Electron transfer between horse heart and Candida krusei cytochromes c in the free and bound states

Electron transfer between horse heart and Candida krusei cytochromes c in the free and phosvitin-bound states was examined by difference spectrum and stopped-flow methods. The difference spectra in the wavelength range of 540–560 nm demonstrated that electrons are exchangeable between the cytochromes c of the two species. The equilibrium constants of the electron transfer reaction for the free and phosvitin-bound forms, estimated from these difference spectra, were close to unity at 20°C in 20 mM Tris-HCl buffer (pH 7.4). The electron transfer rate for free cytochrome c was (2–3) · 10⁴ M^?1 · s^?1 under the same conditions. The transfer rate for the bound form increased with increase in the binding ratio at ratios below half the maximum, and was almost constant at higher ratios up to the maximum. The maximum electron exchange rate was about 2 · 10⁶ M^?1 · s^?1, which is 60–70 times that for the free form at a given concentration of cytochrome c. The activation energy of the reaction for the bound cytochrome c was equal to that for the free form, being about 10 kcal/mol. The dependence of the exchange rate on temperature, cytochrome c concentration and solvent viscosity suggests that enhancement of the electron transfer rate between cytochromes c on binding to phosvitin is due to increase in the collision frequency between cytochromes c concentrated on the phosvitin molecule.     

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.     

The kinetics of photooxidation of c-type cytochromes by Rhodospirillum rubrum reaction centers.

The kinetics of photooxidation of c-type cytochromes from horse heart, Rhodospirillum rubrum, and Rhodopseudomonas capsulata by purified reaction centers from R. rubrum have been investigated. The kinetic mechanism was found to be complex with a second-order step (complex formation) followed by a rate limiting first-order step. Based on studies of the reaction as a function of pH, ionic strength, and detergent concentration, it appears that the complex formation step is largely electrostatically controlled with only portions of the surfaces of the interacting molecules participating. Further, the first-order process observed at high cytochrome concentration appears to result from solvent reorganization and/or a conformational change following complex formation. Based on data analysis in terms of outersphere electron transfer, it is proposed that another first-order process exists which is not rate limiting and is the electron transfer step. Finally, it was found that the detergent concentration can have a profound effect on both the oxidation-reduction potential of the cytochromes and the kinetics of photooxidation. These results limit the detergent concentration range over which experiments can be conducted and interpreted.     

Effects of protein-protein interactions on electron transfer: docking and electron transfer calculations for complexes between flavodoxin and c-type cytochromes

 Theoretical studies of protein-protein association and electron transfer were performed on the binary systems formed by Desulfovibrio vulgaris Hildenborough (D. v. H.) flavodoxin and D. v. H. cytochrome c ₅₅₃ and by flavodoxin and horse heart cytochrome c. Initial structures for the complexes were obtained by rigid-body docking and were refined by MD to allow for molecular flexibility. The structures thus obtained were analysed in terms of their relative stability through the calculation of excess energies. Electrostatic, van der Waals and solvation energy terms showed all to have significant contributions to the stability of complexes. In the best association solutions found for both cytochromes, these bind to different zones of flavodoxin. The binding site of flavodoxin observed for cytochrome c is in accordance with earlier works [27]. The various association modes found were characterised in terms of electron transfer using the Pathways model. For complexes between flavodoxin and horse heart cytochrome c, some correlation was observed between electron tunnelling coupling factors and conformation energy; the best conformation found for electron transfer corresponded also to the best one in terms of energy. For complexes between flavodoxin and cytochrome c ₅₅₃ this was not the case and a lower correlation was observed between electron tunnelling coupling factors and excess energies. These results are in accordance with the differences in the experimental dependence of electron transfer rates with ionic strength observed between these two cases. Received: 29 December 1998 / Accepted: 22 March 1999     

Oxidation of c-Type Cytochromes by the Membrane-Bound Cytochrome Oxidase (Cytochrome aa(3)) of Blue-Green Algae

