Use of specific trifluoroacetylation of lysine residues in cytochrome c to study the reaction with cytochrome b5, cytochrome c1, and cytochrome oxidase

The preparation, purification, and characterization of four new derivatives of cytochrome c trifluoroacetylated at lysines 72, 79, 87, and 88 are reported. The redox reaction rates of these derivatives with cytochrome b5, cytochrome c1 and cytochrome oxidase indicated that the interaction domain on cytochrome c for all three proteins involves the lysines immediately surrounding the heme crevice. Modification of lysines 72, 79, 87 had a large effect on the rate of all three reactions, while modification of lysine 88 had a very small effect. Even though lysines 87 and 88 are adjacent to one another, lysine 87 is at the top left of the heme crevice oriented towards the front of cytochrome c, while lysine 88 is oriented more towards the back. Since the interaction sites for cytochrome c1 and cytochrome oxidase are essentially identical, cytochrome c probably undergoes some type of rotational diffusion during electron transport.     

Observations on iron uptake, iron metabolism, cytochrome c content, cytochrome a content and cytochrome c-oxidase activity in regenerating rat liver

1. Differential and density-gradient centrifugation were used to fractionate mitochondria and fluffy layer from normal and regenerating rat liver. The iron, cytochrome a and cytochrome c contents and cytochrome c-oxidase activity were studied as well as the uptake of (59)Fe into protein and cytochrome c. 2. A certain degree of heterogeneity was evident between the heavy-mitochondrial and light-mitochondrial fractions, and in their behaviour during liver regeneration. 3. The specific content of light-mitochondrial iron and cytochrome a was 1.3-1.4 times that of heavy mitochondria. Changes in cytochrome c-oxidase activity closely followed those of cytochrome a content during liver regeneration, but not for light mitochondria after 10 days. 4. Radioactive iron ((59)Fe) was most actively taken up by well-washed light mitochondria during early liver regeneration. After 22 days fluffy layer became preferentially labelled. This substantiates the view that fluffy layer partially represents broken-down mitochondria. 5. During early regeneration, light-mitochondrial fractions separated along a density gradient were about 3 times as radioactive, and showed distinct heterogeneity of (59)Fe-labelling, in contrast with near homogeneity for heavy mitochondria. 6. Immediately after partial hepatectomy fractions corresponding to density 1.155 were 5-10 times as radioactive as particles of greater density. The radioactivity decreased sharply after 6 days. 7. These particles of low density possessed higher NADH-cytochrome c-reductase (1.5-5-fold) and succinate-dehydrogenase (1.1-2-fold) activities than typical mitochondrial fractions. Their succinate-cytochrome c-reductase and cytochrome c-oxidase activities were slightly lower. 8. The results are discussed in relation to mitochondrial morphogenesis, and a possible route from submitochondrial particles is suggested.     

Comparison of the binding sites on cytochrome c for cytochrome c oxidase, cytochrome bc1, and cytochrome c1. Differential acetylation of lysyl residues in free and complexed cytochrome c

The isolated complexes of ferricytochrome c with cytochrome c oxidase, cytochrome c reductase (cytochrome bc1 or complex III), and cytochrome c1 (a subunit of cytochrome c reductase) were investigated by the method of differential chemical modification (Bosshard, H.R. (1979) Methods Biochem. Anal. 25, 273-301). By this method the chemical reactivity of each of the 19 lysyl side chains of horse cytochrome c was compared in free and in complexed cytochrome c and binding sites were deduced from altered chemical reactivities of particular lysyl side chains in complexed cytochrome c. The most important findings follow. 1. The binding sites on cytochrome c for cytochrome c oxidase and cytochrome c reductase, defined in terms of the involvement of particular lysyl residues, are indistinguishable. The two oxidation-reduction partners of cytochrome c interact at the front (exposed heme edge) and top left part of the molecule, shielding mainly lysyl residues 8, 13, 72 + 73, 86, and 87. The chemical reactivity of lysyl residues 22, 39, 53, 55, 60, 99, and 100 is unaffected by complex formation while the remaining lysyl residues in positions 5, 7, 25, 27, 79, and 88 are somewhat less reactive in the complexed molecule. 2. When bound to cytochrome c reductase or to the isolated cytochrome c1 subunit of the reductase the same lysyl side chains of cytochrome c are shielded. This indicates that cytochrome c binds to the c1 subunit of the reductase during the electron transfer process.     

