A tale of two charges: distinct roles for an acidic and a basic amino acid in the structure and function of cytochrome c.

Cytochrome c is a small electron transport protein found in the intermembrane space of mitochondria. As it interacts with a number of different physiological partners in a specific fashion, its structure varies little over eukaryotic evolutionary history. Two highly conserved residues found within its sequence are those at positions 13 and 90 (numbering is based on the standard horse cytochrome c); with single exceptions, residue 13 is either Lys or Arg, and residue 90 is either Glu or Asp. There have been conflicting views on the roles to be ascribed to these residues, particularly residue 13, so the functional properties of a number of site-directed mutants of Saccaromyces cerevisiae iso-1 cytochrome c have been examined. Results indicate that the two residues do not interact specifically with each other; however, residue 13 (Arg) is likely to be involved in interactions between cytochrome c and other electrostatically oriented physiological partners (intermolecular), whereas residue 90 (Asp) is involved in maintaining the intrinsic structure and stability of cytochrome c (intramolecular). This is supported by molecular dynamics simulations carried out for these mutants where removal of the negative charge at position 90 leads to significant shifts in the conformations of neighboring residues, particularly lysine 86. Both charged residues appear to exert their effects through electrostatics; however, biological activity is significantly more sensitive to substitutions of residue 13 than of residue 90.     

Protein engineering of cytochrome c by semisynthesis: substitutions at glutamic acid 66

We have used protein semisynthesis to prepare four analogues of horse cytochrome c, in which the glutamic acid residue at position 66 has been removed and replaced by norvaline, glutamine, lysine and, as a methodological control, glutamic acid. This residue is quite strongly conserved in mitochondrial cytochrome c, and forms part of a cluster of acidic residues that occurs in all cytochromes c but whose function is obscure. Comparative studies of the physical and biochemical properties of the analogues have now disclosed two specific roles for Glu66 in the protein. It contributes significantly to the stabilization of the active conformation of the protein, probably by salt bridge formation, and it appears to participate in the redox-state-dependent ATP-binding site of cytochrome c. Our results also support two general views of the role of surface charged residues in cytochrome c, namely that their disposition influences both redox potential, through the electrostatic field felt at the redox centre, and the kinetics of electron transfer, through the dipole moment they generate.     

Cytochrome c and cytochrome c oxidase interactions: the effects of ionic strength and hydrostatic pressure studied with site-specific modifications of cytochrome c.

Seven cytochromes c, in which individual lysines have been modified to the propylthiobimane derivatives, have been prepared. These derivatives were also converted to the porphyrin cytochromes c by treatment with HF. The properties of both types of modified proteins were studied in their reactions with cytochrome c oxidase. The results show that lysines 25, 27, 60, 72, and 87 do not contribute a full charge to the binding interaction with the oxidase. These five residues, with the exception of the lysine-60 derivative, on the front surface of the protein and contain the solvent-accessible edge of the heme prosthetic group. By contrast, lysines 8 and 13 at the top of the front surface do contribute a full charge to the binding interaction with the oxidase. The removal of the positive charge on any one lysine weakens the binding to cytochrome c oxidase by at least 1 kcal (1 cal = 4.1868 J). The presence of bimane at lysines 13 and 87 clearly forces the separation of the cytochrome c and oxidase, but this does not occur with the other complexes. The bimane-modified lysine-13 protein, and to a lesser extent that modified at lysine 8, show the interesting effect of enhanced complex formation with cytochrome c oxidase when subjected to pressure, possibly because of entrapment of water at the newly created interface of the complex. Our observations indicate that the two proteins of the cytochrome c - cytochrome oxidase complex have preferred, but not obligatory, spatial orientations and that interaction occurs without either protein losing significant portions of its hydration shell.     

Role of specific lysine residues in binding cytochrome c2 to the Rhodobacter sphaeroides reaction center in optimal orientation for rapid electron transfer

The role of specific lysine residues in facilitating electron transfer from Rhodobacter sphaeroides cytochrome c2 to the Rb. sphaeroides reaction center was studied by using six cytochrome c2 derivatives each labeled at a single lysine residue with a carboxydinitrophenyl group. The reaction of native cytochrome c2 at low ionic strength has a fast phase with a half-time of 0.6 microseconds that has been assigned to the reaction of bound cytochrome c2 [Overfield, R.E., Wraight, C.A., & DeVault, D. (1979) FEBS Lett. 105, 137]. Modification of lysine-55 did not affect the half-time of this phase but decreased the apparent binding constant by a factor of 2. The derivatives modified at lysines-10, -88, -95, -97, -99, -105, and -106 surrounding the heme crevice did not show any detectable fast phase but only slow second-order phases due to the reaction of solution cytochrome c2. These lysines thus appear to be involved in binding cytochrome c2 to the reaction center in an optimal orientation for electron transfer. The involvement of lysines-95 and -97 is especially significant, since they are located in an extra loop comprising residues 89-98 that is not present in eukaryotic cytochrome c. The reactions of horse cytochrome c derivatives modified at single lysine amino groups with trifluoroacetyl or [(trifluoromethyl)phenyl]carbamoyl were also studied. The derivatives modified at lysines-22, -55, -88, and -99 far removed from the heme crevice had nearly the same half-times for the fast phase as native cytochrome c, 6 microseconds.(ABSTRACT TRUNCATED AT 250 WORDS)     

Definition of cytochrome c binding domains by chemical modification. II. Identification and properties of singly substituted carboxydinitrophenyl cytochromes c at lysines 8, 13, 22, 27, 39, 60, 72, 87, and 99.

