The conformation of eukaryotic cytochrome c around residues 39, 57, 59 and 74.

1H-n.m.r. studies of horse, tuna, Candida krusei and Saccharomyces cerevisiae cytochromes c showed that each of the proteins contains a similar cluster of residues at the bottom of the protein that assists in shielding the haem from the solvent. The relative positions of the residues forming these clusters vary continuously with temperature, and they change with the change in protein redox state. This conformational heterogeneity is discussed with reference to the conformational flexibility of cytochrome c around residues 57, 59 and 74. Spectroscopic measurements of pKa values for Lys-55 (horse and tuna cytochromes c) and His-33 and His-39 (C. krusei and S. cerevisiae cytochromes c) are in excellent agreement with expectations based on chemical-modification studies of horse cytochrome c. [Bosshard & Zürrer (1980) J. Biol. Chem. 255, 6694-6699] and on the X-ray-crystallographic structure of tuna cytochrome c [Takano & Dickerson (1981) J. Mol. Biol. 153, 79-94, 95-115].     

Structural studies of eukaryotic cytochrome c modified at methionine-65.

1H n.m.r. spectra were recorded in both oxidation states for the following species: tuna cytochrome c, tuna [carboxymethylmethionine-65]cytochrome c, horse cytochrome c and horse [homoserine-65]cytochrome c. The experiments give the assignments of the singlet methyl resonances of methionine-65 and the N-terminal acetyl group. The modification at methionine-65 is shown to cause an extremely small structural perturbation to one part of the molecule close to the site of modification.     

13C-NMR spectroscopy of acetyltyrosyl-guanidinated horse heart cytochrome c

The tyrosine residues of guanidinated horse heart cytochrome c have been specifically acetylated by reaction with N-[1-13C]acetylimidazole (90 atom%). Acetylation was monitored by 13C-NMR spectroscopy. The tyrosine residues were found to show widely varying reactivities ranging from one that is completely and exclusively acetylated at low reagent concentration (residue 67) to one that is acetylated only when the protein is unfolded (residue 97). Homogeneous derivatives were prepared containing one (either residue 67 or 97), three 48, 67 and 74), or four (residues 48, 67, 74 and 97) O-[1-13C]acetyl groups. 13C-NMR spectra of selected derivatives were obtained at pH 5.8, in the presence of cyanide ion, in the ferrous and ferric oxidation states, and after denaturation with 6M guanidine hydrochloride. The O-[1-13C]acetyltyrosyl resonances gave chemical shift values ranging from 171.8 to 176.0 ppm. These resonances were assigned to specific groups based on the known order of reactivity of the tyrosyl side chains toward N-acetylimidazole. The chemical shift of O-[1-13C]acetyltyrosyl 67 was found to be particularly sensitive to changes in protein structure. The proximity of this group to the heme makes it subject to distance-dependent paramagnetic and ring current effects. Acetylation of tyrosyl 74 gives rise to a pH-dependent equilibrium between conformers in the ferric state and a conformation change in the ferrous state. Acetylation of this residue also leads to an absorbance decrease at 695 nm that can be related to the 13C-NMR-detected conformational equilibrium. Addition of cyanide ion abolished this equilibrium.     

Semi-synthetic analogs of cytochrome C at positions 67 and 74.

New semi-synthetic peptide analogs of horse heart cytochrome $\text{c}$ have been prepared by replacing tyrosine at position 67 by phenylalanine and L-parafluorophenylalanine. The former has 56% biological activity in the succinate oxidase system whereas the parafluorophenylalanine analog is totally inactive. The tyrosine at position 74 has also been replaced by leucine. It shows a high level of biological activity (58%), which demonstrates that the presence of an aromatic residue at this position is not required for electron transfer.     

The semisynthesis of fragments corresponding to residues 66-104 of horse heart cytochrome c.