Direct electrochemistry of protein-protein complexes involving cytochrome c, cytochrome b5, and plastocyanin

The direct electrochemistry of the cytochrome c/cytochrome b5 and cytochrome c/plastocyanin complexes has been investigated at edge-plane graphite and modified gold electrode surfaces, which are selective for one of the two components of the complex. Electrochemical response of one protein at an otherwise electrostatically unfavorable electrode surface was achieved in the presence of the other protein, and the calculated heterogeneous electron-transfer rate constant and diffusion coefficient were found to be in good agreement with the values determined previously from the electrochemistry of the individual proteins [Armstrong, F. A., Hill, H. A. O., & Walton, N. J. (1988) Acc. Chem. Res. 21, 407 and references therein]. A dynamic model of the protein-protein-electrode ternary complex is proposed to explain the promotion effect, and this model is supported by a study comparing the electrochemical responses of covalent and electrostatic cytochrome c/plastocyanin complexes. It is also suggested that the behavior of protein-protein complexes at electrode surfaces could be related to that of the complexes associated with biological membranes.     

Steady-state redox behavior of cytochrome c, cytochrome a, and CuA of cytochrome c oxidase in intact rat liver mitochondria.

We have examined the steady-state redox behavior of cytochrome c (Fec), Fea, and CuA of cytochrome c oxidase during steady-state turnover in intact rat liver mitochondria under coupled and uncoupled conditions. Ascorbate was used as the reductant and TMPD (N,N,N',N'-tetramethyl-1,4-phenylenediamine) as the redox mediator. After elimination of spectroscopic interference from the oxidized form of TMPD, we found that Fea remains significantly more oxidized than previously thought. During coupled turnover, CuA always appears to be close to redox equilibrium with Fec. By increasing the amount of TMPD, both centers can be driven to fairly high levels of reduction while Fea remains relatively oxidized. The reduction level at Fea is close to a linear function of the enzyme turnover rate, but the levels at Fec and CuA do not keep pace with enzyme turnover. This behavior can be explained in terms of a redox equilibrium among Fec, CuA, and Fea, where Fea is the electron donor to the oxygen reduction site, but only if Fea has an effective Em (redox midpoint potential) of 195 mV. This is too low to be accounted for on the basis of nonturnover measurements and the effects of the membrane potential. However, if there is no equilibrium, the internal CuA----Fea electron-transfer rate constant must be slow in the time average (about 200 s-1). Other factors which might contribute to such a low Em are discussed. In the presence of uncoupler, this situation changes dramatically. Both Fec and CuA are much less reduced; within the resolution of our measurements (about 10%), we were unable to measure any reduction of CuA. Fea and CuA remain too oxidized to be in redox equilibrium with Fec during steady-state turnover. Furthermore, our results indicate that, in the uncoupled system, the (time-averaged) internal electron-transfer rate constants in cytochrome oxidase must be of the order of 2500 s-1 or higher. When turnover is slowed by azide, the relative redox levels at Fea and Fec are much closer to those predicted from nonturnover measurements. In presence of uncouplers, Fea is always more reduced than Fec, but in the absence of uncouplers, the two centers track together. Unlike the uninhibited, coupled system, the redox behavior here is consistent with the known effect of the electrical membrane potential on electron distribution in the enzyme. Interestingly, in these circumstances (azide and uncoupler present), Fea behaves as if it were no longer the kinetically controlling electron donor to the bimetallic center.     

A new membrane-bound b-type cytochrome, cytochrome b-558, from photosynthetically grown Rhodopseudomonas sphaeroides

A new membrane-bound b-type cytochrome, cytochrome b-558, was removed from chromatophore membranes of photosynthetically grown Rhodopseudomonas sphaeroides strain R-26 by deoxycholate-cholate extraction. The cytochrome was purified by ammonium sulfate fractionation and ion-exchange chromatography. Cytochrome b-558 had absorption maxima at 280 and 405 nm in the oxidized form, and at 558, 528, and 420 nm in the reduced form. It had a midpoint potential of--130 mV at pH 7.0. The minimal molecular weight of this protein was 42,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and it contained one mole heme per mole of protein. The isoelectric point was 8.5. The electrophoretic pattern of heme-carrying proteins and the redox potentiometry showed that cytochrome b-558 was present in membranes from wild type, strain R-26, and strain GA grown photosynthetically, but not from any strain grown aerobically.     

