Phylogenetic implications of the amino acid sequence of cytochrome c from Enteromorpha intestinalis

The amino acid sequence of Epiteromorpha cytochrome c has been added to an affinity tree relating the cytochrome c sequences of animals, plants, fungi, protozoans and one bacterium, cytochrome c₂ from Rhodospirillum. The Enteromorpha sequence lies on the line of descent of the higher plant sequences; it is not closely related to the cytochrome c of the photosynthetic protozoan, Euglena. The distribution of ε-N-trimethyllysine in cytochrome c is discussed.     

Nucleotide sequence of the gene encoding the 45-kilodalton subunit of alcohol dehydrogenase from Acetobacter aceti

The gene encoding the 45 kilodalton subunit of alcohol dehydrogenase from Acetobacter aceti was found to exist in the 3′ flanking region of adhA which encodes the dehydrogenase subunit of the alcohol dehydrogenase. The amino acid sequence of the 45-kilodalton subunit, which was deduced from the DNA sequence, seemed to consist of three mono-heme cytochrome c polypeptide chains joined to form a single tri-heme cytochrome c. The cytochrome c-552 from Thermus aquaticus, which had been reported to be a unique mono-heme cytochrome c, and the 45-kilodalton subunit were found to have considerable similarity in their amino acid sequences.     

Cloning,Sequencing and Functional Expression in Escherichia coli of dmc Gene Encoding Periplasmic Tetraheme Cytochrome c3from Desulphovibrio desulphuricans M6

A small soluble protein, periplasmic tetraheme cytochrome c₃, was purified fromDesulphovibrio desulfuricans M6 and its gene, dmc, was cloned and the complete nucleotide sequence determined. The purity index of purified cytochrome c₃was 3.3 and the molecular weight was determined as 14.5 kDa by SDS-PAGE. It was found that the 387 bp of dmc gene encoded 21 amino acids of hydrophobic signal peptide and 107 residues of apoprotein. The nucleotide sequence and the predicted amino acid sequence of dmc showed 76% and 83% identities to those of 13 kDa cytochrome c₃from D. desulphuricans ATCC 27774, respectively.dmc gene was functionally expressed in aerobically grown Escherichia coli BL-21(DE3) by co-expressing eightccm genes which were reported to be involved in cytochrome c maturation. The molecular weight of overexpressed holocytochrome c₃was identical to that of the original protein. Visible spectrum of dithionite-reduced form exhibited typical characteristics of c -type cytochromes. In addition, the redox potential was measured to −340 mV by cyclic voltammetry.     

The amino acid sequence of cytochrome c from Enteromorpha intestinalis

Proposed amino acid sequence of cytochrome c from Enteromorpha intestinalis is presented. The cytochrome is a basic protein, homologous with higher plant and animal cytochromes c of mitochondrial origin. Peptides from chymotryptic and tryptic digests were analysed by the dansyl-phenylisothiocyanate method and aligned by comparison with other homologous cytochromes c. The proposed sequence consists of a single polypeptide chain of 111 residues which is acetylated at its N-terminus. The sequence contains one residue of the unusual amino acid ?-N-trimethyllysine in position 80.     

A Cytochrome c from a Lupanine-Transforming Pseudomonas putida Strain Is Expressed in Escherichia coli during Aerobic Cultivation and Efficiently Exported and Assembled in the Periplasm

We have cloned, sequenced, and heterologously expressed a periplasmic cytochrome c from a lupanine-utilizing Pseudomonas putida strain. Aerobic batch cultivation of Escherichia coli TB1 harboring the cytochrome c gene placed downstream of the lac promoter in pUC9 vector resulted in significant production of the holo-cytochrome c in the periplasm (~4 mg of hemoprotein/liter of culture). The recombinant cytochrome c was purified to homogeneity and was found to be functional in accepting electrons from lupanine hydroxylase while catalyzing hydroxylation of lupanine. Comparison of the N-terminal amino acid sequence of the isolated cytochrome c with that deduced from the DNA sequence indicated that the signal sequence was processed at the bond position predicted by the SigPep program. The molecular size of the cytochrome c determined by electrospray mass spectrometry (9,595) was in precise agreement with that predicted from the nucleotide sequence.     

