The preparation of fully N-epsilon-acetimidylated cytochrome c.

We have re-examined the acetimidylation and subsequent deprotection of cytochrome c by published methods in the light of recent findings on the tendency of protein acetimidylation reactions to yield side products of differing net charges. We find that the protection methods do indeed yield a mixture of products, some of which have considerably diminished biological activity. Our observations support a postulated mechanism for the generation of side products, and we have been able to identify the major factor responsible for their formation by published methods. The deprotection method appears to be free of side reactions. We describe a new procedure for acetimidylation that will produce fully N-epsilon-acetimidylated cytochrome c. This derivative, lacking detectable side products and having good biological activity, is useful for structure-function studies and as an intermediate in the semisynthesis of cytochrome c analogues.     

Redox potential of the cytochrome c in the flavocytochrome p-cresol methylhydroxylase

The redox potential of the cytochrome c in 5 flavocytochrome c proteins, all p-cresol methylhydroxylases purified from species of Pseudomonas, was measured. All gave similar values ranging from 226-250 mV. Two of the enzymes, from Pseudomonas putida NC1B 9866 and NC1B 9869, were resolved into their flavoprotein and cytochrome subunits and the redox potentials of the isolated cytochrome c subunits measured. The values for these were 60-70 mV below those for the whole enzymes but, in both cases, reconstitution of active enzyme by addition of the flavoprotein subunit restored the original potential.     

Amino acid sequence, crystallization and structure determination of reduced and oxidized cytochrome c6 from the green alga Scenedesmus obliquus.

Cytochrome c6from the unicellular green alga Scenedesmus obliquus was sequenced, crystallized in its reduced and oxidized state and the three-dimensional structure of the protein in both redox states was determined by X-ray crystallography. Reduced cytochrome c6crystallized as a monomer in the space group P 21212, whereas the oxidized protein crystallized as a dimer in the space group P 3121. The structures were solved by molecular replacement and refined to 1. 9 and 2.0 A, respectively.Comparison of the structures of both redox states revealed only slight differences on the protein surface, whereas a distortion along the axis between the heme iron and its coordinating Met61 residue was observed. No redox-dependent movement of internal water molecules could be detected. The high degree of similarity of the surfaces and charge distributions of both redox states, as well as the dimerization of cytochrome c6as observed in the oxidized crystal, is discussed with respect to its biological relevance and its implications for the reaction mechanisms between cytochrome c6and its redox partners. The dimer of oxidized cytochrome c6may represent a molecular structure occurring in a binary complex with cytochrome b6f. This assembly might be required for the correct orientation of cytochrome c6with respect to its redox partner cytochrome b6f, facilitating the electron transfer within the complex. If the dimerization is not redox-dependent in vivo, the almost identical surfaces of both redox states do not support a long range differentiation between reduced and oxidized cyt c6, i.e. a random collision model for the formation of an electron transfer complex must be assumed.     

Complexity in the redox titration of the dihaem cytochrome c4

Redox titration of the dihaem, two domain cytochromes c4 from Pseudomonas aeruginosa, Pseudomonas stutzeri and Azotobacter vinelandii showed complex behaviour indicative of the presence of two redox components. In the case of the P. stutzeri cytochrome c4, two spectroscopically distinct components were present during the redox titration. In contrast, cytochrome c-554(548) from a halophilic Paracoccus species is a stable dimer of a monohaem cytochrome which shows close homology to cytochrome c4, but does not show complexity in its redox titration. The presence of chemically distinct haem environments or anti-cooperative interactions between identical haem groups are two possible explanations for the redox complexity of cytochrome c4. The simple redox titration of cytochrome c-554(548) shows that haems situated relatively close together need not interact, but direct cleavage, separation and study of the domains will be necessary to decide whether they do or do not interact in the case of cytochrome c4.     

Methionine sulfoxide cytochrome c.