Respiratory particles containing an aa₃-type cytochrome oxidase were prepared from Anacystis nidulans, Synechocystis 6714, Synechococcus lividus, Anabaena variabilis, Nostoc sp. strain MAC, Nostoc muscorum, and Mastigocladus laminosus. Oxidation of c-type cytochromes by membrane preparations of the different blue-green algae was observed using purified cytochromes from horse heart, Candida krusei, tuna, Saccharomyces oviformis, Rhodospirillum rubrum, Rhodospirillum molischianum, Rhodopseudomonas palustris, Rhodocyclus purpureus, Paracoccus denitrificans, Anacystis nidulans, Anabaena variabilis, Euglena gracilis, and Scenedesmus obliquus. Rapid oxidations were consistently observed with the mitochondrial c-type cytochromes (horse heart cytochrome c reacts most rapidly) and with cytochromes c₂ from Rhodopseudomonas palustris and Rhodocyclus purpureus; in contrast, the cytochrome c₂ from Rhodospirillum rubrum and the plastidic cytochromes from E. gracilis and Scendesmus obliquus were inactive with all membrane preparations. All reactions were inhibited by low concentrations of KCN, NaN₃, and CO, and they were activated by Tween 80, thus indicating participation of the terminal oxidase. The results are discussed in view of the spectral similarities between the terminal oxidase of blue-green algae and the mitochondrial aa₃-type cytochrome oxidase of plants and other eukaryotes.     

A Brownian dynamics study: the effect of a membrane environment on an electron transfer system

During the past few years, three-dimensional crystal structures of many of the important integral membrane proteins responsible for the bioenergetic processes of photosynthesis and respiration have been determined. Moreover, a few crystal structures of protein-protein complexes have become available that characterize the interaction between those membrane proteins and the electron carrier protein cytochrome c. Here, we address the association kinetics for binding of cytochrome c to cytochrome c oxidase (COX) from Paracoccus denitrificans by Brownian dynamics simulations. The effects of ionic strength and protein mutations were studied for two different cytochrome c species: the positively charged, dipolar horse heart cytochrome c and the negatively charged physiological electron transfer partner cytochrome c(552). We studied association toward "naked" COX and toward membrane-embedded COX where the membrane is represented as an uncharged DPPC bilayer modeled in atomistic detail. For the nonnatural association toward "naked" COX, the association rates are >100 times larger for horse heart cytochrome c than for cytochrome c(552). Interestingly, the presence of the lipid bilayer leads to a dramatic decrease of the association rate of horse heart cytochrome c, but slightly enhances association of cytochrome c(552), leading to very similar association rates of both proteins to membrane-embedded COX. This finding from computational modeling studies may reflect the optimization of surface patches and of the total net charge on electron transfer pairs in nature.     

Cytochrome c interactions with membranes. A photoaffinity-labeled cytochrome c.

The aryl azide, 2,4-dinitro-5-fluorophenylazide, was reacted with horse heart cytochrome c to give a photoaffinity-labeled derivative of this heme protein. The modified cytochrome c, with one to two dinitroazidophenyl groups per mole of the enzyme, has a half-reduction potential the same (± 10 mV) as native cytochrome c. The dissociation constant for the modified cytochrome c from cytochrome c-depleted mitochondrial membranes and the apparent K_m for the reaction with cytochrome c oxidase were each five to six times greater than the values for native cytochrome c. Irradiation of cytochrome c-depleted mitochondrial membranes supplemented with an excess of photoaffinity-labeled cytochrome c resulted in covalent binding of the derivative to the mitochondrial membranes. Fractionation of the irradiated mitochondria in the presence of detergents and salts followed by chromatography on agarose, Bio-Gel A, showed that labeled cytochrome c was bound covalently to cytochrome c oxidase in a 1:1 molar complex. The covalently linked cytochrome c-cytochrome c oxidase complex was active in mediating the electron transfer between N,N,N′,N′-tetramethyl-p-phenylenediamine/ascorbate and the oxidase.     

