Characterisation of ionisations that influence the redox potential of mitochondrial cytochrome c and photosynthetic bacterial cytochromes c2

Several cytochromes c₂ from the Rhodospirillaceae show a pH dependence of redox potential in the physiological pH range which can be described by equations involving an ionisation in the oxidised form (pK_o) and one in the reduced form (pK_r). These cytochromes fall into one of two groups according to the degree of separation of pK_o and pK_r. In group A, represented here by the Rhodomicrobium vannielii cytochrome c₂, the separation is approx. one pH unit and the ionisation is that of a haem propionic acid. Members of this group are unique among both cytochromes c₂ and mitochondrial cytochromes c in lacking the conserved residue Arg-38. We propose that the role of Arg-38 is to lower the pK of the nearby propionic acid, so that it lies out of the physiological pH range. Substitution of this residue by an uncharged amino acid leads to a raised pK for the propionic acid. In group B, represented here by Rhodopseudomonas viridis cytochrome c₂, the separation between pK_o and pK_r is approx. 0.4 pH unit and the ionisable group is a histidine at position 39. This was established by NMR spectroscopy and confirmed by chemical modification. Only a few other members of the cytochrome c₂/mitochondrial cytochrome c family have a histidine at this position and of these, both Crithidia cytochrome c-557 and yeast cytochrome c were found to have a pH-dependent redox potential similar to that of Rps. viridis cytochrome c₂. Using Coulomb's law, it was found that the energy required to separate pK_o and pK_r could be accounted for by simple electrostatic interactions between the haem iron and the ionisable group.     

Redox potentials of the photosynthetic bacterial cytochromes c2 and the structural bases for variability.

The cytochromes c2 of the Rhodospirillaceae show a much greater variation in redox potential and its pH dependence than the mitochondrial cytochromes c that have been studied. It is proposed that the range of redox potential for cytochromes c2 functioning as the immediate electron donor to photo-oxidised bacteriochlorophyll may be 345-395 mV at pH 5. Closely related cytochromes c2 with different redox potentials show patterns of amino acid substitution which are consistent with changes in hydrophobicity near the haem being at least a partial determinant of redox potential. More distantly related cytochromes are difficult to compare because of the large number of amino acid substitutions and the probability that there are subtle changes in overall peptide chain folding. The redox potential versus pH curves can be analysed in terms of either one ionisation in the oxidised form or two in the oxidised form and one in the reduced. The pK in the oxidised form at higher pH values can be correlated with the pK for the disappearance or shift of the near infrared absorption band located near 695 nm. The structural bases of these ionisations are not known but the possible involvement of the haem propionate residues is discussed.     

The two cytochromes c in the facultative methylotroph Pseudomonas AM1

It was previously suggested that there is only one soluble cytochrome c in Pseudomonas AM1, having a molecular weight of 20000, a redox midpoint potential of about +260mV and a low isoelectric pint [Anthony (1975) Biochem. J. 146, 289–298; Widdowson & Anthony (1975) Biochem. J. 152, 349–356]. A more thorough examination of the soluble fraction of methanol-grown Pseudomonas AM1 has now revealed the presence of two different cytochromes c. These were both purified to homogeneity by acid treatment, ion-exchange chromatography, gel filtration, chromatography on hydroxyapatite and preparative isoelectric focusing. Molecular weights were determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis; midpoint redox potentials were determined directly by using platinum and calomel electrodes; isoelectric points were estimated by electrophoresis and by the behaviour of the two cytochromes on ion-exchange celluloses. The more abundant cytochrome c_H (λ_max. 550.5nm) had a low molecular weight (11000), a midpoint potential of about +294mV and a high isoelectric point, not being adsorbed on DEAE-cellulose in 20mm-Tris/HCl buffer, pH8.0. The less abundant cytochrome c_L (λ_max. 549nm) was about 30% of the total; it had a high molecular weight (20900), a midpoint potential of about +256mV and a low isoelectric point, binding strongly to DEAE-cellulose in 20mm-Tris/HCl buffer, pH8.0. The pH-dependence of the midpoint redox potentials of the two cytochromes c were very similar. There were four ionizations affecting the redox potentials in the pH range studied (pH4.0–9.5), two in the oxidized form (pK values about 3.5 and 5.5) and two in the reduced form (pK values about 4.5 and 6.5), suggesting that the ionizing groups involved may be the two propionate side chains of the haem. Neither of the cytochromes c was present in mutant PCT76, which was unable to oxidize or grow on C₁ compounds, although still able to grow well on multicarbon compounds such as succinate. Whether or not these two cytochromes c have separate physiological functions is not yet certain.     