Cytochrome c has been chemically modified by methylene blue mediated photooxidation. It is established that the methionine residues of the protein have been specifically converted to methionine sulfoxide residues. No oxidation of any other amino acid residues or the cysteine thioether bridges of the molecule occurs during the photooxidation reaction. The absorbance spectrum of methionine sulfoxide ferricytochrome c at neutrality is similar to that of the unmodified protein except for an increase in the extinction coefficient of the Soret absorbance band and for the complete loss of the ligand sensitive 695 nm absorbance band in the spectrum of the derivative. The protein remains in the low spin configuration which implies the retention of two strong field ligands. Spin state sensitive spectral titrations and model studies of heme peptides indicate that the sixth ligand is definitely not provided by a lysine residue but may be methionine-80 sulfoxide coordinated via its sulfur atom. Circular dichroism spectra indicate that the heme crevice of methionine sulfoxide ferri- and ferrocytochrome c is weakened relative to native cytochrome c. The redox potential of methionine sulfoxide cytochrome c is 184 mV which is markedly diminished from the 260 mV redox potential of native cytochrome c. The modified protein is equivalent to native cytochrome c as a substrate for cytochrome oxidase and is not autoxidizable at neutral pH but is virtually inactive with succinate-cytochrome c reductase. These results indicate that the major role of the methionine-80 in cytochrome c is to preserve a closed hydrophobic heme crevice which is essential for the maintainance of the necessary redox potential.     

A comparative structural and functional analysis of cytochrome cM cytochrome c6 and plastocyanin from the cyanobacterium Synechocystis sp. PCC 6803

Cytochrome cM is a new c-class photosynthetic haem protein whose physiological role is still unknown. It has been proposed previously that cytochrome cM can replace cytochrome c6 and plastocyanin in transferring electrons between the two membrane complexes cytochrome b6-f and photosystem I in organisms growing under stress conditions. The experimental evidence herein provided allows us to discard such a hypothesis. We report a procedure to overexpress cytochrome cM from the cyanobacterium Synechocystis sp. PCC 6803 in Escherichia coli cells in mg quantities. This has allowed us to perform a comparative laser flash-induced kinetic analysis of photosystem I reduction by the three metalloproteins from Synechocystis. The bimolecular rate constant for the overall reaction is up to 100 times lower with cytochrome cM than with cytochrome c6 or plastocyanin. In addition, the redox potential value and surface electrostatic potential distribution of cytochrome cM are quite different from those of cytochrome c6 and plastocyanin. These findings strongly indicate that cytochrome cM cannot be recognised by and interact with the same redox partners as the other two metalloproteins.     

Stimulation of the respiratory chain of rat liver mitochondria between cytochrome c1 and cytochrome c by glucagon treatment of rats

Mitochondria from glucagon-treated rats oxidize succinate, but not ascorbate plus tetramethylphenylenediamine, faster in the uncoupled state than do control mitochondria. The rate of O(2) uptake in the presence of both substrates is equal to the sum of the rates of the O(2) uptake in the presence of either substrate alone. It is concluded that the mitochondrial respiratory chain is limited at some point between cytochromes b and c and that this step is regulated by glucagon. Measurement of the cytochrome spectra under uncoupled conditions in the presence of succinate and rotenone demonstrates a crossover between cytochromes c and c(1) when control mitochondria are compared with those from glucagon-treated rats, cytochrome c being more oxidized and cytochrome c(1) more reduced in control mitochondria. Under conditions where pyruvate metabolism is studied the control mitochondria are generally more oxidized than those from glucagon-treated rats, the redox state of cytochrome b-566 correlating with the rate of pyruvate metabolism in sucrose medium. However, when the redox state of the mitochondria is taken into account, a crossover between cytochromes c and c(1) is again apparent. The spectra of the b cytochromes are complex, but cytochrome b-562 appears to become more reduced relative to cytochrome b-566 in mitochondria from glucagon-treated rats than in control mitochondria. This can be explained by the existence of a more alkaline matrix in glucagon-treated rats, the redox potential for cytochrome b being pH-sensitive. It is concluded that glucagon stimulates electron flow between cytochromes c(1) and c. The physiological significance of these findings is discussed.     