Subtle Change in the Charge Distribution of Surface Residues May Affect the Secondary Functions of Cytochrome c

Although the primary function of cytochrome c (cyt c) is electron transfer, the protein caries out an additional secondary function involving its interaction with membrane cardiolipin (CDL), its peroxidase activity, and the initiation of apoptosis. Whereas the primary function of cyt c is essentially conserved, its secondary function varies depending on the source of the protein. We report here a detailed experimental and computational study, which aims to understand, at the molecular level, the difference in the secondary functions of cyt c obtained from horse heart (mammalian) and Saccharomyces cerevisiae (yeast). The conformational landscape of cyt c has been found to be heterogeneous, consisting of an equilibrium between the compact and extended conformers as well as the oligomeric species. Because the determination of relative populations of these conformers is difficult to obtain by ensemble measurements, we used fluorescence correlation spectroscopy (FCS), a method that offers single-molecule resolution. The population of different species is found to depend on multiple factors, including the protein source, the presence of CDL and urea, and their concentrations. The complex interplay between the conformational distribution and oligomerization plays a crucial role in the variation of the pre-apoptotic regulation of cyt c observed from different sources. Finally, computational studies reveal that the variation in the charge distribution at the surface and the charge reversal sites may be the key determinant of the conformational stability of cyt c.     

The NT-26 cytochrome c552 and its role in arsenite oxidation

Arsenite oxidation by the facultative chemolithoautotroph NT-26 involves a periplasmic arsenite oxidase. This enzyme is the first component of an electron transport chain which leads to reduction of oxygen to water and the generation of ATP. Involved in this pathway is a periplasmic c-type cytochrome that can act as an electron acceptor to the arsenite oxidase. We identified the gene that encodes this protein downstream of the arsenite oxidase genes (aroBA). This protein, a cytochrome c₅₅₂, is similar to a number of c-type cytochromes from the α-Proteobacteria and mitochondria. It was therefore not surprising that horse heart cytochrome c could also serve, in vitro, as an alternative electron acceptor for the arsenite oxidase. Purification and characterisation of the c₅₅₂ revealed the presence of a single heme per protein and that the heme redox potential is similar to that of mitochondrial c-type cytochromes. Expression studies revealed that synthesis of the cytochrome c gene was not dependent on arsenite as was found to be the case for expression of aroBA.     

Characteristic temperature dependences of respiratory and photosynthetic electron-transport activities in membrane preparations from Anacystis nidulans grown at different temperatures

Electron-transport activities supported by seven different electron donor/acceptor couples in the light and in the dark, respectively, were measured in particle preparations of the cyanobacterium (blue-green alga) Anacystis nidulans after growth at 40, 30 and 25°C. The Arrhenius plots of the photosynthetic electron-transport reactions between ascorbate (plus 2,6-dichlorophenolindophenol (DCIP)) and NADP⁺, diphenylcarbazide and DCIP, diaminodurene and benzyl viologen (O₂), and the plot of the photooxidation of reduced horse heart cytochrome c showed a single discontinuity at approx. 24–25, 15–17 and 10–13°C in membranes derived from cells grown at 40, 30 and 25°C, respectively. By contrast, the dark respiratory electron-transport reactions between NADPH, ascorbate (plus DCIP) or reduced horse heart cytochrome c and oxygen, and the reduction by horse heart cytochrome c of the aa₃-type terminal oxidase as followed directly by dual-wavelength spectrophotometry, all gave Arrhenius plots distinguished by two distinct breaks: The break at the higher temperature corresponded to the break also found in the Arrhenius plots of the photosynthetic reactions while an additional discontinuity was observed at 17–18, 8–9 and 5–6°C in membranes prepared from cells grown at 40, 30 and 25°C, respectively. The temperatures at which the discontinuities in the Arrhenius plots occurred depended on the temperature at which the cells had been grown; they were independent, however, of the specific electron donors and acceptors employed. The characteristic features in the Arrhenius plots of respiratory and photosynthetic electron-transport reactions are discussed in terms of lipid-phase transitions in the cytoplasmic and the intracytoplasmic (thylakoid) membranes of A. nidulans. Implications for possibly distinct sites of the respiratory and photosynthetic electron-transport systems in A. nidulans will be mentioned.     