We describe the N epsilon-acetimidylation of horse heart cytochrome c with retention of biological activity, the cleavage of the modified protein by CNBr, the separation of the fragments, and their further side-chain protection. We describe the manipulation of the amino acid sequences of the fragments by stepwise semisynthetic methods. We have prepared fragments corresponding to residues 66-78 and 66-79 of the protein, as well as the [Asp66] analogue of fragment 66-79. We have prepared the natural sequence and the [o-fluoro-Phe82] analogue of the fragment corresponding to residues 81-104 of the protein, and the [N epsilon-trifluoroacetyl-Lys79], the [N epsilon-dinitrophenyl-Lys79] and the [S-acetamidomethyl-Cys79] analogues of fragment 79-104, and the [N epsilon-Cbz-Lys81] analogue of fragment 80-104. We have coupled back the fragments of natural sequence to form a semisynthetic fragment corresponding to residues 66-104 of the protein. Modified fragments were also coupled to give analogues of the 66-104-residue sequence. In every case the homoserine residue representing methionine-80 was removed from the C-terminus of the 66-80-residue fragment and replaced by methionine on the N-terminus of the 81-104 residue fragment during the preparation of the fragments for coupling. The semisynthetic fragments are ready for specific deprotection and further coupling. We have coupled one such fragment to the (1-65)-peptide to produce semisynthetic [Hse65]cytochrome c. The product has satisfactory characteristics on chemical analysis, and on assay of its biological activity.     

Time course and site(s) of cytochrome c tyrosine nitration by peroxynitrite

Cytochrome c-dependent electron transfer and apoptosome activation require protein-protein binding, which are mainly directed by conformational and specific electrostatic interactions. Cytochrome c contains four highly conserved tyrosine residues, one internal (Tyr67), one intermediate (Tyr48), and two more accessible to the solvent (Tyr74 and Tyr97). Tyrosine nitration by biologically-relevant intermediates could influence cytochrome c structure and function. Herein, we analyzed the time course and site(s) of tyrosine nitration in horse cytochrome c by fluxes of peroxynitrite. Also, a method of purifying each (nitrated) cytochrome c product by cation-exchange HPLC was developed. A flux of peroxynitrite caused the time-dependent formation of different nitrated species, all less positively charged than the native form. At low accumulated doses of peroxynitrite, the main products were two mononitrated cytochrome c species at Tyr97 and Tyr74, as shown by peptide mapping and mass spectrometry analysis. At higher doses, all tyrosine residues in cytochrome c were nitrated, including dinitrated (i.e., Tyr97 and Tyr67 or Tyr74 and Tyr67) and trinitrated (i.e., Tyr97, Tyr74, and Tyr67) forms of the protein, with Tyr67 well represented in dinitrated species and Tyr48 being the least prone to nitration. All mono-, di-, and trinitrated cytochrome c species displayed an increased peroxidase activity. Nitrated cytochrome c in Tyr74 and Tyr67, and to a lesser extent in Tyr97, was unable to restore the respiratory function of cytochrome c-depleted mitochondria. The nitration pattern of cytochrome c in the presence of tetranitromethane (TNM) was comparable to that obtained with peroxynitrite, but with an increased relative nitration yield at Tyr67. The use of purified and well-characterized mono- and dinitrated cytochrome c species allows us to study the influence of nitration of specific tyrosines in cytochrome c functions. Moreover, identification of cytochrome c nitration sites in vivo may assist in unraveling the chemical nature of proximal reactive nitrogen species.     

The binding of platinum complexes to tuna cytochrome c.

The binding of [PtCl4]2- and cis-[PtCl2(NH3)2] to methionine-65 of tuna cytochrome c was investigated by 1H n.m.r. The modification at methionine-65 is shown to cause an extremely small structural perturbation to the protein at the site of modification.     