Mitochondrial regulation of caspase activation by cytochrome oxidase and tetramethylphenylenediamine via cytosolic cytochrome c redox state

Cytochrome c release from mitochondria induces caspase activation in cytosols; however, it is unclear whether the redox state of cytosolic cytochrome c can regulate caspase activation. By using cytosol isolated from mammalian cells, we find that oxidation of cytochrome c by added cytochrome oxidase stimulates caspase activation, whereas reduction of cytochrome c by added tetramethylphenylenediamine (TMPD) or yeast lactate dehydrogenase/cytochrome c reductase blocks caspase activation. Scrape-loading of cells with this reductase inhibited caspase activation induced by staurosporine. Similarly, incubating intact cells with ascorbate plus TMPD to reduce intracellular cytochrome c strongly inhibited staurosporine-induced cell death, apoptosis, and caspase activation but not cytochrome c release, indicating that cytochrome c redox state can regulate caspase activation. In homogenates from healthy cells cytochrome c was rapidly reduced, whereas in homogenates from apoptotic cells added cytochrome c was rapidly oxidized by some endogenous process. This oxidation was prevented if mitochondria were removed from the homogenate or if cytochrome oxidase was inhibited by azide. This suggests that permeabilization of the outer mitochondrial membrane during apoptosis functions not just to release cytochrome c but also to maintain it oxidized via cytochrome oxidase, thus maximizing caspase activation. However, this activation can be blocked by adding TMPD, which may have some therapeutic potential.     

Expression of a cytochrome c2 isozyme restores photosynthetic growth of Rhodobacter sphaeroides mutants lacking the wild-type cytochrome c2 gene

Deletion of the cytochrome c2 gene in the purple bacterium Rhodobacter sphaeroides renders it incapable of phototrophic growth (strain cycA65). However, suppressor mutants which restore the ability to grow phototrophically are obtained at relatively high frequency (1-10 in 10(7)). We examined two such suppressors (strains cycA65R5 and cycA65R7) and found the expected complement of electron transfer proteins minus cytochrome c2: SHP, c', c551.5, and c554. Instead of cytochrome c2 which elutes from DEAE-cellulose between SHP and cytochrome c', at about 50 mM ionic strength in wild-type extracts, we found a new high redox potential cytochrome c in the mutants which elutes with cytochrome c551.5 at about 150 mM ionic strength. The new cytochrome is more acidic than cytochrome c2, but is about the same size or slightly smaller (13,500 Da). The redox potential of the new cytochrome from strain cycA65R7 (294 mV) is about 70 mV lower than that of cytochrome c2. The 280 nm absorbance of the new cytochrome is smaller than that of cytochrome c2, which suggests that there is less tryptophan (the latter has two residues). In vitro kinetics of reduction by lumiflavin and FMN semiquinones show that the reactivity of the new cytochrome is similar to that of cytochrome c2, and that there is a relatively large positive charge (+2.6) at the site of reduction, despite the overall negative charge of the protein. This behavior is characteristic of cytochromes c2 and unlike the majority of bacterial cytochromes examined. Fourteen out of twenty-four of the N-terminal amino acids of the new cytochrome are identical to the sequence of cytochrome c2. The N-termini of the cycA65R5 and cycA65R7 cytochromes were the same. The kinetics and sequence data indicate that the new protein may be a cytochrome c2 isozyme, which is not detectable in wild-type cells under photosynthetic growth conditions. We propose the name iso-2 cytochrome c2 for the new cytochrome produced in the suppressor strains.     

Control of proteoliposomal cytochrome c oxidase: the partial reactions

The steady-state spectroscopic behaviour and the turnover of cytochrome c oxidase incorporated into proteoliposomes have been investigated as functions of membrane potential and pH gradient. The respiration rate is almost linearly dependent on [cytochrome c2+] at high flux, but while the cytochrome a redox state is always dependent on the [cytochrome c2+] steady state, it reaches a maximum reduction level less than 100% in each case. The maximal aerobic steady-state reduction level of cytochrome a is highest in the presence of valinomycin and lowest in the presence of nigericin. The proportion of [cytochrome c2+] required to achieve 50% of maximal reduction of cytochrome a varies with the added ionophores; the apparent redox potential of cytochrome a is most positive in the fully decontrolled system (plus valinomycin and nigericin). At low levels of cytochrome a reduction, the rate of respiration is no longer a linear function of [cytochrome c2+], but is dependent upon the redox state of both cytochromes a and c. That is, proteoliposomal oxidase does not follow Smith-Conrad kinetics at low cytochrome c reduction levels, especially in the controlled states. The control of cytochrome oxidase turnover by delta pH and by delta psi can be explained either by an allosteric model or by a model with reversed electron transfer between the binuclear centre and cytochrome a. Other evidence suggests that the reversed electron transfer model may be the correct one.     

