The oxidation mechanisms of thiosulphate and sulphide in Chlorobium thiosulphatophilum: Roles of cytochrome c-551 and cytochrome c-553

A thiosulphate-cytochrome c reductase was highly purified from Chlorobium thiosulphatophilum and its properties were studied. The enzyme catalyses reduction with Na₂S₂O₃ of c cytochromes, including cytochrome c-551 of the bacterium. Cytochrome c (555, C. thiosulphatophilum) does not react directly with the enzyme at an appreciable rate but stimulates greatly the reduction by the enzyme of cytochrome c-551 with Na₂S₂O₃. The reduction of c cytochromes catalysed by the enzyme is strongly inhibited by cyanide and sulphite.Cytochrome c (553, C. thiosulphatophilum), a c-type cytochrome with covalently bound flavin, was found to catalyse reduction with sulphide of c cytochromes, including cytochrome c-555. The reaction is strongly inhibited by cyanide. Cyanide seems to combine strongly with cytochrome c-553 probably at the flavin moiety. Thus, the absorption spectrum attributable to flavin of the haemoprotein is changed on addition of cyanide, and neither the original spectrum nor the activity reappears even after the cyanide-treated cytochrome has been subjected to gel filtration with a Sephadex G-25 column or to isoelectric focusing.     

Submolecular unfolding units of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> cytochrome <Emphasis Type="Italic">c</Emphasis>-551

Hydrogen exchange rates for backbone amide protons of oxidized Pseudomonas aeruginosa cytochrome c-551 (P. aeruginosa cytochrome c) have been measured in the presence of low concentrations of the denaturant guanidine hydrochloride. Analysis of the data has allowed identification of submolecular unfolding units known as foldons. The highest-energy foldon bears similarity to the proposed folding intermediate for P. aeruginosa cytochrome c. Parallels are seen to the foldons of the structurally homologous horse cytochrome c, although the heme axial methionine-bearing loop has greater local stability in P. aeruginosa cytochrome c, in accord with previous folding studies. Regions of low local stability are observed to correspond with regions that interact with redox partners, providing a link between foldon properties and function. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Electron transfer kinetics between rhus vernicifera stellacyanin and cytochrome c (horse heart cytochrome c and pseudomonas cytochrome c551)

The electron transfer reactions between Rhus vernicifera stellacyanin and either horse heart cytochrome c or Pseudomonas aeruginosa cytochrome c₅₅₁ were investigated by rapid reaction techniques. The time course of electron transfer is monophasic under all conditions, and thus consistent with a simple formulation of the reaction. Both stopped-flow and temperature-jump experiments yield equilibrium constants in reasonable agreement with values calculated from the redox potentials. The differences in reaction rate between the two cytochromes and stellacyanin are discussed in terms of the Marcus theory.     

Discovery and characterization of electron transfer proteins in the photosynthetic bacteria

Research on photosynthetic electron transfer closely parallels that of other electron transfer pathways and in many cases they overlap. Thus, the first bacterial cytochrome to be characterized, called cytochrome c ₂, is commonly found in non-sulfur purple photosynthetic bacteria and is a close homolog of mitochondrial cytochrome c. The cytochrome bc ₁ complex is an integral part of photosynthetic electron transfer yet, like cytochrome c ₂, was first recognized as a respiratory component. Cytochromes c ₂ mediate electron transfer between the cytochrome bc ₁ complex and photosynthetic reaction centers and cytochrome a-type oxidases. Not all photosynthetic bacteria contain cytochrome c ₂; instead it is thought that HiPIP, auracyanin, Halorhodospira cytochrome c551, Chlorobium cytochrome c555, and cytochrome c ₈ may function in a similar manner as photosynthetic electron carriers between the cytochrome bc ₁ complex and reaction centers. More often than not, the soluble or periplasmic mediators do not interact directly with the reaction center bacteriochlorophyll, but require the presence of membrane-bound intermediates: a tetraheme cytochrome c in purple bacteria and a monoheme cytochrome c in green bacteria. Cyclic electron transfer in photosynthesis requires that the redox potential of the system be delicately poised for optimum efficiency. In fact, lack of redox poise may be one of the defects in the aerobic phototrophic bacteria. Thus, large concentrations of cytochromes c ₂ and c′ may additionally poise the redox potential of the cyclic photosystem of purple bacteria. Other cytochromes, such as flavocytochrome c (FCSD or SoxEF) and cytochrome c551 (SoxA), may feed electrons from sulfide, sulfur, and thiosulfate into the photosynthetic pathways via the same soluble carriers as are part of the cyclic system. This revised version was published online in August 2006 with corrections to the Cover Date.     