Solid-phase synthesis of the native sequence of horse heart cytochrome c-(66-104)-nonatriacontapeptide and of a C-terminal carboxyamide analog selectively modified at Met-80

The peptide corresponding to the (66-104) sequence of horse heart cytochrome c and its carboxyamide analog, selectively modified at the critical Met80 residue, have been synthesized by stepwise solid-phase methods on PAM and BHA resins respectively. The correctness of the growing peptide chain as well as the homogeneity of the final products have been monitored by several analytical methods including quantitative Edman degradation. After HF cleavage both peptides were purified by semipreparative HPLC. The overall yields were 24% for the native (66-104) and 10% for the carboxyamide analog. The homogeneity of the purified synthetic peptides have been determined by different criteria including HPLC, amino acid composition, Edman degradation, electrophoresis, and tryptic peptide mapping. The synthetic fragments have been utilized for preliminary semisynthesis experiments with the native [Hse greater than 65] (1-65)H heme-sequence.     

Complexation which facilitates rejoining of horse cytochrome c apofragment [Homoser-lactone65](1-65) or [Homoser-lactone65] (23-65) to apofragment (66-104).

We have shown that two CNBr fragments of horse apocytochrome c, [Homoser-lactone65](1-65) and (66-104), bind to the ferric heme fragment (1-25)H to form a non-productive three-fragment complex, and that when the heme of this complex has been kept reduced for 48 h at 25 degrees, the peptide bond between residues 65 and 66 is restored with a yield of 24% or more. We have also shown that another CNBr fragment [Homoser-lactone65](23-65), but not [Homoser-lactone65](39-65), similarly rejoins to fragment (66-104) in the presence of the ferrous heme fragment with 25% or more yield. For complex of ferro-heme fragment [Hse-lacton65](1-65)H and apofragment (66-104) of horse cytochrome c, which restores the peptide bond between residues 65 and 66 (located on the left side of the heme) (cf. Harbury, H.A. (1978) in Semisynthetic Peptides and Proteins (Offord, R.E. & DiBello, C., eds.), pp. 73-89, Academic Press, New York). Corradin & Harbury have suggested that axial ligation of methionine 80 to the heme (on the left side) is important. Consistent with their idea, fragment [Hse80](66-104) was found not to link to [Hse-lactone65](1-65) in the presence of ferro(1-25)H. Furthermore, the present studies indicate that the interaction involving residues 26 to 38 (on the right side) is also important for such a conformation which assists in the rejoining of the two apofragments. Combining these two ideas, we suggest that restoration of the peptide bond between residues 65 and 66 reflects the structural integrity of these complexes in the reduced form. Thus, the present reaction system can be used not only for chemical synthesis of [Homoser65] apocytochrome c but also to extend amino acid substitution studies of cytochrome c to residues 1 to 64.     

Ultraviolet resonance Raman spectra of cytochrome c conformational states

Ultraviolet resonance Raman (UV RR) spectra are reported for ferricytochrome c from tuna and horse heart at pH 1.6, 7, 10, and 13, representing distinct conformational states of the protein (states II, III, IV, and V, respectively). The spectra were obtained with pulsed laser excitation at 200 and 218 nm, via H2 Raman shifting the fourth harmonic output of a pulsed YAG laser. At these deep UV wavelengths, strong enhancement is observed for vibrational modes associated with tryptophan, tyrosine, and phenylalanine side chains and with the amide groups of the polypeptide backbone. The amide I peak frequency is consistent with a dominant contribution from alpha-helical regions, although a broad high-frequency tail reflects a variety of unordered conformations. The peak frequency is 12 cm-1 higher for cytochrome c from tuna than from horse, suggesting a less tightly wound structure, which is consistent with the lower denaturation temperature previously reported for the tuna protein. The amide I peak broadens when native protein (state III) is converted to the low- or high-pH forms (states II and IV), reflecting some disordering of the polypeptide chain, but the peak frequencies are unshifted, establishing that the alpha-helical segments are not completely unfolded in these states. Raising the pH to 13 (state V), however, does produce a frequency upshift, reflecting helix unfolding. The amide II and III frequencies are likewise consistent with a dominant alpha-helix contribution in the native proteins; they gain intensity, and amide III is shifted to a lower frequency, in states II and IV, consistent with partial disordering.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nitration of solvent-exposed tyrosine 74 on cytochrome c triggers heme iron-methionine 80 bond disruption. Nuclear magnetic resonance and optical spectroscopy studies

