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.     

Shifts of bacteriochlorophyll and carotenoid absorption bands linked to cytochrome c-555 photooxidation in Chromatium

Shifts in the absorption bands of bacteriochlorophyll and carotenoids in Chromatium vinosum chromatophores were measured after short actinic flashes, under various conditions. The amplitude of the bacteriochlorophyll band shift correlated well with the amount of cytochrome c-555 that was oxidized by P₈₇₀⁺ after a flash. No bacteriochlorophyll band shift appeared to accompany the photooxidation of P₈₇₀ itself, nor the oxidation of cytochrome c-552 by P₈₇₀⁺. The carotenoid band shift also correlated with cytochrome c-555 photooxidation, although a comparatively small carotenoid shift did occur at high redox potentials that permitted only P₈₇₀ oxidation.

The results explain earlier observations on infrared absorbance changes that had suggested the existence of two different photochemical systems in Chromatium. A single photochemical system accounts for all of the absorbance changes.

Previous work has shown that the photooxidations of P₈₇₀ and cytochrome c-555 cause similar changes in the electrical charge on the chromatophore membrane. The specific association of the band shifts with cytochrome c-555 photooxidation therefore argues against interpretations of the band shifts based on a light-induced membrane potential.     

Study of electrogenic electron transfer steps in chromatophore membrane of Chromatium vinosum by the response of merocyanin dye

1. Electrogenic steps in photosynthetic cyclic electron transport in chromatophore membrane of Chromatium vinosum were studied by measuring absorption changes of added merocyanin dye and of intrinsic carotenoid.

2. The change in dye absorbance was linear with the membrane potential change induced either by light excitation or by application of diffusion potential by adding valinomycin in the presence of K⁺ concentration gradient.

3. It was estimated that chromatophore membrane became 40–60 mV and 110–170 mV inside positive upon single and multiple excitations with single-turnover flashes, respectively, from the responses of the dye and the carotenoid.

4. Electron transfers between cytochrome c-555 or c-552 and reaction center bacteriochlorophyll dimer (BChl₂) and between BChl₂ and the primary electron acceptor were concluded to be electrogenic from the redox titration of the dye response.

5. No dye response which corresponded to the change of redox level of cytochrome b was observed in the titration curve. Addition of antimycin A slightly decreased the dye response.

6. The dye response was decreased under phosphorylating conditions.

7. From the results obtained localization of the electron transfer components in chromatophore membrane is discussed.     

Redistribution of electric charge accompanying photo-synthetic electron transport in Chromatium

The isoionic pH of Chromatium chromatophores is 5.2±0.1. At pH 7.7, the net charge on the chromatophore is approx. −1·10⁴. If a change in this charge accompanies the oxidation of an electron carrier, the midpoint redox potential (E_m) of that carrier should be a function of the solution ionic strength (I). of that carrier should be a function of the solution ionic strength (I).

The E_m values of P870 and cytochrome c-555 increase strongly with increasing I at low values of I. The E_m of cytochrome c-552 also increases with increasing I, though not so strongly. These effects probably cannot be attributed to an influence of I on the activity coefficient of a dissociable ion. We conclude that, when either P870 or cytochrome c-555 loses an electron, no specific ions (including protons) are bound or released in significant amounts, and the absolute value of the charge on the chromatophore decreases.

The E_m values of the primary and secondary electron acceptors, X and Y, do not depend on I. Because these E_m values have been shown previously to depend on pH, we conclude that the uptake of a proton keeps the charge on the chromatophore constant when either X or Y accepts an electron. This means that the primary and secondary electron transfer reactions in Chromatium result in a net decrease in the charge on the photosynthetic membrane. They do not result in the translocation of protons across the membrane.

The E_m of the soluble flavocytochrome c-552 from Chromatium depends only weakly on I, but depends strongly on the pH. The uptake of a proton appears to keep the net charge on this cytochrome constant upon reduction.     

Nature of photochemical reactions in chromatophores of Chromatium D. II. Quantum yield of photooxidation of cytochromes in Chromatium chromatophores

Initial rates of the light-induced absorption decrease in Chromatium chromatophores due to the oxidation of cytochromes were measured under various experimental conditions. The initial rate in the presence of 10 mM potassium ferrocyanide and 50 μM potassium ferricyanide was about one-half to two-thirds of that in the presence of 30 mM ascorbate or in a medium with a redox potential (E_h) of − 78 mV.

Light-minus-dark difference spectrum indicated that, in the presence of 10 mM ferrocyanide and 50 μM ferricyanide, only cytochrome c-555 was photooxidized. In the presence of 30 mM ascorbate or at E_h values lower than about 0 mV, both cytochrome c-555 and cytochrome c-552 were photooxidized. The quantum yield of cytochrome c-555 photooxidation was calculated to be about 0.4.