The effect of complex-formation with polyanions on the redox properties of cytochrome c.

1. The stable complex formed between mammalian cytochrome c and phosvitin at low ionic strength was studied by partition in an aqueous two-phase system. Oxidized cytochrome c binds to phosvitin with a higher affinity than reduced cytochrome c. The difference was equivalent to a decrease of the redox potential by 22 mV on binding. 2. Complex-formation with phosvitin strongly inhibited the reaction of cytochrome c with reagents that react as negatively charged species, such as ascorbate, dithionite, ferricyanide and tetrachlorobenzoquinol. Reaction with uncharged reagents such as NNN'N'-tetramethylphenylenediamine and the reduced form of the N-methylphenazonium ion (present as the methylsulphate) was little affected by complex-formation, whereas oxidation of the reduced cytochrome by the positively charged tris-(phenanthroline)cobalt(III) ion was greatly stimulated. 3. A similar pattern of inhibition and stimulation of reaction rates was observed when phosvitin was replaced by other macromolecular polyanions such as dextran sulphate and heparin, indicating that the results were a general property of complex-formation with polyanions. A weaker but qualitatively similar effect was observed on addition of inositol hexaphosphate and ATP. 4. It is suggested that the effects of complex-formation with polyanions on the reactivity of cytochrome c with redox reagents are mainly the result of replacing the positive charge on the free cytochrome by a net negative charge. Any steric effects on polyanion binding are small in comparison with such electrostatic effects.     

Novel cyanide inhibition at cytochrome c1 of Rhodobacter capsulatus cytochrome bc1

Oxidized cytochrome c(1) in photosynthetic bacterium Rhodobacter capsulatus cytochrome bc(1) reversibly binds cyanide with surprisingly high, micromolar affinity. The binding dramatically lowers the redox midpoint potential of heme c(1) and inhibits steady-state turnover activity of the enzyme. As cytochrome c(1), an auxiliary redox center of the high-potential chain of cytochrome bc(1), does not interact directly with the catalytic quinone/quinol binding sites Q(o) and Q(i), cyanide introduces a novel, Q-site independent locus of inhibition. This is the first report of a reversible inhibitor that manipulates the energetics and electron transfers of the high-potential redox chain of cytochrome bc(1), while maintaining quinone substrate catalytic sites in an intact form.     

Kinetic characterization of the interaction between cytochrome oxidase and cytochrome c

The mechanism of electron transfer catalyzed by cytochrome oxidase was investigated by monitoring the reaction of cytochrome oxidase with cytochrome c under carefully controlled anaerobic conditions. The kinetics of the reaction were examined by varying conditions of ionic strength, inhibitor binding, and oxidation-reduction potential. An analogue of cytochrome c in which the iron atom was replaced with cobalt was used to probe the effect of redox potential on the reaction. Under conditions of low ionic strength, there is very rapid oxidation of cytochrome c and reduction of oxidase which occurs at a rate of 3 X 10(7) M-1 s-1. The number of electrons transferred exhibit a hyperbolic dependence on the concentration of cytochrome c reaching a maximum of 2 electrons transferred at the highest concentration of reduced cytochrome c employed. The total number of electrons transferred was always observed to be distributed equally between cytochrome a and a second acceptor which appears to be the associated copper center; electron transfer to cytochrome a3 did not occur in the absence of oxygen. Substitution of cytochrome c by the cobalt analogue (which represents a decrease in oxidation-reduction potential of about 400 mV) yielded identical results indicating that the origin of the lack of reactivity of cytochrome a3 is of a kinetic nature. The effect of increasing the ionic strength on the reaction was 2-fold: a marked decrease in reaction rate and the appearance of biphasic kinetics with the amplitude of the very fast absorbance changes at 605 nm decreasing from 80% to 40% of the total anticipated from static absorbance measurements. Each of the two phases accounted for a maximum of 1 electron at the highest ionic strength employed. These results are simulated in terms of a sample kinetic reaction scheme involving a two-step electron transfer at one binding site.     