Comparison of low oxidoreduction potential cytochrome c553 from Desulfovibrio vulgaris with the class I cytochrome c family

The cytochrome c553 from Desulfovibrio vulgaris (DvH c553) is of importance in the understanding of the relationship of structure and function of cytochrome c due to its lack of sequence homology with other cytochromes, and its abnormally low oxido-reduction potential. In evolutionary terms, this protein also represents an important reference point for the understanding of both bacterial and mitochondrial cytochromes c. Using the recently determined nuclear magnetic resonance (NMR) structure of the reduced protein we compare the structural, dynamic, and functional characteristics of DvH c553 with members of both the mitochondrial and bacterial cytochromes c to characterize the protein in the context of the cytochrome c family, and to understand better the control of oxido-reduction potential in electron transfer proteins. Despite the low sequence homology, striking structural similarities between this protein and representatives of both eukaryotic [cytochrome c from tuna (tuna c)] and prokaryotic [Pseudomonas aeruginosa c551 (Psa c551)] cytochromes c have been recognized. The previously observed helical core is also found in the DvH c553. The structural framework and hydrogen bonding network of the DvH c553 is most similar to that of the tuna c, with the exception of an insertion loop of 24 residues closing the heme pocket and protecting the propionates, which is absent in the DvH c553. In contrast, the Psa c551 protects the propionates from the solvent principally by extending the methionine ligand arm. The electrostatic distribution at the recognized encounter surface around the heme in the mitochondrial cytochrome is reproduced in the DvH c553, and corresponding hydrogen bonding networks, particularly in the vicinity of the heme cleft, exist in both molecules. Thus, although the cytochrome DvH c553 exhibits higher primary sequence homology to other bacterial cytochromes c, the structural and physical homology is significantly greater with respect to the mitochondrial cytochrome c. The major structural and functional difference is the absence of solvent protection for the heme, differentiating this cytochrome from both reference cytochromes, which have evolved different mechanisms to cover the propionates. This suggests that the abnormal redox potential of the DvH c553 is linked to the raised accessibility of the heme and supports the theory that redox potential in cytochromes is controlled by heme propionate solvent accessibility.     

Inhibition of Respiration in Prototheca zopfii by Light

Irradiation of cells of Prototheca zopfii with blue light inhibited the respiratory capacity of the cells. The inhibition of respiration was correlated with a photodestruction of cytochrome c(551), cytochrome b(559), and cytochrome a₃. Cytochrome c(549), cytochrome b(555), and cytochrome b(564) were unaffected by the irradiation treatment. The α-band of reduced cytochrome a was shifted from 599 to 603 nm by irradiation, an effect similar to that observed when methanol was added to nonirradiated cells. The presence of oxygen was required during irradiation for both photoinhibition of respiration and photodestruction of the cytochromes. Cytochrome a₃ was protected against photodestruction by cyanide. Photodestruction of these same cytochromes also occurred when washed mitochondria of P. zopfii were irradiated.     