The amino acid sequence of Nitrosomonas europaea cytochrome c-552

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

Cytochrome c is transformed from anti- to pro-oxidant when interacting with truncated oncoprotein prothymosin α

Many apoptotic signals are known to induce release to cytosol of cytochrome c, a small mitochondrial protein with positively charged amino acid residues dominating over negatively charged ones. On the other hand, in this group, it was shown that prothymosin α (PT), a small nuclear protein where 53 of 109 amino acid residues are negatively charged, is truncated to form a protein of 99 amino acid residues which accumulates in cytosol during apoptosis [FEBS Lett. 467 (2000) 150]. It was suggested that positively charged cytochrome c and negatively charged truncated prothymosin α (tPT), when meeting in cytosol, can interact with each other. In this paper, such an interaction is shown. (1) Formation of cytochrome c⋅tPT complex is demonstrated by a blot-overlay assay. (2) Analytical centrifugation of solution containing cytochrome c and tPT reveals formation of complexes of molecular masses higher than those of these proteins. The masses increase when the cytochrome c/tPT ratio increases. High concentration of KCl prevents the complex formation. (3) In the complexes formed, cytochrome c becomes autoxidizable; its reduction by superoxide or ascorbate as well as its operation as electron carrier between the outer and inner mitochondrial membranes appear to be inhibited. (4) tPT inhibits cytochrome c oxidation by H₂O₂, catalyzed by peroxidase. Thus, tPT abolishes all antioxidant functions of cytochrome c which, in the presence of tPT, becomes in fact a pro-oxidant. A possible role of tPT in the development of reactive oxygen species- and cytochrome c-mediated apoptosis is discussed.     

The oxidation-reduction kinetics of the reaction of cytochrome c1 with non-physiological redox agents

The kinetics of the oxidation-reduction reactions of cytochrome c₁ with ascorbate, ferricyanide, triphenanthrolinecobalt(III) and N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) have been examined using the stopped-flow technique. The reduction of ferricytochrome c₁ by ascorbic acid is investigated as a function of pH. It is shown that at neutral and alkaline pH the reduction of the protein is mainly performed by the doubly deprotonated form of ascorbate. From the ionic-strength-dependence studies of the reactions of cytochrome c₁ with ascorbate, ferricyanide and triphenanthrolinecobalt(III), it is demonstrated that the reaction rate is governed by electrostatic interactions. The second-order rate constants for the reaction of cytochrome c₁ with ascorbate, ferricyanide, TMPD and triphenanthrolinecobalt(III) are 1.4·10⁴, 3.2·10³, 3.8·10⁴ and 1.3·10⁸ M^?1·s^?1 (pH 7.0, I = 0, 10°C), respectively. Application of the Debye-Hückel theory to the the ionic-strength-dependence studies of these redox reactions of cytochrome c₁ yielded for ferrocytochrome c₁ and ferricytochrome c₁ a net charge of ?5 and ?4, respectively. The latter value is close to that of ?3 for the oxidized enzyme, calculated from the amino acid sequence of the protein. This implies that not a local charge on the surface of the protein, but the overall net charge of cytochrome c₁ governs the reaction rate with small redox molecules.     

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.     

The amino acid sequence of cytochrome c from Allium porrum L. (leek)

The amino acid sequence of leek cytochrome c was determined with 0.4μmol of protein. The sequence was deduced solely from a chymotryptic digest. The cytochrome was homologous with other plant cytochromes c of mitochondrial origin. Leek cytochrome c has an N-acetylated `tail' as compared with mammalian cytochrome c, and two residues of ε-N-trimethyl-lysine. Unlike other plant cytochromes c, leek cytochrome c has glutamic acid or glutamine in position 11, leucine in position 20 and alanine in position 51. 4-Hydroxyproline partially substitutes for proline in position 79. Experimental details are given in a supplementary paper that has been deposited as Supplementary Publication SUP 50012, at the National Lending Library for Science and Technology, Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1973) 131, 5.     

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.     

A Gene Encoding a Cytochrome c Oxidase-Like Protein Is Located Closely to the Cytochrome c-553 Gene in the Anaerobic Bacterium,Desulfovibrio vulgaris (Miyazaki F)

The gene encoding cytochrome c-553 from Desulfovibrio vulgaris (Miyazaki F) was cloned using a synthetic oligodeoxyribonucleotide probe. The nucleotide sequence indicated that cytochrome c-553 was synthesized as a precursor protein with an NH₂-terminal signal sequence of 23 residues. In the cloned DNA fragment, there are three other open reading frames whose products have 191, 157, 541 amino acid residues, respectively. The putative ORF-4 product is highly homologous with the cytochrome c oxidase subunit I from various organisms.     