pH dependence of the oxidation-reduction potential of cytochrome c2

The pH dependence of the spectra and of the oxidation-reduction potential of three cytochromes c₂, from Rhodopseudomonas capsulata, Rhodopseudomonas sphaeroides and Rhodomicrobium vannielii, were studied. A single alkaline pK was observed for the spectral changes in all three ferricytochromes. In Rps. capsulata cytochrome c₂ this spectroscopic pK corresponds to the pK observed in the dependence of oxidation-reduction potential on pH. For the other two cytochromes the oxidation-reduction potential showed a complex dependency on pH which can be fitted to theoretical curves involving three ionizations. The third ionization corresponds to the ionization observed in the spectroscopic studies but the first two occur without changes in the visible spectra.The possible structural bases for these ionizations are discussed.     

The pH dependence of the oxidation-reduction midpoint potential of cytochromes c2 in vivo

A recent report by Pettigrew et al. [Biochim. Biophys. Acta 430, (1976), 197–208] has examined the pH dependence of the oxidation-reduction midpoint potential of cytochromes c₂ in vitro. In media of low ionic strength, these workers identified several pKs on the oxidized forms of the cytochromes, and in some cases there were also pKs on the reduced species. In this work we examine the pH dependence of the midpoint potentials of the cytochromes in situ, attached to the chromatophore membrane. Under these conditions no pK values are detected, and we conclude that in vivo there is no net change in the protonation of cytochrome c₂ during oxidation or reduction.     

pH Variation of midpoint potential for three photosynthetic bacterial cytochromes c′. A link between physical and functional properties

Midpoint redox potential (E_M) versus pH curves are reported over the pH range 5 to 10 for the cytochromes c′ from three species of purple photosynthetic bacteria: Rhodospirillum rubrum, Rhodopseudomonas palustris and Chromatium vinosum. In each case, theoretical curves are fitted to the data and pK values for the reduced (pH 5–5.5) and oxidized (pH 8–8.5) forms of the protein are found to influence the midpoint redox potentials. The oxidized form pK values in each case are found to correlate with previously determined pK values for variation in physical and/or spectroscopic properties. This correlation of functional and physical observables is discussed in terms of a possible mechanism of control of midpoint redox potential through heme iron-ligand bonding as moderated by the protein conformation in response to solution conditions. The reduced form pK values are discussed in terms of a mechanism which would alter the polarity of the heme environment, thereby influencing redox potentials.     

Cytochrome C553 from Desulfovibrio,vulgaris: Potentiometric characterization by optical and EPR studies

Cytochrome c₅₅₃ is a monohaemic c type cytochrome isolated from the sulfate reducing bacteria $\text{Desulfovibrio}$,$\text{vulgaris}$. Its midpoint potential value, determined by optical, EPR and polarographic studies is significantly lower than the midpoint potentials reported for other monohaemic cytochromes c (+ 10 mV instead of + 290 mV). In an attempt to study correlations between amino acid sequence, haem iron coordination and haem exposure in cytochromes c, cytochrome c₅₅₃ is compared with mitochondrial and bacterial c type cytochromes.     

Equilibrium and kinetic measurements of the redox potentials of cytochromes c2 in vitro and in vivo

The equilibrium oxidation-reduction mipoint potential (E_m) of isolated Rhodopseudomonas sphaeroides cytochrome c₂ exhibits a pH-dependent behavior which can be ascribed to a pK on the oxidized form at pH 8.0 (Pettigrew et al. (1975) Biochim. Biophys. Acta 430, 197–208). However, as with mammalian cytochrome c (Brandt, K.G., Parks, P.C., Czerlinski, G.H. and Hess, G.P. (1966) J. Biol. Chem. 241, 4180–4185) this pK can more properly be attributed to the combination of a pK beyond pH 11, and a slow conformational change of the ferricytochrome. This has been demonstrated by resolving the E_m of cytochrome c₂ before and after the conformational change. The E_m of the unaltered form is essentially pH independent between pH 7 and 11.5, and the lower equilibrium E_m is due solely to the conformational change. In vivo the conformational change is prevented by the binding of the cytochrome c₂ to the photochemical reaction center, and the cytochrome exhibits an essentially pH-independent E_m from pH 5 to 11. The alkaline transition thus has little physiological significance, and it is unlikely that the redox reactions of cytochrome c₂ in vivo involve protons.     