Commonly stabilized cytochromes c from deep-sea Shewanella and Pseudomonas

Two cytochromes c₅ (SBcytc and SVcytc) have been derived from Shewanella living in the deep-sea, which is a high pressure environment, so it could be that these proteins are more stable at high pressure than at atmospheric pressure, 0.1 MPa. This study, however, revealed that SBcytc and SVcytc were more stable at 0.1 MPa than at higher pressure. In addition, at 0.1–150 MPa, the stability of SBcytc and SVcytc was higher than that of homologues from atmospheric-pressure Shewanella, which was due to hydrogen bond formation with the heme in the former two proteins. This study further revealed that cytochrome c₅₅₁ (PMcytc) of deep-sea Pseudomonas was more stable than a homologue of atmospheric-pressure Pseudomonas aeruginosa, and that specific hydrogen bond formation with the heme also occurred in the former. Although SBcytc and SVcytc, and PMcytc are phylogenetically very distant, these deep-sea cytochromes c are commonly stabilized through hydrogen bond formation.     

Electron donation from membrane-bound cytochrome c to the photosynthetic reaction center in whole cells and isolated membranes of Heliobacterium gestii

The reaction between membrane-bound cytochrome c and the reaction center bacteriochlorophyll g dimer P798 was studied in the whole cells and isolated membranes of Heliobacterium gestii. In the whole cells, the flash-oxidized P798⁺ was rereduced in multiple exponential phases with half times (t _1/2s) of 10 s, 300 s and 4 ms in relative amplitudes of 40, 35 and 25%, respectively. The faster two phases were in parallel with the oxidation of cytochrome c. In isolated membranes, a significantly slow oxidation of the membrane-bound cytochrome c was detected with t _1/2 = 3 ms. This slow rate, however, again became faster with the addition of Mg²⁺. The rate showed a high temperature dependency giving apparent activation energies of 88.2 and 58.9 kJ/mol in the whole cells and isolated membranes, respectively. Therefore, membrane-bound cytochrome c donates electrons to the P798⁺ in a collisional reaction mode like the reaction of water-soluble proteins. The rereduction of the oxidized cytochrome c was suppressed by the addition of stigmatellin both in the whole cells and isolated membranes. This indicates that the electron transfer from the cytochrome bc complex to the photooxidized P798⁺ is mediated by the membrane-bound cytochrome c. The multiple flash excitation study showed that 2–3 hemes c were connected to the P798. By the heme staining after the SDS-PAGE analysis of the membraneous proteins, two cytochromes c were detected on the gel indicating apparent molecular masses of 17 and 30 kDa, respectively. The situation resembles the case in green sulfur bacteria, that is, the membrane-bound cyotochrome c _z couples electron transfer between the cytochrome bc complex and the P840 reaction center complex.This revised version was published online in October 2005 with corrections to the Cover Date.     

Unexpected Elevated Production of Aquifex aeolicus Cytochrome c 555 in Escherichia coli Cells Lacking Disulfide Oxidoreductases

Mutant strains of Escherichia coli lacking DsbA, DsbB, or DsbD (proteins required for disulfide bond formation in the periplasm) did not produce mitochondrial or chloroplast cytochromes c, as previously observed for bacterial ones. Unexpectedly, however, cytochrome c ₅₅₅ (AA c ₅₅₅) from a hyperthermophile, Aquifex aeolicus, was produced in the E. coli periplasm without Dsb proteins, three times more than with them. These results indicate that the Dsb proteins are not necessarily required for AA c ₅₅₅ production in E. coli, possibly because of hyperthermophilic origin compared with the others.     

Complex formation between Chlorobium limicola f. thiosulfatophilumc-type cytochromes

It has been possible to demonstrate, using affinity chromatography, that Chlorobium flavocytochrome c-553 forms an electrostatically stabilized complex with Chlorobium cytochrome c-555. The binding site for cytochrome c-555 appears to be located on the heme-containing subunit of flavocytochrome c-553. This complex appears to be involved in the flavocytochrome c-553-catalyzed transfer of electrons from sulfide to cytochrome c-555. Complex formation has also been demonstrated between Chlorobium cytochromes c-555 and c-551, two components involved in the oxidation of thiosulfate by this green sulfur bacterium. Affinity chromatography data also suggest the possibility that the cytochrome binding sites on the Chlorobium flavocytochrome c-553 and on flavocytochrome c-552 from the purple sulfur bacterium Chromatium vinosum may be similar.     