Cytochrome c, a mitochondrial electron transfer protein containing a hexacoordinated heme, is involved in other physiologically relevant events, such as the triggering of apoptosis, and the activation of a peroxidatic activity. The latter occurs secondary to interactions with cardiolipin and/or post-translational modifications, including tyrosine nitration by peroxynitrite and other nitric oxide-derived oxidants. The gain of peroxidatic activity in nitrated cytochrome c has been related to a heme site transition in the physiological pH region, which normally occurs at alkaline pH in the native protein. Herein, we report a spectroscopic characterization of two nitrated variants of horse heart cytochrome c by using optical spectroscopy studies and NMR. Highly pure nitrated cytochrome c species modified at solvent-exposed Tyr-74 or Tyr-97 were generated after treatment with a flux of peroxynitrite, separated, purified by preparative high pressure liquid chromatography, and characterized by mass spectrometry-based peptide mapping. It is shown that nitration of Tyr-74 elicits an early alkaline transition with a pKa = 7.2, resulting in the displacement of the sixth and axial iron ligand Met-80 and replacement by a weaker Lys ligand to yield an alternative low spin conformation. Based on the study of site-specific Tyr to Phe mutants in the four conserved Tyr residues, we also show that this transition is not due to deprotonation of nitro-Tyr-74, but instead we propose a destabilizing steric effect of the nitro group in the mobile Omega-loop of cytochrome c, which is transmitted to the iron center via the nearby Tyr-67. The key role of Tyr-67 in promoting the transition through interactions with Met-80 was further substantiated in the Y67F mutant. These results therefore provide new insights into how a remote post-translational modification in cytochrome c such as tyrosine nitration triggers profound structural changes in the heme ligation and microenvironment and impacts in protein function.     

Structure-function relationships in the ribosomal protein L12 in the archaeon Sulfolobus acidocaldarius.

A series of mutant L12 ribosomal proteins was prepared by site-directed mutations in the L12 protein gene of the archaeon Sulfolobus acidocaldarius. The mutant protein genes were overexpressed in Escherichia coli, and the products purified and incorporated into ribosomal cores which had been ethanol extracted to remove wild-type L12 protein. Measurements were made to determine if the mutation affected the binding of the L12 protein to the ribosome core or affected the translational activity of the resulting ribosome. Changing tyrosine [3] or tyrosine [5], conserved in all archaea and present in all eukarya in positions [3] and [7], to phenylalanine had no effect on binding or translational activity while changes to glycine significantly reduced binding and translational activity. Changing the single arginine [37] residue, conserved in almost all archaeal and eukaryal L12 proteins, to lysine, glutamic acid, glutamine, or glycine had no effect on binding to the core and had little or no significant effect on translational activity. The same was true when lysine [39], conserved in all archaeal L12 proteins, was changed to arginine, glutamic acid, glutamine, or glycine. Changing phenylalanine [104], the penultimate amino acid at the C-terminal end, which is conserved in all archaeal and eukaryal L12 proteins, to tyrosine or glycine had no effect on binding but lowered the translational activity by 60 and 75%, respectively, suggesting that this amino acid plays an important role in translation. Deletion of the highly charged region in the C-terminal domain, which is present in all archaeal and eukaryal L12 proteins, decreased transitional activity by 50%, suggesting this region is also involved in factor interactions.     

Mutants of human insulin-like growth factor II: expression and characterization of analogs with a substitution of TYR27 and/or a deletion of residues 62-67.