Acetone-inducible cytochrome P-450: purification, catalytic activity, and interaction with cytochrome b5

A procedure was developed for the purification of an acetone-inducible form of cytochrome P-450 (P-450ac) to electrophoretical homogeneity from liver microsomes of acetone-treated rats. The P-450ac preparation containing 16.0 to 16.5 nmol P-450/mg protein moved as a single protein band with an estimated molecular weight of 52,000 upon gel electrophoresis in the presence of sodium dodecyl sulfate. The ferric P-450ac showed an absorption maximum at 394 nm at 25 degrees C, suggesting that it exists mainly in the high-spin form. It also existed in the low-spin form, especially at lower temperatures, as indicated by the absorption maximum in the 412-nm region. Upon reconstitution with NADPH: cytochrome P-450 reductase and phospholipid, P-450ac efficiently catalyzed both the demethylation and denitrosation of N-nitrosodimethylamine (NDMA) showing Vmax values of 23.8 and 2.3 nmol min-1 nmol P-450-1, respectively. The catalytic activity of P-450ac was greatly affected by cytochrome b5 which decreased the Km values of these reactions by a factor of 10 and increased the Vmax values. Cytochrome b5 appeared to interact with P-450 at a molar ratio of 1:1 and an intact cytochrome b5 structure was required for such interaction. Among the substrates studied, the demethylation of NDMA was affected the most by cytochrome b5 and showed the highest rate. P-450ac also catalyzed the oxygenation of N-nitrosomethylethylamine and aniline and the activity was enhanced slightly by cytochrome b5. Cytochrome b5 did not enhance the P-450ac-catalyzed metabolism of other drug substrates such as benzphetamine, aminopyrine, and ethylmorphine. P-450ac appeared to be similar in property to the previously studied rat P-450et (ethanol-inducible), rat P-450j (isoniazid-inducible), and rabbit P-450LM3a (ethanol-inducible). These P-450 species represent a new class of P-450 isozymes that are important in the metabolism of many endobiotics and xenobiotics.     

Chemical modification of cytochrome b5, cytochrome c and myoglobin with diethylpyrocarbonate

Cytochrome b5 is required for the cytochrome P-450 LM2 catalyzed oxidation of the anesthetic methoxyflurane. The ability of cytochrome b5 to support methoxyfluorane oxidation is affected by treatment with diethylpyrocarbonate, a reagent that at neutral pH is relatively specific for histidine residues. This inactivation of cytochrome b5 is reversed with hydroxylamine, which also suggests but does not prove histidine involvement. The studies reported in this paper were undertaken to determine whether histidine modification was involved in the decrease in effectiveness of cytochrome b5, or whether the inactivation could be attributed to modification of another amino acid. Our experiments demonstrate that diethylpyrocarbonate inactivates detergent-solubilized cytochrome b5 by modifying the axial histidines and displacing the heme. Because of the unexpected ease with which diethylpyrocarbonate displaced the heme from cytochrome b5, this same process was investigated in two other hemoproteins, cytochrome c and myoglobin. Diethylpyrocarbonate could not dissociate the heme from cytochrome c, whereas the heme was lost from myoglobin even more readily than from cytochrome b5.     

A double-alpha c-type cytochrome, cytochrome c-555, 549, from an extreme thermophile, Thermus thermophilus HB8.

A "double-alpha" c-type cytochrome, cytochrome c-555, 549, was isolated from the membrane fraction of an extreme thermophile, Thermus thermophilus HB8, and highly purified by chromatographies on DEAE-cellulose and Sephadex G-75 and by isoelectric focusing. The absorption maxima were at 554.8, 548.6, 522, and 417 nm in the reduced form, and at 528, 409, and 360 nm in the oxidized form. The double alpha-peak of this cytochrome was enhanced at liquid nitrogen temperature. The cytochrome contained one heme c group per protein molecule. The isoelectric point, midpoint redox potential and molecular weight were pH 4.0, +0.206 V and about 10,000, respectively. Cytochrome c-555, 549 is highly thermostable.     