The amino acid sequence of Nitrosomonas europaea cytochrome c-552

The complete amino acid sequence of cytochrome c-552 derived from the chemoautotrophic ammonia-oxidizing bacterium Nitrosomonas europaea was determined. The cytochrome consisted of 81 amino acid residues, and its molecular weight was calculated to be 9098 including heme c. Although the sequence of cytochrome c-552 was highly homologous to those of cytochromes c-551, which were known as the electron-donating components to dissimilatory nitrite reductase in pseudomonads, cytochrome c-552 differed from cytochrome c-551 in two points: (1) the sequence of cytochrome c-552 was shorter by two amino acid residues than that of cytochrome c-551 at the N-terminus and (2) one amino acid insertion was present in cytochrome c-552.     

Functional and Structural Comparisons between Prokaryotic and Eukaryotic aa3-Type Cytochrome c Oxidases from an Evolutionary Point of View

The specificities for cytochrome c of the aa3-type cytochromec oxidase were studied with enzymes derived from Thiobacillusnovellas, Nitrobacter agilis, Paracoccus denitrificans and thecow in reaction with the cytochromes c from 5 prokaryotes and7 eukaryotes. The T. novellus enzyme reacted most rapidly withthe cytochromes c of Candida krusei, tuna and bonito as wellas T. novellus cytochrome c; the specificity for cytochromec of the N. agilis enzyme was similar to that of the T. novellusenzyme. The bovine enzyme reacted rapidly with all the eukaryoticcytochromes c tested. The P. denitrificans enzyme showed a specificitysimilar to that of the bovine enzyme, except that it reactedrapidly with P. denitrificans cytochrome c, while the bovineenzyme reacted with it very poorly. All four kinds of enzymesshowed an extremely limited reaction with Pseudomonas aeruginosacytochrome c. The amino acid composition of subunit I of the N. agilis enzymeresembled that of the bovine enzyme, while the compositionsof their subunits II were different. On the basis of these results,an evolutionary relationship between bacterial and eukaryoticenzymes was discussed. (Received May 21, 1981; Accepted August 20, 1981)     

Antigenic diversity of cytochromes c3 from the anaerobic,sulfate-reducing bacteria,Desulfovibrio

An indirect enzyme-linked immunoadsorption assay (ELISA) was developed for cytochrome c₃ using antisera to the cytochromes fromDesulfovibrio africanus Benghazi, Desulfovibrio vulgaris Hildenborough andDesulfovibrio salexigens British Guiana. The ELISA system was used to test for cross-reactions between these antisera and the heterologous antigens. In contrast to previous experiments using the Ouchterlony technique, all of the cytochromes c₃ tested exhibited some degree of cross-reaction. Considerable variation was seen in cross-reactions for cytochromes c₃ from differing strains ofD. desulfuricans. This observation raises questions about the taxonomic relatedness of these strains. No cross-reaction was seen with eukaryotic cytochrome c or withD. vulgaris cytochrome c₅₅₃. The data demonstrate that cytochrome c₃ is capable of undergoing nonprecipitating cross-reactions, and thus may not be as immunologically unique as was once thought.Abbreviations ELISA Enzyme-linked immunoadsorption assay     

Function and properties of a soluble c-type cytochrome c-551 in secondary photosynthetic electron transport in whole cells of Chromatium vinosum as studied with flash spectroscopy