Site-directed mutagenesis of a phenylalanine residue strictly conserved in cytochromes c 3

 Reduction of the haems in tetrahaem cytochromes c ₃ is a cooperative process, i.e., reduction of each of the haems depends on the redox states of the other haems. Furthermore, electron transfer is coupled to proton transfer (redox-Bohr effect). Two of its haems and a strictly conserved nearby phenylalanine residue, F20, in Desulfovibrio vulgaris (Hildenborough) cytochrome c ₃ form a structural motif that is present in all cytochromes c ₃ and also in cytochrome c oxidase. A putative role for this phenylalanine residue in the cooperativity of haem reduction was investigated. Therefore, this phenylalanine was replaced, with genetic techniques, by isoleucine and tyrosine in D. vulgaris (Hildenborough) cytochrome c ₃. Cyclic voltammetry studies revealed a small increase (30 mV) in one of the macroscopic redox potentials in the mutated cytochromes. EPR showed that the main alterations occurred in the vicinity of haem I, the haem closest to residue 20 and one of the haems responsible for positive cooperativities in electron transfer of D. vulgaris cytochrome c ₃. NMR studies of F20I cytochrome c ₃ demonstrated that the haem core architecture is maintained and that the more affected haem proton groups are those near the mutation site. NMR redox titrations of this mutated protein gave evidence for only small changes in the relative redox potentials of the haems. However, electron/electron and proton/electron cooperativity are maintained, indicating that this aromatic residue has no essential role in these processes. Furthermore, chemical modification of the N-terminal amino group of cytochrome c ₃ backbone, which is also very close to haem I, had no effect on the network of cooperativities. Received: 25 June 1996 / Accepted: 26 August 1996     

Soluble cytochromes ofRhodopseudomonas marina

Three acidicc-type cytochromes (c-552,c-550 andc′) were purified from the soluble fraction ofRhodopseudomonas marina. Cytochromec′ is a high-spin cytochrome capable of binding carbon monoxide reversibly to its reduced form. It occurs as a dimer with anM_r of 36700 (estimated by gel filtration) while the monomer has anM_r of 17800 (determined by SDS-acrylamide gel electrophoresis). Cytochromec′ has a midpoint redox potential of +73 mV and an isoelectric point at pH 4.3. Cytochromesc-550 andc-552 are typical low-spin cytochromes. Cytochromec-550 has anM_r of 12500, an isoelectric point at pH 4.5 and a negative redox potential of −163 mV. The molecular properties of cytochromec-552 are as follows:M_r, 18000; isoelectric point, pH 5.4; redox potential, +283 mV.     

Studies of cytochrome synthesis in rat liver

The incorporation of radioactive amino acids and of δ-amino[2,3-³H₂]laevulinate into rat liver cytochromes b₅ and c and cytochrome oxidase has been examined with and without protein-synthesis inhibitors. Cycloheximide promptly inhibits labelling of both haem and protein for cytochrome c in parallel fashion. Although incorporation of ¹⁴C-labelled amino acid into microsomal cytochrome b₅ is also rapidly inhibited, cycloheximide incompletely inhibits haem labelling of cytochrome b₅ and cytochrome a+a₃, and inhibition occurs only after repeated antibiotic injections. The possibility of apo-protein pools, or of haem exchange, with a rapidly renewed `free' haem pool, is considered. Consistent with this model is the observation of non-enzymic haem exchange in vitro between cytochrome b₅ and methaemoglobin. Chloramphenicol, injected intravenously over 5h, results in a 20–40% decrease in incorporation of δ-amino[2,3-³H₂]laevulinate into haem a+a₃ and haem of cytochromes b₅ and c. With the dosage schedule of chloramphenicol studied, amino acid labelling of total liver protein and of cytochrome c was not inhibited. Similarly, ferrochelatase activity was not decreased.     

In situ characterisation of photosynthetic electron transport in Rhodopseudomonas capsulata

1. The effects of varying the ambient oxidation/reduction potential on the redox changes of cytochromes c, cytochromes b and P605 induced by a laser flash in chromatophores from Rhodopseudomonas capsulata Ala Pho⁺ have been investigated.2. The appearance and attenuation of the changes with varying ambient redox potential show that, of the cytochromes present, cytochromes c with Em₇ = 340 mV and 0 mV, and cytochrome b, Em₇ = 60 mV were concerned with photosynthetic electron flow.3. The site of action of antimycin was shown to be between cytochrome b₆₀ and a component, as yet unidentified, called Z.4. The appearance or attenuation of laser-induced changes of cytochromes c₀ and b₆₀ on redox titration was dependent on pH, but no effect of pH on the cytochrome c₃₄₀ titration was observed.5. The dependence on ambient redox potential of the laser-induced bleaching at 605 nm enabled identification of the mid-point potentials of the primary electron donor (Em₇ = 440 mV) and acceptor (Em₇ = ?25 mV).6. The interrelationship of these electron carriers is discussed with respect to the pathway of cyclic electron flow.     