The results obtained in the present study are compared with other investigators' and the possibility of the presence of two types of associations between the cytochromes and reaction-center bacteriochlorophyll is discussed.     

The cytochromes of Chlorobium thiosulfatophilum

Three c-type cytochromes (c-551, c-553, c-555) have been isolated and characterized from a strain of the green photosynthetic bacterium Chlorobium thiosulfatophilum. These cytochromes are atypical when compared to horse heart cytochrome c in many properties, among them: oxidation-reduction potential at pH 7.0 (c-551, 135 mV; c-553, 98 mV; c-555, 145 mV), molecular weight (c-551, 45000–60000; c-553, 50000; c-555, 10000) and isolelectric point (c-551, 6.0; c-553, 6.7). No protoheme was detected in whole cells or cell-free extracts.     

NADH dehydrogenase subunit 1 and cytochrome c oxidase subunit I sequences compared for members of the genus Taenia (Cestoda)

Nine members of the genus Taenia (Taenia taeniaeformis, Taenia hydatigena, Taenia pisiformis, Taenia ovis, Taenia multiceps, Taenia serialis, Taenia saginata, Taenia solium and the Asian Taenia) were characterised by their mitochondrial NADH dehydrogenase subunit 1 gene sequences and their genetic relationships were compared with those derived from the cytochrome c oxidase subunit I sequence data. The extent of inter-taxon sequence difference in NADH dehydrogenase subunit 1 (5.9–30.8%) was usually greater than in cytochrome c oxidase subunit I (2.5–18%). Although topology of the phenograms derived from NADH dehydrogenase subunit 1 and cytochrome c oxidase subunit I sequence data differed, there was concordance in that T. multiceps, T. serialis (of canids), T. saginata and the Asian Taenia (of humans) were genetically most similar, and those four members were genetically more similar to T. ovis and T. solium than they were to T. hydatigena and T. pisiformis (of canids) or T. taeniaeformis (of cats). The NADH dehydrogenase subunit 1 sequence data may prove useful in studies of the systematics and population genetic structure of the Taeniidae.     

Reducibility of cytochromes b in aerobic beef-heart mitochondria treated with antimycin

Under anaerobic conditions cytochrome b in beef-heart mitochondria is partially reduced in the presence of NADH, whereas other cytochromes are completely reduced. Addition of antimycin together with oxygen under these conditions causes an immediate reduction of cytochromes b-558, b-562 and b-566 and oxidation of cytochrome c. During the subsequent transient aerobic steady state cytochromes b-558 and b-566 are rapidly re-oxidized without changes in redox state of cytochrome c, but cytochrome b-562 remains reduced. When oxygen is consumed by the leak through or around the antimycin-inhibition site, cytochrome b-562 becomes oxidized with concomitant reduction of cytochrome c.

The cytochromes b in lyophilized beef-heart mitochondria are more readily accessible to electrons from NADH, and in the presence of antimycin and NADH a complete and stable reduction is obtained under both aerobic and anaerobic conditions. Gradual addition of rotenone under these conditions causes re-oxidation of cytochromes b in which oxidation of cytochromes b-558 and b-566 precedes that of cytochrome b-562.

It is concluded that (1) the effect of antimycin in the presence of oxygen involves all three cytochromes b, (2) the reducibility of the cytochromes b in the aerobic steady state of antimycin-treated mitochondria is dependent upon the potential of the substrate redox couple registered on the cytochromes, and (3) the midpoint potential of cytochrome b-562 in the presence of antimycin is higher than that of cytochrome b-558 or b-566.     

The effect of iron-hexacyanide binding on the determination of redox potentials of cytochromes and copper proteins

The midpoint redox potentials of Pseudomonas aeruginosa cytochrome c-551 and Rhodopseudomonas viridis cytochrome c₂ were measured as a function of pH in the presence of Euglena cytochrome c-558 and the results compared with those obtained in the presence of ferro-ferricyanide. The pattern of pH dependence observed for the two bacterial cytochromes was the same whether it was measured by equilibrium with another redox protein or with the inorganic redox couple. Thus, the pH dependence of redox potential is not a consequence of pH-dependent ligand binding. The midpoint potential of Ps. aeruginosa azurin was measured as a function of pH using both ferro-ferricyanide mixtures and redox equilibrium with horse cytochrome c or Rhodopseudomonas capsulata cytochrome c₂. In this case also the pattern of pH dependence obtained did not vary with the redox system used and it closely resembled that of Ps. aeruginosa cytochrome c-551. This is consistent with the observation that the equilibrium between cytochrome c-551 and azurin is relatively independent of pH. An equation was derived which described pH-dependent ligand binding and which can produce theoretical curves to fit the experimental pH dependence of redox potential for both cytochrome and azurin. However, the pronounced effect on such curves produced by varying the ligand association constants, and the insensitivity of the experimental data to changes in ionic strength, suggest that ligand binding effects do not account for the pH dependence of redox potential.     