Five structural analogs of human insulin-like growth factor II (IGF II), [Leu27]IGF II, [Glu27]IGF II, des(62-67)IGF II, des(62-67)[Leu27]IGF II and des(62-67)[Glu27]IGF II were constructed by site-directed mutagenesis and expressed as protein A fusion proteins in E. coli BL21 pLysS cells, cleaved with CNBr and purified by affinity chromatography and HPLC. These mutants were tested for their binding affinities to type 1 and type 2 IGF receptors, to IGF binding protein-3 (IGFBP-3) and for their stimulation of thymidine incorporation into DNA. [Leu27]IGF II exhibits an affinity to the type 2 IGF receptor close to that of wild-type IGF II, but has lost completely the affinity to the type 1 IGF receptor. The results further suggest that the D domain, which is close to Tyr27, forms part of the binding region for the type 1 IGF receptor.     

Implication of Ca2+-Dependent Protein Tyrosine Phosphorylation in Carbachol-Induced Phospholipase D Activation in Rat Pheochromocytoma PC12 Cells

Abstract: The mechanism for carbachol (CCh)-induced phospholipase D (PLD) activation was investigated in [³H]palmitic acid-labeled pheochromocytoma PC12 cells with respect to the involvement of protein tyrosine phosphorylation and Ca²⁺. PLD activity was assessed by measuring the formation of [³H]phosphatidylbutanol in the presence of 0.3% butanol. Pretreatment of cells with the tyrosine kinase inhibitors herbimycin A, genistein, and tyrphostin inhibited PLD activation by CCh. Western blot analysis revealed several apparent tyrosine-phosphorylated protein bands (111, 91, 84, 74, 65–70, 44, and 42 kDa) in PC12 cells treated with CCh. Phosphorylation of the 111-, 91-, 84-, and 65–70-kDa proteins peaked within 1 min, and their time-dependent changes seemingly correlated with that of PLD activation. Others (74, 44^MAPK, and 42^MAPK kDa) were phosphorylated rather slowly, and maximal tyrosine phosphorylation was observed at 2 min. Herbimycin A inhibited PLD activity and tyrosine phosphorylation of four proteins (111, 91, 84, and 65–70 kDa) in a preincubation time- and concentration-dependent fashion. In Ca²⁺-free buffer, CCh-induced [³H]phosphatidylbutanol formation and protein tyrosine phosphorylation were abolished. A Ca²⁺ ionophore, A23187, caused PLD activation and tyrosine phosphorylation of four proteins of 111, 91, 84, and 65–70 kDa only in the presence of extracellular Ca²⁺. Extracellular Ca²⁺ dependency for CCh-induced PLD activation was well correlated with that for tyrosine phosphorylation of the four proteins listed above, especially the 111-kDa protein. These results suggest that Ca²⁺-dependent protein tyrosine phosphorylation is closely implicated in CCh-induced PLD activation in PC12 cells.     

Inhibition of the cytochrome c/cytochrome c oxidase system by cytochrome c derivatives and related fragments

The oxidation of ferrocytochrome c mediated by cytochrome c oxidase was investigated in the presence of ferricytochrome c, trifluoroacetyl-cytochrome c, the heme fragments Hse65-[1-65] and Hse80-[1-80] and their respective porphyrin derivatives, as well as carboxymethylated apoprotein and related fragments, polycations, salts and neutral additives. The inhibition of the redox reaction by salts and neutral molecules, even if in theoretical agreement with their effect on electrostatic interactions, may alternatively be interpreted in terms of hydrophobicity. The latter can account for the inhibitory properties of trifluoroacetylated ferricytochrome c, similar to those of ferricytochrome c. On the assumption that the inhibitory properties of some of the investigated derivatives monitor their binding affinities to the cytochrome c oxidase at the cytochrome c binding sites, the experimental results do not confirm a primarily electrostatic character for the cytochrome c/cytochrome c oxidase association process. Strong indication was found that the cytochrome c C-terminal sequence is critically involved in the complex formation. Conformational studies by circular dichroism measurements and IR spectroscopy in solution and in solid state respectively, show that some of the derivatives examined may possibly acqkuire in the binding process to the oxidase, as secondary structure similar to that present in the native cytochrome c.     

Effect of aromatic acids on protein synthesis in subcellular preparations from the rat brain.