Changes in the absorption spectrum induced by 10-μs flashes and continuous light of various intensities were studied in whole cells of Chromatium vinosum.This paper describes the role and function of a soluble c-type cytochrome, c-551, which was surprisingly found to act in many ways similar to the cytochrome c-420 in Rhodospirillum rubrum, described in a previous paper [1].After the photooxidation of the membrane bound high potential cytochrome c-555 by a 10-μs flash, (the low potential cytochrome c-552 was kept permanently in the oxidized state) the oxidation of c-551 is observed ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 0.3}\text{ms}$). From a careful analysis of the absorbance difference spectrum and the kinetics it is concluded that there is approximately 0.6–0.7 c-551 per reaction center and that essentially all the c⁺-555 is reduced via the cytochrome c-551. The oxidized-reduced difference spectrum of c-551 shows peaks at 551 and 421.5 nm. The reduction of c⁺-551 following the flash-induced oxidation is strongly inhibited by HOQNO, but only slightly by antimycin A.Cytochrome c-551 reduces only the oxidized high potential cytochrome c-555, which is probably located on the outside of the membrane, on the opposite side of the primary acceptor. The low potential cytochrome c-552 does not show any detectable interaction with cytochrome c-551. After the cells have been sonicated, no c-551 is photooxidized and at least part of the cytochrome occurs in the solution.Analysis of the reduction kinetics of c⁺-551 in the absence and presence of external donors suggests that c⁺-551 is partly reduced via a cyclic pathway, which is blocked by addition of o-phenanthroline, and partly via a non-cyclic pathway. The non-cyclic reduction rate of c⁺-551 (k = 6 s^?1) is increased approximately 5–10 times upon thiosulphate addition, suggesting a role for c-551 between the final donor pool and the oxidized membrane bound c-type cytochromes.     

Resonance Raman fingerprinting of multiheme cytochromes from the cytochrome c 3 family

Resonance Raman (RR) spectroscopy was used to investigate conformational characteristics of the hemes of several ferricytochromes of the cytochrome c ₃ family, electron transfer proteins isolated from the periplasm and membranes of sulfate-reducing bacteria. Our analysis concentrated on the low-frequency region of the RR spectra, a fingerprint region that includes vibrations for heme-protein C–S bonds [ν(C_aS)]. It has been proposed that these bonds are directly involved in the electron transfer process. The three groups of tetraheme cytochrome c ₃ analyzed, namely Type I cytochrome c ₃ (TpIc ₃s), Type II cytochrome c ₃ (TpIIc ₃s) and Desulfomicrobium cytochromes c ₃, display different frequency separations for the two ν(C_aS) lines that are similar among members of each group. These spectral differences correlate with differences in protein structure observed among the three groups of cytochromes c ₃. Two larger cytochromes of the cytochrome c ₃ family display RR spectral characteristics for the ν(C_aS) lines that are closer to TpIIc ₃ than to TpIc ₃. Two other multiheme cytochromes from Desulfovibrio that do not belong to the cytochrome c ₃ family display ν(C_aS) lines with reverse relative areas in comparison with the latter family. This RR study shows that the small differences in protein structure observed among these cytochrome c ₃ correlate to differences on the heme–protein bonds, which are likely to have an impact upon the protein function, making RR spectroscopy a sensitive and useful tool for characterizing these cytochromes.     

A Novel function of cytochrome C (555, Chlorobium thiosulfatophilum) in oxidation of thiosulfate

Thiosulfate-cytochrome c-551 reductase derived from $\text{Chlorobium}\text{thiosulfatophilum}$ has been highly purified. The enzyme reduces cytochrome $\text{c}\text{-551 of}\text{C}\text{.}\text{thiosulfatophilum}$ in the presence of thiosulfate while cytochrome $\text{c}$-555 of the organism is not reduced by the enzyme. Cytochrome $\text{c}$-555 reacts with the enzyme at an appreciable rate only in the presence of cytochrome $\text{c}$-551. However, the reduction rate of cytochrome $\text{c}$-551 by the enzyme is greatly enhanced on addition of a catalytic amount of cytochrome $\text{c}$-555. Therefore, cytochrome $\text{c}$-555 seems to function as an effector on thiosulfate-cytochrome $\text{c}$-551 reductase as well as it acts as the electron donor to the light-excited chlorobium chlorophylls.     