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.     

Orientation of the hemes of high potential cytochromes relative to photosynthetic membranes, as shown by the linear dichroism of oriented preparations

The orientations of high potential cytochromes with respect to photosynthetic membranes was investigated in spinach chloroplasts and in Rhodopseudomonas viridis. The general approach consists in detection with polarized light of photoinduced absorbance changes related to the oxidation of the cytochromes. The orientation of cytochrome c-558 was measured at room temperature in chromatophores and whole cells of Rps. viridis, oriented on glass slides and in a magnetic field, respectively. The orientation of cytochrome b-559 of green plants was detected at 77 K in magnetically oriented chloroplasts. In both cases the dichroic ratio for the band shows that the heme plane makes an angle greater than 35°C with the membrane plane. Moreover, the dichroic ratio is not constant throughout the and β bands, for both cytochrome c-558 and b-559. Linear dichroism spectra of oriented pure horse heart cytochrome c and cytochrome c₂ of Rhodopseudomonas sphaeroides in stretched polyvinyl alcohol films show that the variations of the dichroic ratio in the and β bands can be explained by the occurrence of x- and y-polarized transitions absorbing at slightly different wavelengths.     

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.     

木霉属3个中国新记录种

报道了采自我国黑龙江、吉林及西藏的木霉属Trichoderma 3个中国新记录种：内生木霉Trichoderma endophyticum、意大利木霉T. italicum和酒色木霉T. vinosum。首次发现近深绿木霉T. paratroviride的有性阶段,提供了上述种的详细描述及宏观和微观特征的图示,并探讨了其分类地位。     

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.     

Properties of the reaction center of the thermophilic purple photosynthetic bacterium Chromatium tepidum

Reaction centers were purified from the thermophilic purple sulfur photosynthetic bacterium Chromatium tepidum. The reaction center consists of four polypeptides L, M, H and C, whose apparent molecular masses were determined to be 25, 30, 34 and 44 kDa, respectively, by polyacrylamide gel electrophoresis. The heaviest peptide corresponds to tightly bound cytochrome. The tightly bound cytochrome c contains two types of heme, high-potential c-556 and low-potential c-553. The low-potential heme is able to be photooxidized at 77 K. The reaction center exhibits laser-flash-induced absorption changes and circular dichroism spectra similar to those observed in other purple photosynthetic bacteria. Whole cells contain both ubiquinone and menaquinone. Reaction centers contain only a single active quinone; chemical analysis showed this to be menaquinone. Reaction center complexes without the tightly bound cytochrome were also prepared. The near-infrared pigment absorption bands are red-shifted in reaction centers with cytochrome compared to those without cytochrome.     

The redox properties of the cytochromes of purified Complex III

Purified Complex III from beef heart contains two b cytochromes: a high-potential (E_{m 7.2} = +93 mV) cytochrome b-562 which can be enzymatically reduced, and a low-potential (E_{m 7.2} = −34 mV) cytochrome b-565 which is reduced only by dithionite. The two components each contribute approximately 50% to the total cytochrome b of Complex III. Cytochrome c₁ of Complex III titrates with a half-reduction potential of +232 mV.     

The genetic analysis of F1 hybrid larvae between female Trichinella spiralis and male Trichinella britovi

Hybrids between female Trichinella spiralis and male Trichinella britovi were constructed. Then, hybrid genotype was characterized by DNA markers including mitochondrial cytochrome c oxidase subunit I (CO I) gene, the gene encoding the 43-kDa excretory–secretory (ES) protein, and genomic DNA fragments specific for T. spiralis and T. britovi identified from random amplified polymorphism DNA (RAPD). PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of the mitochondrial CO I gene revealed that all hybrids carried a T. spiralis pattern. The same analysis of the gene encoding the 43-kDa ES protein showed that each hybrid carried both T. spiralis and T. britovi gene type simultaneously. In the analysis of genomic DNA using RAPD-derived PCR primers, some hybrids carried T. spiralis and T. britovi-specific RAPD markers, while others carried the RAPD marker of T. spiralis only.     