The incorporation of [3H]phenylalanine, [3H]tyrosine, and [3H]tryptophan into protein and amino acyl-tRNA was studied in cell-free preparations from rat brain. Tyrosine and tryptophan inhibited the incorporation of phenylalanine into protein, and tyrosine inhibited the incorporation of phenylalanine and tryptophan into amino acyl-tRNAs. In most cases, homogentisate, phenylpyruvate, and phenyllactate inhibited the incorporation of phenylalanine, tyrosine, and tryptophan into protein and amino acyl-tRNAs, and the incorporation of phenylalanine into polyphenylalanine. All other protein amino acids, and phenylacetate, salicylate, and benzoate were wholly ineffectual. The results suggest that the formation of amino acyl-tRNAs may have been the step which was affected most by the inhibitors. The incorporation data at different concentrations of the aromatic amino acids were fitted to the simple Michaelis equation. Homogentisate and phenylpyruvate generally tended to reduce both Km and V in the incorporation of aromatic amino acids into protein and amino acyl-tRNAs, even if V decreased more than Km.     

Circular-dichroic spectra of vasopressin analogues and their cyclic fragments.

The circular dichroic spectra of [Arg8]vasopressin, [Mpr1, Arg8]vasopressin, [Mpr1, D-Arg8]-vasopressin, pressinamide, deaminopressinamide, tocinamide, deaminotocinamide, [Leu4, D-Arg8]-vasotocin, [Mpr1, Leu4, D-Arg8]vasotocin and [Phe2, Lys8]vasopressin have been studied. All these substances showed a characteristic positive dichroic band at about 225 nm due to the presence of tyrosine in sequence position 2. The intensity of this band was affected by interactions between the tyrosine side-chain and other structural elements in the molecule, such as the Na-amino group, the side-chain of phenylalanine in position 3 and the linear C-terminal peptide. Analysis of the response of this band to structural modifications of the molecule and change in the solvent (particularly comparing neutral aqueous solutions with hexafluoroacetone solutions) allowed some conformational conclusions. The linear C-terminal tripeptide is probably situated over the cyclic portion of the molecule both in vasopressin and oxytocin substances. Its steric interaction with the tyrosine side-chain seems to be particularly efficient in molecules containing D-arginine in position 8. In the vasopressin series the stacking interaction of neighbouring aromatic amino acid residues furthermore limits the conformational freedom of the tyrosine side-chain and also probably distorts the dihedral angles of residues 1-3 in comparison with oxytocin. The interactions of phenylalanine and arginine with tyrosine relatively decrease the conformational effects of the primary amino group. Consequently the local conformation of vasopressin in the region of the tyrosine residue is more rigid and less sensitive to changes in medium than that of oxytocin. The circular dichroic spectra did not show any basic conformational differences in the backbone peptide chain of oxytocin and vasopressin substances. A weak negative disulphide band at about 290 nm could be observed in the spectra of both series of substances.     

In vivo regulation of phenylalanine hydroxylation to tyrosine, studied using enrichment in apoB-100

Phenylalanine hydroxylation is necessary for the conversion of phenylalanine to tyrosine and disposal of excess phenylalanine. Studies of in vivo regulation of phenylalanine hydroxylation suffer from the lack of a method to determine intrahepatocyte enrichment of phenylalanine and tyrosine. apoB-100, a hepatic export protein, is synthesized from intrahepatocyte amino acids. We designed an in vivo multi-isotope study, [(15)N]phenylalanine and [2H2]tyrosine to determine rates of phenylalanine hydroxylation from plasma enrichments in free amino acids and apoB-100. For independent verification of apoB-100 as a reflection of enrichment in the intrahepatocyte pool, [1-(13)C]lysine was used as an indicator amino acid (IAA) to measure in vivo changes in protein synthesis in response to tyrosine supplementation. Adult men (n = 6) were fed an amino acid-based diet with low phenylalanine (9 mg.kg(-1).day(-1), 4.54 mumol.kg(-1).,h(-1)) and seven graded intakes of tyrosine from 2.5 (deficient) to 12.5 (excess) mg.kg(-1).day(-1). Gas chromatography-quadrupole mass spectrometry did not detect any tracer in apoB-100 tyrosine. A new and more sensitive method to measure label enrichment in proteins using isotope ratio mass spectrometry demonstrated that phenylalanine hydroxylation measured in apoB-100 decreased linearly in response to increasing tyrosine intake and reached a break point at 6.8 mg.kg(-1).day(-1). IAA oxidation decreased with increased tyrosine intake and reached a break point at 6.0 mg.kg(-1).day(-1). We conclude: apoB-100 is an accurate and useful measure of changes in phenylalanine hydroxylation; the synthesis of tyrosine via phenylalanine hydroxylation is regulated to meet the needs for protein synthesis; and that plasma phenylalanine does not reflect changes in protein synthesis.     