Characterization of cytochrome 579, an unusual cytochrome isolated from an iron-oxidizing microbial community

A novel, soluble cytochrome with an unusual visible spectral signature at 579 nm (Cyt(579)) has been characterized after isolation from several different microbial biofilms collected in an extremely acidic ecosystem. Previous proteogenomic studies of an Fe(II)-oxidizing community indicated that this abundant red cytochrome could be extracted from the biofilms with dilute sulfuric acid. Here, we found that the Fe(II)-dependent reduction of Cyt(579) was thermodynamically favorable at a pH of >3, raising the possibility that Cyt(579) acts as an accessory protein for electron transfer. The results of transmission electron microscopy of immunogold-labeled biofilm indicated that Cyt(579) is localized near the bacterial cell surface, consistent with periplasmic localization. The results of further protein analysis of Cyt(579), using preparative chromatofocusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, revealed three forms of the protein that correspond to different N-terminal truncations of the amino acid sequence. The results of intact-protein analysis corroborated the posttranslational modifications of these forms and identified a genomically uncharacterized Cyt(579) variant. Homology modeling was used to predict the overall cytochrome structure and heme binding site; the positions of nine amino acid substitutions found in three Cyt(579) variants all map to the surface of the protein and away from the heme group. Based on this detailed characterization of Cyt(579), we propose that Cyt(579) acts as an electron transfer protein, shuttling electrons derived from Fe(II) oxidation to support critical metabolic functions in the acidophilic microbial community.     

The interactions of cyanobacterial cytochrome c6 and cytochrome f,characterized by NMR

During oxygenic photosynthesis, cytochrome c(6) shuttles electrons between the membrane-bound complexes cytochrome bf and photosystem I. Complex formation between Phormidium laminosum cytochrome f and cytochrome c(6) from both Anabaena sp. PCC 7119 and Synechococcus elongatus has been investigated by nuclear magnetic resonance spectroscopy. Chemical-shift perturbation analysis reveals a binding site on Anabaena cytochrome c(6), which consists of a predominantly hydrophobic patch surrounding the heme substituent, methyl 5. This region of the protein was implicated previously in the formation of the reactive complex with photosytem I. In contrast to the results obtained for Anabaena cytochrome c(6), there is no evidence for specific complex formation with the acidic cytochrome c(6) from Synechococcus. This remarkable variability between analogous cytochromes c(6) supports the idea that different organisms utilize distinct mechanisms of photosynthetic intermolecular electron transfer.     

The interactions of cytochrome c and porphyrin cytochrome c with cytochrome c oxidase. The resting, reduced and pulsed enzymes

Cytochrome c oxidase forms tight binding complexes with the cytochrome c analog, porphyrin cytochrome c. The behaviour of the reduced and pulsed forms of the oxidase with porphyrin cytochrome c have been followed as functions of ionic strength; this behaviour has been compared with that of the resting oxidase [Kornblatt, Hui Bon Hoa and English (1984) Biochemistry 23, 5906-5911]. All forms of the cytochrome oxidase studied bind one porphyrin cytochrome c per 'functional' cytochrome oxidase (two heme a); it appears as though porphyrin cytochrome c and cytochrome c compete for the same site on the oxidase. The resting enzyme binds cytochrome c 8 times more strongly than porphyrin cytochrome c; the reduced enzyme, in contrast, binds the two with almost equal affinity. In all three cases, resting, pulsed and reduced, the heme-to-porphyrin distance is estimated to be about 3 nm. The tight-binding complexes formed between cytochrome oxidase and porphyrin cytochrome c can be dissociated by salt. Debye-Hückel analysis of salt titrations indicate that the resting enzyme and the reduced enzyme are similar in that the product of the interaction charges on the two proteins is about -14. The product of the charges for the pulsed enzyme is -25, indicating that on average another positive and negative charge take part in the interaction of the two proteins. While there is one tight binding site for cytochrome c per two heme a, cytochrome c is able to 'communicate' with four heme a. In the absence of cytochrome c, electron transfer from tetramethylphenylenediamine to the oxidase to oxygen results in the conversion of the resting form to the 'oxygenated'; in the presence of cytochrome c, the same electron transfer results in the appearance of the 'pulsed' form. Cytochrome c titrations of the enzyme show that a ratio of only one cytochrome c to four heme a is sufficient to convert all the oxidase to the 'pulsed' form. Porphyrin cytochrome c, like cytochrome c, catalyzes the same conversion with the same stoichiometry. The binding data and salt effects indicate that major structural alterations occur in the oxidase as it is converted from the resting to the partially reduced and subsequently to the pulsed form.     