Studies on well-coupled Photosystem I-enriched subchloroplast vesicles — characteristics and reinterpretation of single-turnover cyclic electron transfer

The contributions of ferredoxin, P-700, plastocyanin and the cytochromes c-554, and b-563 to single-turnover electron transfer in Photosystem (PS) I-enriched subchloroplast vesicles were deconvoluted by fitting the literature-derived spectra of these components to the observed absorption data at a series of wavelengths, according to a linear least-squares method. The obtained corresponding residuals showed that the applied component spectra were satisfactory. The deconvoluted signals of cytochromes c-554 and b-563 differed in some cases significantly from the classical dual-wavelength signals recording at 554–545 nm and 563–575 (or −572) nm, due to interference from other electron-transferring components. KCN, DNP-INT (2-iodo-6-isopropyl-3-methyl-2′,4,4′-trinitrodiphenyl ether), DBMIB (2,5-dibromo-3-methyl-6-isopropyl-p-benzo-quinone) and antimycin A all inhibited electron transfer, although antimycin and DBMIB inhibited only after a few turnovers of the cytochrome bf complex. Fast flash-induced reduction of cytochrome b-563 exclusively reflected oxidant-induced reduction. Fast electron flow from cytochrome c-554 to plastocyanin and P-700 resulted in an apparent rereduction of cytochrome c-554 that was slower than the reduction of cytochrome b-563. Model simulations indicate that under highly oxidizing conditions for the Rieske FeS centre and reducing conditions for cytochrome b-563, the semiquinone at the Q_z site cannot only reduce cytochrome b-563, but can also oxidize cytochrome b-563 and reduce the Rieske FeS centre. The effect of 10 μM gramicidin D was evaluated in order to determine the contributions by electrochromic absorption changes around 518 nm. Gramicidin left electron transfer, monitored in the 550–600 nm range, unchanged. The gramicidin-sensitive (membrane potential-associated) signal at 518 nm differed from the signals recorded in the absence of gramicidin at 518 nm or 518–545 nm, due to spectral interference from electron-transferring components in the latter signals. KCN, DBMIB and antimycin A affected both the fast and slow components of the electrochromic signal, but did not proportionally affect the initial electron transfer from P-700 to ferredoxin (charge separation in PS I). Not only the slow (10–100 ms) component of the 518 nm absorption change, but also part of the fast (less than 1 ms) component appears to minitor electrogenic events in the cytochrome bf complex.     

Respiratory cytochromes of Euglena gracilis

1. 1. Difference spectra of whole cells and of a particulate fraction of a streptomycin-bleached strain of Euglena gracilis showed the presence of a b-type cytochrome, cytochrome b (561 Euglena), and an a-type cytochrome, cytochrome a-type (609 Euglena). The cytochromes were characterized by pyridine hemochromogen formation and were found associated with a particulate fraction enriched with mitochondria.

2. 2. Both b-type and a-type cytochromes were reduced by succinate, oxidized by oxygen and reacted with a soluble c-type cytochrome, cytochrome c-type (556 Euglena), in reversible oxidation-reduction reactions. The steady-state level of reduction for each cytochrome was 92, 22 and 5% of the anaerobic level for the b-type, c-type and a-type cytochrome, respectively.

3. 3. Oxidation of c-type and a-type cytochromes was completely inhibited by cyanide, although respiration of a particulate fraction was only 60% inhibited by the same concentration of cyanide. Antimycin A inhibited respiration by up to 70% but completely inhibited reduction of the c-type cytochrome.

4. 4. The data suggest that electron transfer in the respiratory pathway of Euglena involves the b-, c- and a-type cytochrome in a direct sequence. The cyanide and antimycin A-insensitive oxidation pathway is considered to involve a more direct oxidation of the b-type cytochrome.

Rate of electron transfer between plastocyanin, cytochrome ƒ, related proteins and artificial redox reagents in solution

The rate of electron transfer between reduced cytochrome ƒ and plastocyanin (both purified from parsley) has been measured as k = 3.6 · 10⁷ M⁻¹ · s⁻¹, at 298 °K and pH 7.0, with activation parameters ΔH^‡ = 44 kJ · mole⁻¹ and ΔS^‡ = +46 J · mole⁻¹ · °K⁻¹. Replacement of cytochrome ƒ with red algal cytochrome c-553, Pseudomonas cytochrome c-551 and mammalian cytochrome c gave rates at least 30 times slower: k = 5 · 10⁵, 7.5 · 10⁵ and 1.0 · 10⁶ M⁻¹ · s⁻¹, respectively.

Similar measurements made with azurin instead of plastocyanin gave k = 6 · 10⁶ and approx. 2 · 10⁷ M⁻¹ · s⁻¹ for reaction of reduced azurin with cytochrome ƒ and algal cytochrome respectively.

Rate constants of 115 and 80 M⁻¹ · s⁻¹ were found for reduction of plastocyanin by ascorbate and hydroquinone at 298 °K and pH 7.0. The rate constants for the oxidation of plastocyanin, cytochrome ƒ, Pseudomonas cytochrome c-551 and red algal cytochrome c-553 by ferricyanide were found to be between 3 · 10⁴ and 8 · 10⁴ M⁻¹ · s⁻¹.

The results are discussed in relation to photosynthetic electron transport.