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.     

Purification and Characterization of Microsomal Cytochrome b(5) and NADH Cytochrome b(5) Reductase from Pisum sativum

In this communication we document the reproducible protocols for the purification of milligram quantities of cytochrome b₅ and NADH-cytochrome b₅ reductase from the microsomal fraction of Pisum sativum. The cytochrome b₅ component of this NADH linked electron transport chain was found to have a molecular mass of 16,400 daltons and the reductase a molecular mass of 34,500 daltons. These components could be reconstituted into a functional NADH oxidase activity active in the reduction of exogenous cytochrome c or ferricyanide. In the latter assay the purified reductase exhibited a turnover number of 22,000 per minute. The amino-terminal amino acid sequence of the cytochrome b₅ component was determined by sequential Edmund degredation, thus providing crucial information for the efficient cloning of this central protein of plant microsomal electron transfer.     

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.     

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.     

The "cytochrome b5 fold": structure of a novel protein superfamily

Selective proteolysis allows the isolation of a heme-binding fragment spectrally similar to microsomal cytochrome b₅ from both baker's yeast flavocytochrome b₂ (a flavohemoprotein) and liver sulfite oxidase (a molybdoprotein). The amino acid sequences of these two fragments have been published separately (Guiard &; Lederer, 1976,1979). We present in this paper an alignment of those sequences with that of microsomal cytochrome b₅. The structural consequences of the similarity between the three primary structures are discussed in the light of the cytochrome b₅ three-dimensional model (Mathews et al., 1971,1972,1975; Mathews &; Czerwinski, 1976).It is concluded that the three heme-binding proteins are in all probability the products of a divergent evolution from a common ancestor and that they must present a basically similar backbone with some surface alterations. We propose to name this backbone the “cytochrome b₅ fold”. The comparison of the three proteins suggests hypotheses concerning the molecular surface areas involved in the recognition of cytochrome c (the common acceptor) and of the respective reductase (flavo- or molybdoprotein).In addition, our results suggest that at some point in evolution, several copies of an initial hemoprotein gene were formed in the cellular genome. Subsequently, one copy was fused with the gene for another function: a flavoreductase in yeast cells or a molybdoreductase in hepatic cells.     

The use of a water-soluble carbodiimide to cross-link cytochrome c to plastocyanin

A water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, has been used to cross-link horse heart cytochrome c to spinach chloroplast plastocyanin. The complex was formed in yields up to 90%, and was found to have a stoichiometry of 1 mol plastocyanin per mol cytochrome c. The cytochrome c in the complex was fully reducible by ascorbate and potassium ferrocyanide, and had a redox potential only 25 mV less than that of native cytochrome c. The complex was nearly completely inactive towards succinate-cytochrome c reductase and cytochrome c oxidase, suggesting that the heme crevice region of cytochrome c was blocked. We propose that the carbodiimide promoted the formation of amide cross-links between lysine amino groups surrounding the heme crevice of cytochrome c and complementary carboxyl groups on plastocyanin. It is of interest that the high-affinity site for cytochrome c binding on bovine heart cytochrome c oxidase has recently been found to involve a sequence of subunit II with some homology to the copper-binding sequence of plastocyanin.     

Purification and some properties of cytochrome C553(550) isolated from Desulfovibrio desulfuricans Norway.

A new c-type cytochrome containing a single heme group, cytochrome c_553(550) has been purified from $\text{Desulfovibrio desulfuricans}$ (Norway strain) and some of its properties have been investigated. It has an isoelectric point of 6.6 and a higher redox potential than cytochrome c₃ isolated from the same bacteria. Its molecular weight was estimated to be 9,200 by gel filtration. The main absorption peaks are at 553, 522.5 and 417 nm in the reduced form and at 690, 529, 411, 357 and 280 nm in the oxidized form. The asymmetric α band of the reduced state is similar to the one reported for socalled “split α” cytochromes c. The cytochrome contains 86 amino acid residues with 5 methionine, two cysteine and two histidine residues. The N terminal sequence of $\text{D. desulfuricans}$ Norway cytochrome c_553(550) presents no evident homology with that of $\text{Desulfovibrio vulgaris}$ Hildenborough cytochrome c₅₅₃.