Sensitive thin layer peptide mapping is employed to establish the identity and the homogeneity of eight singly substituted 4-carboxy-2,6-dinitrophenyl derivatives of horse cytochrome c. Seven of the components, all of greater than 95% homogeneity, are modified at lysyl residues 13, 72, 87, 8, 27, 39, and 60. The eighth component is a mixture of derivatives at lysines 22 and 99. The positions of the modified residues were confirmed by the amino acid analysis and Edman sequential degradation of the CDNP-peptides. Physiochemical properties characteristic of cytochrome c are unchanged in the chemically modified products examined. These properties, that include the proton NMR spectrum, are sensitive probes of the polypeptide organization surrounding the heme prosthetic group. The lack of any discernable changes indicates that modification of the epsilon-amino groups on the surface of cytochrome c does not perturb the overall structure of the protein. The widespread distribution of the modifications on the surface of the molecule, together with the homogeneity and native conformation of the CDNP-derivatives make them well suited for assessing the effects of changes in the charge topography on the electron transfer activity of cytochrome c.     

Use of specific lysine modifications to locate the reaction site of cytochrome c with cytochrome oxidase.

The reaction of cytochrome c with trifluoromethylphenyl isocyanate was carried out under conditions which led to the modification of a small number of the 19 lysines. Extensive ion-exchange chromatography was used to separate and purify six different derivatives, each modified at a single lysine residue, lysines 8, 13, 27, 72, 79, and 100, respectively. The only modifications which affected the activity of cytochrome c with cytochrome oxidase (EC 1.9.3.1) were those of lysines immediately surrounding the heme crevice, lysines 13, 27, 72, and 79, and also lysine 8 at the top of the heme crevice. In each case, the modified cytochrome c had the same maximum velocity as that of native cytochrome c, but an increased Michaelis constant for high affinity phase of the reaction. This supports the hypothesis that the cytochrome oxidase reaction site is located in the heme crevice region, and the highly conserved lysine residues surrounding the heme crevice are important in the binding.     

Synthesis and characterization of singly modified (carboxyferrocenyl)cytochrome c derivatives.

Carbodiimide-activated coupling chemistry has been used to covalently attach 1,1'-dicarboxyferrocene (dcFc) to the epsilon-amine of surface lysine residues of horse heart cytochrome c. Conditions have been found that optimize the production of singly modified (dcFc)cytochrome c derivatives and the presence of one free carboxylate per modification site allows separation and purification of about 10 of these derivatives by cation-exchange chromatography. Reversed-phase HPLC tryptic peptide mapping techniques have been used to identify the attachment sites of eight pure (dcFc)cytochrome c derivatives (at lysines 7, 8, 13, 25, 60, 72, 73, and 100). Through-space distances from these lysines to the nearest heme edge span the 6-16 A range and these derivatives should prove useful in exploring the distance dependence of long-range intramolecular electron transfer in cytochrome c.     

Binding of arylazidocytochrome c to yeast cytochrome c peroxidase

Cytochrome c derivatives modified with a photoactivatable arylazido group in selected lysine residues were irradiated in the presence of cytochrome c peroxidase (EC 1.11.1.5). A derivative modified at lysine 13 was able to cross-link to the enzyme and inhibit electron transfer activity. Complete inhibition of cytochrome c peroxidase activity was obtained when 1 mol of cytochrome c was covalently bound per mol of cytochrome c peroxidase. Chemical cleavage of the covalent complex has been used for a preliminary characterization of the site of cross-linking of cytochrome c to cytochrome c peroxidase. This linkage site was localized to the NH2 terminal part of cytochrome c peroxidase including residues 1-51.     