Probing stability and dynamics of proteins by protease digestion. II: Identification of the initial chymotryptic cleavage sites of homologous cytochromes c

Three homologous cytochromes c from horse, rabbit and tuna were subjected to chymotryptic digestion and their initial cleavage sites were identified. The sites in oxidized cytochromes c are the COOH-terminal sides of Tyr-48, Phe-46 and Tyr-46 for horse, rabbit and tuna cytochromes c, respectively. The results show that the chymotrypsin attacks a single site in each protein; the sites are located at the almost identical position on the polypeptide chain. Through the time-course studies of digestion, it was found that the three cytochromes c have different chymotrypsin-susceptibility at the initial cleavage site in the order of horse less than rabbit less than tuna. Studies on chymotryptic digestion of tuna cytochrome c in the reduced form revealed that the haem-reduction does not alter the initial cleavage site but increases the resistance to the proteolysis at the site. The uniqueness of the initial cleavage site in each cytochrome c species suggests that the protease susceptibility reflects some overall properties of the protein. At the same time, it was clarified that the initial cleavage site is also affected by a neighboring region by the fact that another potential cleavage site is located near the site in question. In order to elucidate the initial cleavage site, several physical properties of tuna cytochrome c molecule deduced from the X-ray 3D structure, accessible surface area, temperature factor, effective hydrophobicity and electrostatic potential, were compared with the experimental results and it was concluded that these properties given by a residue have no direct relationship with the chymotrypsin susceptibility.     

Chemical modification of the haem propionate of cytochrome c. A re-evaluation of the reaction of cytochrome c with a water-soluble carbodi-imide.

Horse heart and tuna heart cytochromes c were treated with the water-soluble carbodi-imide 1-(3-dimethylaminopropyl)-3-ethylcarbodi-imide. When the reaction is followed spectroscopically two kinetic phases are apparent. Alteration of the reactivity of the proteins with such ligands as CO, however, occurs in a single phase identical with the faster phase detected spectroscopically. The modified proteins both show spectroscopic and redox properties identical with those described for the tuna heart cytochrome c derivative by Timkovich [Biochem. J. (1980) 185, 47-57]. The use of radiolabelled carbodi-imide identifies two or three sites of reactivity. However, the addition of glycine methyl ester to the reaction mixture leads to the addition of nine glycine moieties in the case of the horse protein and seven in the case of the tuna protein, indicating a larger number of reactive sites than previously reported. A further set of reaction sites was identified by peptide mapping of the modified proteins, and these sites take part in intramolecular reactions leading to internal cross-linking and the formation of an enzymically indigestible 'core particle'. The haem group was identified as a site of reaction with the carbodi-imide, and is as a consequence covalently linked to the peptide by a bond in addition to the thioether bonds normally present. In the light of these findings, the alterations in the properties of the tuna protein, subsequent to reaction with the carbodi-imide, which have been previously explained in structural terms, must be re-evaluated. This study also highlights the importance of internal cross-link formation, which can occur by intramolecular nucleophilic attack, a process that has often been overlooked by investigators employing carbodi-imide modification of carboxylate groups in proteins.