Pseudomonas aeruginosa cytochrome c551 denaturation by five systematic urea derivatives that differ in the alkyl chain length

Reversible denaturation of Pseudomonas aeruginosa cytochrome c₅₅₁ (PAc₅₅₁) could be followed using five systematic urea derivatives that differ in the alkyl chain length, i.e. urea, N-methylurea (MU), N-ethylurea (EU), N-propylurea (PU), and N-butylurea (BU). The BU concentration was the lowest required for the PAc₅₅₁ denaturation, those of PU, EU, MU, and urea being gradually higher. Furthermore, the accessible surface area difference upon PAc₅₅₁ denaturation caused by BU was found to be the highest, those by PU, EU, MU, and urea being gradually lower. These findings indicate that urea derivatives with longer alkyl chains are stronger denaturants. In this study, as many as five systematic urea derivatives could be applied for the reversible denaturation of a single protein, PAc₅₅₁, for the first time, and the effects of the alkyl chain length on protein denaturation were systematically verified by means of thermodynamic parameters.     

Parallel electron donation pathways to cytochrome cz in the type I homodimeric photosynthetic reaction center complex of Chlorobium tepidum

We studied the regulation mechanism of electron donations from menaquinol:cytochrome c oxidoreductase and cytochrome c-554 to the type I homodimeric photosynthetic reaction center complex of the green sulfur bacterium Chlorobium tepidum. We measured flash-induced absorption changes of multiple cytochromes in the membranes prepared from a mutant devoid of cytochrome c-554 or in the reconstituted membranes by exogenously adding cytochrome c-555 purified from Chlorobium limicola. The results indicated that the photo-oxidized cytochrome c_z bound to the reaction center was rereduced rapidly by cytochrome c-555 as well as by the menaquinol:cytochrome c oxidoreductase and that cytochrome c-555 did not function as a shuttle-like electron carrier between the menaquinol:cytochrome c oxidoreductase and cytochrome c_z. It was also shown that the rereduction rate of cytochrome c_z by cytochrome c-555 was as high as that by the menaquinol:cytochrome c oxidoreductase. The two electron-transfer pathways linked to sulfur metabolisms seem to function independently to donate electrons to the reaction center.     

The electron transport chain ofStreptomyces erythreus: Possible functions of cytochromeaa 3 and a novel green pigment

The cytochromes of the bacteriumStreptomyces erythreus have been investigated. Membrane-bounda-, b-, andc-type cytochromes were found together with a green pigment, which was found in both a soluble and membrane-bound form. Cells containing the green pigment exhibited cyanide-insensitive oxygen uptake. The CO-binding pigments included cytochromea ₃, ab-type cytochrome, cytochrome P450, and the green pigment. Photodissociation spectra at various low temperatures, in the presence or absence of oxygen, revealed cytochromeaa ₃ to be the predominant cytochrome terminal oxidase. The green pigment was capable of electron transport; the relationship of the pigment to the remainder of the electron transport chain remains to be ascertained.     

Potentiometric and optical resolution of cytochromes c and c 1 in purified mitochondria from higher plant tissue,solanum tuberosum

Mitochondrial cytochromes c and c ₁ have long been considered indistinguishable from a potentiometric point of view. By combining rapid scan spectrometry to run redox titrations with numerical analysis using a generalized Nernst equation, it was possible to resolve cytochrome c and c ₁ midpoint potentials in yeast and mushroom mitochondria. In the reported work, this approach has been applied to purified mitochondria from higher plant tissue (Solanum tubersosum L.). The rapid scan spectrometric technique provided clear evidence of reversible base line changes monitored by redox potential changes. The basic mechanism responsible for this modification in the mitochondria optical properties remains to be defined. However, we suggest that this phenomenon could play a regulatory role in the overall electron transfer process. It is necessary to make an initial correction of the recorded spectra prior to numerical analysis. When this is done, two midpoint potential values are resolved by running analyses in the 550–555 nm range: 283±3 m V and 213±11 m V. They are identical to the ones found for cytochromes c and c ₁ in yeast and mushroom mitochondria. The individual difference bands were resolved by running analyses at each wavelength of the corrected spectra, the resolved midpoint potentials being kept fixed. This approach, the only one to date which has succeeded in resolving mitochondrial cytochrome c and c ₁ midpoint potentials, is discussed with respect to other methods. Limitations are pointed out.Abbreviation Mops morpholinopropane sulfonate