Isolation and properties of cytochrome c-553, cytochrome c-550, and cytochrome c-549, 554 from Nitrobacter agilis

Three c-type cytochromes isolated from Nitrobacter agilis were purified to apparent homogeneity: cytochrome c-553, cytochrome c-550 and cytochrome c-549, 554. Their amino acid composition and other properties were studied. Cytochrome c-553 was isolated as a partially reduced form and could not be oxidized by ferricyanide. The completely reduced form of the cytochrome had absorption maxima at 419, 524 and 553 nm. It had a molecular weight of 25 000 and dissociated into two polypeptides of equal size of 11 500 during SDS gel electrophoresis. The isoelectric point of cytochrome c-553 was pH 6.8. The ferricytochrome c-550 exhibited an absorption peak at 410 nm and the ferrocytochrome c showed peaks at 416, 521 and 550 nm. The molecular weight of the cytochrome estimated by gel filtration and by SDS gel electrophoresis was 12 500. It had an E_m(7) value of 0.27 V and isoelectric point pH 8.51. The N-terminal sequence of cytochrome c-550 showed a clear homology with the corresponding portions of the sequences of other c-type cytochromes. Cytochrome c-549, 554 possessed atypical absorption spectra with absorption peaks at 402 nm as oxidized form and at 419, 523, 549 and 554 nm when reduced with Na₂S₂O₄. Its molecular weight estimated by gel filtration and SDS polyacrylamide gel electrophoresis was 90 000 and 46 000, respectively. The cytochrome had an isoelectric point of pH 5.6. Cytochrome c-549, 554 was highly autoxidizable.     

Circular dichroism of cytochrome oxidase, cytochrome b566, and cytochrome c in beef heart mitochondrial membrane fragments

1. Circular dichroism spectra of the cytochromes in membrane fragments derived from sonicated beef heart mitochondria have been obtained in the wavelength region 400–480 nm in which the major absorbance maxima of the heme prosthetic groups are found.

2. 2. Cytochrome oxidase in the mitochondrial membrane fragments has a band of positive ellipticity at 426 nm in the oxidized form and a pronounced band of positive ellipticity at 445 nm in the reduced form. The reduced-minus-oxidized difference molar ellipticity at 445 nm, Δ[θ]₄₄₅ is 3.0·10⁵ degree·cm⁻²·dmole⁻¹ heme a for membrane-bound oxidase compared to 1.6·10⁵ degree·cm⁻²·dmole⁻¹ heme a for the purified oxidase. The membrane-bound oxidase in the reduced form also appears to have a band of negative ellipticity at 426 nm not found in the purified oxidase.

3. 3. When reduced with succinate in the presence of cyanide and oxygen, cytochrome oxidase in the membrane fragments has a positive band at 442 nm very similar to that observed with the purified oxidase.

4. 4. Cytochrome c, which has a positive band at 426 nm in the purified form when reduced, appears to have a negative band at this wavelength in the mito-chondrial membrane fragments which contributes to the pronounced negative band at 426 nm observed in the membrane fragments reduced with succinate in anaerobiosis. There is no evidence for a contribution to the CD spectra of the membrane fragments from cytochrome c₁ or from cytochrome b₅₆₁ in either the oxidized or the reduced form.