Active site structure and dynamics of cytochrome c3 from Desulfovibrio gigas immobilized on electrodes

Cytochrome c3 from Desulfovibrio gigas is electrostatically adsorbed on Ag electrodes coated with self-assembled monolayers (SAMs) of 11-mercaptoundecanoic acid. The redox equilibria and electron transfer dynamics of the adsorbed four-heme protein are studied by surface enhanced resonance Raman spectroscopy. Immobilization on the coated electrodes does not cause any structural changes in the redox sites. The potential-dependent stationary experiments distinguish the redox potential of heme IV (-0.19 V versus normal hydrogen electrode) from those of the other hemes for which an average value of -0.3 V is determined. Taking into account the interfacial potential drops, these values are in good agreement with the redox potentials of the protein in solution. The heterogenous electron transfer between the electrode and heme IV of the adsorbed cytochrome c3 is analyzed on the basis of time-resolved experiments, leading to a formal electron transfer rate constant of 15 s(-1), which is a factor of 3 smaller than that of the monoheme protein cytochrome c.     

Identification of cytochrome a and a3 in yeast cells.

1) Cells of Saccharomyces cerevisiae have been analysed by single and double-bean spectroscopy. Evidence is given for two components of cytochrome c oxidase in the alpha-region of their absorption spectrum. A rapidly reduceable component with a maximum at 600 nm and a slowly reduceable component with a maximum at 604 nm contribute about equal amounts to the total alpha-absorption of cytochrome c oxidase. 2) The component absorbing at 600 nm was identified as the high-potential component with a redox potential of 340 - 355mV, and the 604-nm component as the low-potential component of cytochrome c oxidase with redox potential of 180 - 190 mV. 3) Both components can be characterized by analysing the reduction kinetics in the presence of carbon monoxide. In the presence of saturating concentrations of carbon monoxide, an oxygen pulse leads to a rapid oxidation and subsequent reduction of cytochrome c oxidase, but the rapid reduction phase at 600 nm completely disappears, demonstrating its identity with cytochrome a3, which, being liganded by carbon monoxide in its reduced state, cannot react any more. The component which becomes oxidized and later reduced in the presence of carbon monoxide -- by definition cytochrome a -- has an absorption maximum at 604 nm. 4) The total extinction change at 604 nm in the presence of carbon monoxide is nearly as high as in its absence, but the reduction occurs in two phases and only the second phase, which contributes 50 - 60% to the total absorbance, corresponds in redox potential and kinetic properties to cytochrome a. Because the redox potential of the first reduction phase is very close to that of the low-potential copper atom of cytochrome c oxidase, it is concluded that the apparent increase in the extinction coefficient of cytochrome a in the presence of carbon monoxide is the result of a strong interaction between the ligand fields of cytochrome a and copper, induced by the binding of carbon monoxide to reduced cytochrome a3.     

Electron transfer reactions of cytochrome c confined within erythrocyte ghosts

We have prepared and characterized resealed erythrocyte ghosts in which the only discernible pigment is cytochrome c. The resealed ghosts have the normal orientation and are free of 'leaky' species; they are stable and can be maintained at 4 degrees C for many days without lysis. The internal cytochrome c participates in redox reactions with both soluble and insolubilized cytochrome c present externally, and with external cytochrome b5. No reaction was observed with plastocyanin, cytochrome c oxidase or NADPH-cytochrome c reductase. A study has been made of the reaction of the internal cytochrome c with the low molecular weight reductants, ascorbate and glutathione. Complex kinetics are observed with both reagents: with ascorbate the results are best explained by assuming the existence, in the membrane, of a redox-active species able to undergo dedimerization. A protein bound disulfide bond would satisfy the requirement.     

The structure of an electron transfer complex containing a cytochrome c and a peroxidase

Efficient biological electron transfer may require a fluid association of redox partners. Two noncrystallographic methods (a new molecular docking program and 1H NMR spectroscopy) have been used to study the electron transfer complex formed between the cytochrome c peroxidase (CCP) of Paracoccus denitrificans and cytochromes c. For the natural redox partner, cytochrome c550, the results are consistent with a complex in which the heme of a single cytochrome lies above the exposed electron-transferring heme of the peroxidase. In contrast, two molecules of the nonphysiological but kinetically competent horse cytochrome bind between the two hemes of the peroxidase. These dramatically different patterns are consistent with a redox active surface on the peroxidase that may accommodate more than one cytochrome and allow lateral mobility.     