Immobilization of cytochrome c on beaded poly(N-acrylylpyrrolidine)

In order to examine a procedure whereby the points of covalent attachment between the components of a protein-polymer conjugate could be determined, horse heart cytochrome c was attached to a beaded copolymer of N-acrylylpyrrolidine, N,N′-bis(acrylyl)-1,2-diaminoethane and N-acrylyl-1,6-aminohexane through a cleavable azo linkage. Studies of protein removed from this conjugate showed that attachment of the polymer to cytochrome occurred predominantly through single lysine residues on the protein surface; lysine-25 was tentatively identified as the residue most extensively utilized in this way. Protein was also linked to the polymer by two lysine residues and a significant amount of protein was irreversibly attached to the polymer under the reaction conditions used.     

Localization of cytochrome b6f complexes implies an incomplete respiratory chain in cytoplasmic membranes of the cyanobacterium Synechocystis sp. PCC 6803

The cytochrome b₆f complex is an integral part of the photosynthetic and respiratory electron transfer chain of oxygenic photosynthetic bacteria. The core of this complex is composed of four subunits, cytochrome b, cytochrome f, subunit IV and the Rieske protein (PetC). In this study deletion mutants of all three petC genes of Synechocystis sp. PCC 6803 were constructed to investigate their localization, involvement in electron transfer, respiration and photohydrogen evolution. Immunoblots revealed that PetC1, PetC2, and all other core subunits were exclusively localized in the thylakoids, while the third Rieske protein (PetC3) was the only subunit found in the cytoplasmic membrane. Deletion of petC3 and both of the quinol oxidases failed to elicit a change in respiration rate, when compared to the respective oxidase mutant. This supports a different function of PetC3 other than respiratory electron transfer. We conclude that the cytoplasmic membrane of Synechocystis lacks both a cytochrome c oxidase and the cytochrome b₆f complex and present a model for the major electron transfer pathways in the two membranes of Synechocystis. In this model there is no proton pumping electron transfer complex in the cytoplasmic membrane.Cyclic electron transfer was impaired in all petC1 mutants. Nonetheless, hydrogenase activity and photohydrogen evolution of all mutants were similar to wild type cells. A reduced linear electron transfer and an increased quinol oxidase activity seem to counteract an increased hydrogen evolution in this case. This adds further support to the close interplay between the cytochrome bd oxidase and the bidirectional hydrogenase.     

The reaction of Euglena gracilis cytochrome c-552 with nonphysio-logical oxidants and reductants.

The reaction of Euglena gracilis cytochrome c-552 (cytochrome f) with the nonphysiological reactants potassium ferrocyanide, potassium ferricyanide, sodium ascorbate, sodium dithionite, and Chromatium vinosum high potential nonheme iron protein was studied by stopped-flow and temperature-jump kinetic methods. The reaction of the purified, water-soluble protein with the reactants was investigated as a function of ionic strength, pH, and temperature. The results demonstrated that reduction and oxidation takes place at a negatively charged site on the cytochrome c-552 surface. Participation of specific amino acid residues in electron transfer is implicated from the pH results. The results obtained for the nonphysiological reactions of cytochrome c-552 are compared with available data for horse heart cytochrome c and Rhodospirillum rubrum cytochrome c₂. The results strongly suggest that Euglena gracilis cytochrome c-552 undergoes nonphysiological oxidation and reduction by a mechanism different from that found for cytochrome c or cytochrome c₂.     