5. 5. Cytochrome b₅₆₆ in the mitochondrial membrane fragments has no detectable CD spectrum in the oxidized form, but has a small positive band at 427 nm and a small negative band at 436 nm in the reduced form. The same CD spectrum is observed with cytochrome b₅₆₆ reduced with succinate in the presence of antimycin A or 2-heptyl-4-hydroxyquinoline-N-oxide. The same increase in positive ellipticity is observed at 427 nm in the mitochondrial membrane fragments, treated with oligomycin to restore energy coupling, when cytochrome b₅₆₆ is reduced with succinate in the energized membrane, as is observed in the inhibitor-treated membrane fragments. The absence of a pronounced conformational change in cytochrome b₅₆₆ on energization, as revealed by its CD spectrum, favors the concept that its reduction by succinate in the energized state is due to reversed electron transport rather than an intrinsic shift in the cytochrome's midpoint redox potential.

Modification of trypsin-solubilized cytochrome b5, apocytochrome b5, and liposome-bound cytochrome b5 by diethylpyrocarbonate

The interactions of diethylpyrocarbonate (DEP) with the various forms of cytochrome b5 were studied to gain a better understanding of the factors that influence the extent of modification of the axial histidines of cytochrome b5. Very low concentrations of DEP were able to decrease the heme binding capacity of apocytochrome b5. Moreover, it was shown that two additional histidines, presumed to be the axial ligands (His 39 and 63), were modified in the apo but not the holo form of a given preparation of cytochrome b5. Trypsin-solubilized bovine cytochrome b5 was resistant to the effects of DEP. A 200-fold molar excess of DEP displaced only 15% of the heme in the trypsin-solubilized protein in contrast to an 84% displacement of the heme in the detergent-solubilized protein. However, detergent-solubilized cytochrome b5 which had been incorporated into phospholipid vesicles exhibited the same reactivity with DEP as did the trypsin-solubilized protein. This is attributed to the fact that the two resistant preparations of cytochrome b5 are monomeric in their respective environments while detergent-solubilized cytochrome b5 is known to exist as an octamer in aqueous solutions. Our studies suggest that dissociation of the octamer to the monomer results in a conformational change that decreases the reactivity of the axial ligands of the hydrophilic heme-containing domain of cytochrome b5. Examination of the cytochrome b5 molecule by computer graphics indicates that a tunnel leads from the surface of the molecule to axial histidine 63 and that axial histidine 39 is buried.     

Biological activity of phenolic compounds. Hepatic cytochrome P-450, cytochrome b5, and NADPH cytochrome c reductase in chicks and rats fed phenolic monomers, polymers, and glycosides

Eight experiments were conducted to determine effects of a phenolic polymer (Kraft wood lignin, Indulin), phenolic glycosides (cane molasses and wood molasses), and phenolic monomers (vanillin, vanillic acid, ferulic acid, and p-coumaric acid) on liver cytochromes P-450, cytochrome b5, and NADPH cytochrome c reductase in chicks and rats. Chicks fed 6.0% lignin had a higher (P less than 0.01) cytochromes P-450 content than did chicks fed 0% fiber, 6.0% wood cellulose (Solka Floc), or 6.0% arenaceous flour. NADPH cytochrome c reductase activity was not affected by treatment. Chicks fed 12.0% wood molasses had a higher (P less than 0.06) cytochromes P-450 level than did chicks fed 0% fiber or 6.0% wood molasses. Cane molasses incorporated at both 6.0 and 12.0% of the diet induced (P less than 0.05) cytochromes P-450 content over those of control-fed birds. Chicks fed 6.0% lignin, with or without antibiotic (bacitracin:neomycin sulfate, 2:1), had a higher (P less than 0.01) cytochromes P-450 level than did chicks fed control diets, with or without antibiotic. Additionally, chicks fed 6.0% lignin had lower (P less than 0.01) intestinal diaminopimelic acid (DAP) levels than did chicks fed 0% fiber. Rats fed 0% fiber, 6.0% wood cellulose, 6.0% arenaceous flour, or 6.0% lignin exhibited no difference in cytochrome level or activity among treatments. Chicks fed 0.5% vanillin, 0.5% vanillic acid, 0.5% ferulic acid, or 0.5% p-coumaric acid had comparable cytochromes level and activity compared with chicks fed no phenolics. Chicks fed 0.5% p-coumaric acid had lower (P less than 0.05) rates of gain than did chicks fed control or other phenolic-containing diets. Rats fed these phenolics had similar cytochromes P-450 content among treatments.