Purification and properties of cytochrome c-553, an electron acceptor for formate dehydrogenase of Desulfovibrio vulgaris, Miyazaki

Cytochrome c-553 of Desulfovibrio vulgaris, Miyazaki, was purified to homogeneity. The absorption spectrum of the ferro form has four peaks at 553, 525, 417 and 317 nm with a plateau near 280 nm, and that of the ferri form has three peaks at 525, 410 and 360 nm with a plateau near 280 nm and a shoulder at 560 nm. The millimolar absorbance coefficient of the α-peak of the ferro form is 23.9. The molecular weight of cytochrome c-553 is 8000, and it contains one heme. Its isoelectric point is rather alkaline, and its standard redox potential is ?0.26 V at pH 7.0. Its amino acid composition is unique; it lacks proline, isoleucine and tryptophan.Ferrocytochrome c-553 does not combine with CO, nor does it transfer electrons directly to various redox carriers such as flavin nucleotides, methylene blue, indigodisulfonate, 5-methylphenazinium methyl sulfate, 1-methoxy-5-methylphenazinium methyl sulfate, viologens and cytochrome c₃, but is oxidized by ferricyanide or by O₂.Cytochrome c-553 can be reduced by formate dehydrogenase of this bacterium in the presence of formate, but not by hydrogenase under H₂. The formate dehydrogenase does not reduce cytochrome c₃ in the presence of formate. The systematic name for formate dehydrogenase of D. vulgaris is, therefore, established as formate:ferricytochrome c-553 oxidoreductase in EC subclass 1.2.2.—.     

Oxidation-reduction potentials and spectral properties of some cytochromes from Thiobacillus versutus (A2)

Cytochromes c-550 (acidic), c-550 (basic), c-551 and c-552.5 from Thiobacillus versutus have been highly purified and characterized. Their spectral properties at 77 K are described. Oxidation-reduction titrations of cytochromes c-550 (acidic) and c-550 (basic) showed them to exhibit Nernst values of n = 1, with single redox centres in the cytochromes, and to have midpoint redox potentials at pH 7.0 (E_m,7) of 290 and 260 mV, respectively. Cytochrome c-551 contained two separately titratable redox components, each giving n = 1. The low potential centre (55% of titratable cytochrome) and the high potential centre (45%) had E_m,7 values of ?115 and +240 mV, espectively. Cytochrome c-552.5 also contained at least two redox centres. One (65% of titratable cytochrome) had n = 1 and E_m,7 = 220mV. The remaining 35% appeared to be a low potential component with an E_m,7 possibly as low as ?215 mV. the roles of these cytochromes in respiratory thiosulphate oxidation are discussed.     

Functional properties of type I and type II cytochromes c3 from Desulfovibrio africanus

Type I cytochrome c₃ is a key protein in the bioenergetic metabolism of Desulfovibrio spp., mediating electron transfer between periplasmic hydrogenase and multihaem cytochromes associated with membrane bound complexes, such as type II cytochrome c₃. This work presents the NMR assignment of the haem substituents in type I cytochrome c₃ isolated from Desulfovibrio africanus and the thermodynamic and kinetic characterisation of type I and type II cytochromes c₃ belonging to the same organism. It is shown that the redox properties of the two proteins allow electrons to be transferred between them in the physiologically relevant direction with the release of energised protons close to the membrane where they can be used by the ATP synthase.     

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.     

A thermodynamic characterisation of the cytochromes of chromatophores from Rhodopseudomonas capsulata

1. The cytochromes of chromatophores from photosynthetically grown Rhodopseudomonas capsulata have been characterised both spectrally, using the carotenoid free mutant Ala Pho⁺, and thermodynamically, using the technique of redox titrations. Five cytochromes were present; two cytochromes b, E′₀ = 60 mV at pH 7.0; and three cytochromes c, E′₀ = 340 mV, $\text{E}\text{t}\text{?}{}_0\text{= 120}\text{mV}$, E′₀ = 0 mV at pH 7.0.2. Redox titrations at different values of pH indicated that the mid point potentials of all the cytochromes varied with pH over some parts of the range between pH 6 and 9, with the possible exception of cytochrome c₃₄₀.3. The effects of succinate and NADH on the steady state reduction of the cytochromes are reported. Succinate could reduce cytochromes c₃₄₀, c₁₂₀ and b₆₀; NADH could reduce cytochromes c₃₄₀, c₁₂₀, b₆₀ and b_?25. Cytochrome c₀ could be reduced by dithionite but not by the other substrates tested.     