<Emphasis Type="Italic">Candida krusei</Emphasis> isolated from fruit juices ultrafiltration membranes promotes colonization of <Emphasis Type="Italic">Escherichia coli</Emphasis> O157:H7 and <Emphasis Type="Italic">Salmonella enterica</Emphasis> on stainless steel surfaces

To clarify the interactions between a common food spoilage yeast and two pathogenic bacteria involved in outbreaks associated with fruit juices, the present paper studies the effect of the interplay of Candida krusei, collected from UF membranes, with Escherichia coli O157:H7 and Salmonella enterica in the overall process of adhesion and colonization of abiotic surfaces. Two different cases were tested: a) co-adhesion by pathogenic bacteria and yeasts, and b) incorporation of bacteria to pre-adhered C. krusei cells. Cultures were made on stainless steel at 25°C using apple juice as culture medium. After 24 h of co-adhesion with C. krusei, both E. coli O157:H7 and S. enterica increased their counts 1.05 and 1.11 log CFU cm², respectively. Similar increases were obtained when incorporating bacteria to pre-adhered cells of Candida. Nevertheless C. krusei counts decreased in both experimental conditions, in a) 0.40 log CFU cm² and 0.55 log CFU cm² when exposed to E. coli O157:H7 and S. enterica and in b) 0.18 and 0.68 log CFU cm², respectively. This suggests that C. krusei, E. coli O157:H7, and S. enterica have a complex relationship involving physical and chemical interactions on food contact surfaces. This study supports the possibility that pathogen interactions with members of spoilage microbiota, such as C. krusei, might play an important role for the survival and dissemination of E. coli O157:H7 and Salmonella enterica in food-processing environments. Based on the data obtained from the present study, much more attention should be given to prevent the contamination of these pathogens in acidic drinks.     

The dynamic complex of cytochrome c6 and cytochrome f studied with paramagnetic NMR spectroscopy

The rapid transfer of electrons in the photosynthetic redox chain is achieved by the formation of short-lived complexes of cytochrome b₆f with the electron transfer proteins plastocyanin and cytochrome c₆. A balance must exist between fast intermolecular electron transfer and rapid dissociation, which requires the formation of a complex that has limited specificity. The interaction of the soluble fragment of cytochrome f and cytochrome c₆ from the cyanobacterium Nostoc sp. PCC 7119 was studied using NMR spectroscopy and X-ray diffraction. The crystal structures of wild type, M58H and M58C cytochrome c₆ were determined. The M58C variant is an excellent low potential mimic of the wild type protein and was used in chemical shift perturbation and paramagnetic relaxation NMR experiments to characterize the complex with cytochrome f. The interaction is highly dynamic and can be described as a pure encounter complex, with no dominant stereospecific complex. Ensemble docking calculations and Monte-Carlo simulations suggest a model in which charge–charge interactions pre-orient cytochrome c₆ with its haem edge toward cytochrome f to form an ensemble of orientations with extensive contacts between the hydrophobic patches on both cytochromes, bringing the two haem groups sufficiently close to allow for rapid electron transfer. This model of complex formation allows for a gradual increase and decrease of the hydrophobic interactions during association and dissociation, thus avoiding a high transition state barrier that would slow down the dissociation process.     