The 1.6A X-ray structure of the unusual c-type cytochrome, cytochrome cL, from the methylotrophic bacterium Methylobacterium extorquens

The structure of cytochrome cL from Methylobacterium extorquens has been determined by X-ray crystallography to a resolution of 1.6 A. This unusually large, acidic cytochrome is the physiological electron acceptor for the quinoprotein methanol dehydrogenase in the periplasm of methylotrophic bacteria. Its amino acid sequence is completely different from that of other cytochromes but its X-ray structure reveals a core that is typical of class I cytochromes c, having alpha-helices folded into a compact structure enclosing the single haem c prosthetic group and leaving one edge of the haem exposed. The haem is bound through thioether bonds to Cys65 and Cys68, and the fifth ligand to the haem iron is provided by His69. Remarkably, the sixth ligand is provided by His112, and not by Met109, which had been shown to be the sixth ligand in solution. Cytochrome cL is unusual in having a disulphide bridge that tethers the long C-terminal extension to the body of the structure. The crystal structure reveals that, close to the inner haem propionate, there is tightly bound calcium ion that is likely to be involved in stabilization of the redox potential, and that may be important in the flow of electrons from reduced pyrroloquinoline quinone in methanol dehydrogenase to the haem of cytochrome cL. As predicted, both haem propionates are exposed to solvent, accounting for the unusual influence of pH on the redox potential of this cytochrome.     

Electron transfer between soluble and immobilized mammalian cytochrome c. Equilibrium and kinetic studies on immobilized cytochrome c.

Horse heart cytochrome c was covalently bound to Sepharose 4B and its redox properties were measured under various experimental conditions. The equilibrium constant for the electron exchange between the oxidized and the reduced form of cytochrome c when one of the two forms was in the semi-solid state and the other one in solution was close to 1. Matrix-bound ferrocytochrome c is very stable to autoxidation and is not oxidized by O2 even in the presence of mammalian cytochrome oxidase. Oxidation occurs if catalytic amounts of soluble cytochrome c are added to the reaction mixture. The rate of oxidation of matrix-bound ferrocytochrome c in the presence of cytochrome oxidase and catalytic amounts of soluble cytochrome c may be correlated with the rate of electron transfer between soluble and matrix-bound cytochrome c. This rate is more than two orders of magnitude lower than that reported for the homonuclear (between identical species) electron transfer in solution.     

Anion and ionic strength effects upon the oxidation of cytochrome c by cytochrome c oxidase

Citrate and other polyanion binding to ferricytochrome c partially blocks reduction by ascorbate, but at constant ionic strength the citrate-cytochrome c complex remains reducible; reduction by TMPD is unaffected. At a constant high ionic strength citrate inhibits the cytochrome c oxidase reaction competitively with respect to cytochrome c, indicating that ferrocytochrome c also binds citrate, and that the citrate-ferrocytochrome c complex is rejected by the binding site at high ionic strength. At lower ionic strengths, citrate and other polyanions change the kinetic pattern of ferrocytochrome c oxidation from first-order towards zero-order, indicating preferential binding of the ferric species, followed by its exclusion from the binding site. The turnover at low cytochrome c concentrations is diminished by citrate but not the Km (apparent non-competitive inhibition) or the rate of cytochrome a reduction by bound cytochrome c. Small effects of anions are seen in direct measurements of binding to the primary site on the enzyme, and larger effects upon secondary site binding. It is concluded that anion-cytochrome c complexes may be catalytically competent but that the redox potentials and/or intramolecular behaviour of such complexes may be affected when enzyme-bound. Increasing ionic strength diminishes cytochrome c binding not only by decreasing the 'association' rate but also by increasing the 'dissociation' rate for bound cytochrome c converting the 'primary' (T) site at high salt concentrations into a site similar kinetically to the 'secondary' (L) site at low ionic strength. A finite Km of 170 microM at very high ionic strength indicates a ratio of K infinity m/K 0 M of about 5000. It is proposed that anions either modify the E10 of cytochrome C bound at the primary (T) site of that they perturb an equilibrium between two forms of bound c in favour of a less active form.