Nine-haem cytochrome c from Desulfovibrio desulfuricans ATCC 27774 : primary sequence determination, crystallographic refinement at 1.8 and modelling studies of its interaction with the tetrahaem cytochrome c 3

A monomeric nine-haem cytochrome c (9Hcc) with 292 amino acid residues was isolated from cells of the sulfate- and nitrate-reducing bacterium Desulfovibrio desulfuricans ATCC 27774 grown under both nitrate- and sulfate-respiring conditions. The nucleotide sequence encoding the 292 residues was determined, allowing the correction of about 10% of the previous primary structure, determined from 1.8?Å electron density maps. The refinement at 1.8?Å resolution of the structural model was completed, giving an R-value of 16.5%. The nine haem groups are arranged into two tetrahaem clusters, located at both ends of the molecule, with Fe-Fe distances and local protein fold very similar to tetrahaem cytochromes c ₃, and the extra haem is located asymmetrically between the two regions. The new primary sequence determination confirmed the 39% sequence homology found between this cytochrome and the C-terminal region (residues 229–514) of the high-molecular-weight cytochrome c (Hmc) from D. vulgaris Hildenborough, providing strong evidence of structural similarity between 9Hcc and the C-terminal region of Hmc. The interaction between 9Hcc and the tetrahaem cytochrome c ₃ from the same organism was studied by modelling methods, and the results suggest that a specific interaction is possible between haem 4 of tetrahaem cytochrome c ₃ and haem 1 or haem 2 of 9Hcc, in agreement with previous kinetic experiments which showed the catalytic effect of the tetrahaem cytochrome c ₃ upon the reduction of 9Hcc by the [NiFe] hydrogenase from D. desulfuricans ATCC 27774. These studies suggest a role for 9Hcc as part of the assembly of redox proteins involved in recycling the molecular hydrogen released by the cell as a result of substrate oxidation.     

Alkaline isomerization of ferricytochrome c: Lysine is not replacing methionine at the sixth co-ordination site of the haem iron

Oxidized cytochrome c is known to undergo a restricted conformational refolding of its haem area at around pH 9. Methionine 80, the sixth ligand of the ferric haem iron in the biologically active neutral conformational state, is replaced by a new strong-field ligand in the biologically inactive alkaline state of the molecule. It had been proposed that a lysine residue, possibly lysine 79. is the new haem ligand.We have tested this proposition by a more direct approach than hitherto employed, namely by measuring the relative chemical reactivity of lysines in the oxidized eytochrome c and in fragment 66–80 cut out of the native molecule. The relative rates of acetylation of lysine 79, measured between pH 7 and pH 11, are virtually identical in the intact molecule and in the haem-free fragment 66–80. Similarly, the rates are also the same for the amidination reaction with isethionylacetimidate. When the relative rates of acetylation and amidination of lysines 72 + 73 were compared there was again no significant difference between the intact molecule and fragment 66–80. These results contradict the involvement of any of the three lysines in the alkaline isomerization, as a haem-bound ?-amino group would be much less reactive than its freely accessible counterpart in fragment 66–80.To corroborate the above finding, the pK value and absolute rate constant of acetylation of lysine 79 were determined and compared with the respective values for lysines 39 and 60. The latter two residues are on the side opposite to the haem pocket and hence unable to bind to the haem iron.The three pK values and rate constants k obey the Brønsted relationship: log κ = α + βpK with β = 0.48, a value characteristic of the acetylation of freely accessible primary amino groups.Taken together, these results oppose an ?-amino: haem iron co-ordination in the alkaline state of oxidized eytochrome c.     

Purification,primary structure,and evolution of cytochrome c-550 from the magnetic bacterium,Magnetospirillum magnetotacticum

Cytochrome c-550 was purified from Magnetospirillum magnetotacticum to an electrophoretically homogeneous state, and some of its properties were determined. The cytochrome showed absorption peaks at 528 and 409 nm in the oxidized form, and at 550, 521, and 414 nm in the reduced form. Its midpoint redox potential at pH 7.0 was determined to be +289 mV. The primary structure of cytochrome c-550 was determined. Cytochrome c is composed of 97 amino acid residues, and its molecular weight was calculated to be 10,873, including heme c. Its primary structure is very similar to those of Rhodospirillum fulvum and Rhodospirillum molischianum cytochromes c ₂, suggesting that M. magnetotacticum is phylogenetically related to photosynthetic bacteria.