Early Bacteriopheophytin Reduction in Charge Separation in Reaction Centers of Rhodobacter sphaeroides

A question at the forefront of biophysical sciences is, to what extent do quantum effects and protein conformational changes play a role in processes such as biological sensing and energy conversion? At the heart of photosynthetic energy transduction lie processes involving ultrafast energy and electron transfers among a small number of tetrapyrrole pigments embedded in the interior of a protein. In the purple bacterial reaction center (RC), a highly efficient ultrafast charge separation takes place between a pair of bacteriochlorophylls: an accessory bacteriochlorophyll (B) and bacteriopheophytin (H). In this work, we applied ultrafast spectroscopy in the visible and near-infrared spectral region to Rhodobacter sphaeroides RCs to accurately track the timing of the electron on B_A and H_A via the appearance of the B_A and H_A anion bands. We observed an unexpectedly early rise of the H_A⁻ band that challenges the accepted simple picture of stepwise electron transfer with 3 ps and 1 ps time constants. The implications for the mechanism of initial charge separation in bacterial RCs are discussed in terms of a possible adiabatic electron transfer step between B_A and H_A, and the effect of protein conformation on the electron transfer rate.     

Thermodynamic properties of triheme cytochrome PpcF from Geobacter metallireducens reveal unprecedented functional mechanism

The bacterium Geobacter metallireducens is highly efficient in long-range extracellular electron transfer, a process that relies on an efficient bridging between the cytoplasmic electron donors and the extracellular acceptors. The periplasmic triheme cytochromes are crucial players in these processes and thus the understanding of their functional mechanism is crucial to elucidate the extracellular electron transfer processes in this microorganism. The triheme cytochrome PpcF from G. metallireducens has the lowest amino acid sequence identity with the remaining cytochromes from the PpcA-family of G. sulfurreducens and G. metallireducens, making it an interesting target for structural and functional studies. In this work, we performed a detailed functional and thermodynamic characterization of cytochrome PpcF by the complementary usage of NMR and visible spectroscopic techniques. The results obtained show that the heme reduction potentials are negative, different from each other and are also modulated by the redox and redox-Bohr interactions that assure unprecedented mechanistic features to the protein. The results showed that the order of oxidation of the hemes in cytochrome PpcF is maintained in the entire physiological pH range. The considerable separation of the hemes' redox potential values facilitates a sequential transfer within the chain of redox centers in PpcF, thus assuring electron transfer directionality to the electron acceptors.     

Occurrence of cytochrome aa3 in Anacystis nidulans

The cytochrome content of membrane fragments prepared from the bluegreen alga (cyanobacterium) Anacystis nidulans was examined by difference spectrophotometry. Two b-type cytochromes and a hitherto unknown cytochrome a could be characterized. In the reduced-minus-oxidised difference spectra the a-type cytochrome showed an α-band at 605 nm and a γ-band at 445 nm. These bands shifted to 590 and 430 nm, respectively, in CO difference spectra. NADPH, NADH and ascorbate reduced the cytochrome through added horse heart cytochrome c as electron mediator. In presence of KCN the reduced-minus-oxidised spectrum showed a peak at 600 nm and a trough at 604 nm. Photoaction spectra of O₂ uptake and of horse heart cytochrome c oxidation by CO-inhibited membranes showed peaks at 590 and 430 nm. These findings are consistent with cytochrome aa₃ being the predominant respiratory cytochrome c oxidase in Anacystis nidulans.     

Proteins of the thermophilic fungus Humicola lanuginosa. II. Some physicochemical properties of a cytochrome c

A cytochrome c from Humicola lanuginosa is unique among eukaryotic cytochromes c in having phenylalanine as Residue 74. This protein has certain properties which differ from those of other cytochromes c to which it is generally similar. The Humicola cytochrome c is as stable as horse heart cytochrome c in urea, but more stable than both horse heart and yeast cytochromes c in acidic and alkaline conditions. Spectrophotometric titration of the four tyrosyl residues of the Humicola protein was nonsigmoidal with a pK_app of 11.4. Solvent perturbation difference spectra indicate that 50% of the tyrosyl residues are exposed to solvent in the native protein, and that the single tryptophanyl and all four tyrosyl residues become exposed in 8 m urea. Certain unusual features in both the optical rotatory dispersion and circular dichroism spectra in the 290-250-nm region are tentatively attributed to the substitution of phenylalanine for tyrosine at position 74.