The cytochrome c oxidase-cytochrome c complex: spectroscopic analysis of conformational changes in the protein-protein interaction domain

Binding to cytochrome c oxidase induces a conformational change in the cytochrome c molecule. This conformational change has been characterized by comparing the binding of native cytochrome c and chemically modified cytochrome c derivatives to bovine cytochrome c oxidase by using absorption, circular dichroism (CD), and magnetic circular dichroism (MCD) spectroscopy. The following derivatives were analyzed: (i) cytochrome c modified at all 19 lysine residues to yield the (N epsilon-acetimidyl)19 cytochrome c, (N epsilon-isopropyl)19 cytochrome c, and (N epsilon,N epsilon-dimethyl)19 cytochrome c; (ii) cytochrome c in which Met65 and Met80 are converted to the methionine sulfoxide; (iii) cytochrome c with a single break in the polypeptide chain at Arg38 or Gly37. The derivatives bind to cytochrome c oxidase at a ratio of one heme c per heme aa3. The association constants are similar to that of native cytochrome c except for (N epsilon-isopropyl)19 and (N epsilon,N epsilon-dimethyl)19 cytochromes c, which bind respectively four times and six times less strongly. The derivatives are good substrates for the cytochrome c oxidase reaction. The spectral changes accompanying the binding of the modified cytochromes c to cytochrome c oxidase are quite different from the spectral changes observed with native cytochrome c. The different optical absorption and MCD changes are explained by a polarity change around the exposed heme edge in the cytochrome c-cytochrome c oxidase complex. The CD changes indicate a conformational rearrangement restricted to the surface area surrounding the exposed heme edge. The rearrangement may involve a movement of the evolutionarily conserved Phe82 out of the vicinity of the heme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Methionine sulfoxide cytochrome c.

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

Role of specific lysine residues in the reaction of Rhodobacter sphaeroides cytochrome c2 with the cytochrome bc1 complex

The reaction of Rhodobacter sphaeroides cytochrome c2 with the Rb. sphaeroides cytochrome bc1 complex was studied by using singly labeled cytochrome c2 derivatives. Cytochrome c2 was treated with chlorodinitrobenzoic acid to modify lysine amino groups to negatively charged carboxydinitrophenyllysines and separated into eight different fractions by ion-exchange chromatography on a Whatman SE 53 (sulfoxyethyl)cellulose column. Peptide mapping studies indicated that six of these fractions were modified at single lysine amino groups. Each of the derivatives had the same Vmax value as native cytochrome c2 in the steady-state reaction with the Rb. sphaeroides cytochrome bc1 complex. However, the Km values of the cytochrome c2 derivatives modified at lysines 10, 55, 95, 97, 99, and 106 were found to be larger than that of native cytochrome c2 by factors of 6, 2, 3, 32, 13, and 8, respectively. These results indicate that lysines located in the sequence 97-106 on the left side of the heme crevice have the greatest involvement in binding the cytochrome bc1 complex. The involvement of lysine 97 is especially significant because it is located in an extra loop comprising residues 89-98 that is not present in eukaryotic cytochrome c.     

Ionization of tyrosine and lysine residues in native and modified horse cytochrome c.

1H-n.m.r. and 13C-n.m.r. spectroscopy of horse cytochrome c and 1H-n.m.r. spectroscopy of the lysine-modified proteins N epsilon-acetimidyl-, N epsilon-amidino-, N epsilon-trifluoroacetyl- and N epsilon-maleyl-cytochrome c have shown that, although the lysine modifications do not greatly perturb the protein structure at pH7 and 27 degrees C, at higher temperature or at alkaline pH some parts of the structure are markedly perturbed. At pH7 and 27 degrees C the region of the protein about Ile-57 is affected in all the modified proteins, though not all to the same degree. N epsilon-Maleylation most seriously affects the protein structure, and the fully maleylated protein is readily unfolded. At 27 degrees C all four of the tyrosine residues of native horse cytochrome c have pKa values above 11, but in N epsilon-acetimidyl-cytochrome c the pKa of one tyrosine residue is 10.2.     

Interaction of cytochrome c with cytochrome c oxidase. Photoaffinity labeling of beef heart cytochrome c oxidase with arylazido-cytochrome c.

Cytochrome c derivatives labeled with a 3-nitrophenylazido group at lysine 13, at lysine 22, or at both residues have been prepared. The interaction of the cytochrome c derivatives with beef heart cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1) in the presence of ultrviolet light results in formation of a covalent complex between cytochrome c and the oxidase. Using the lysine 22 derivative, the polypeptide composition of the oxidase is not modified, nor is its catalytic activity, whereas with the lysine 13 derivative, the gel electrophoretic pattern is altered and the catalytic activity of the complex diminished. The data are consisten with a specfic covalent interaction of the lysine 13 derivative of cytochrome c with the polypeptide of molecular weight 23,700 (Subunit II) of cytochrome c oxidase.     

Soluble, prolonged-acting insulin derivatives. II. Degree of protraction and crystallizability of insulins substituted in positions A17, B8, B13, B27 and B30

It has previously been found that insulins, to which positive charge has been added by substitutions in position B30, thus raising the isoelectric point towards pH 7, had a prolonged action when injected as slightly acidic solutions because such derivatives crystallize very readily upon neutralization. Positive charge has now been added by substituting the B13 and A17 glutamic acid residues with glutamines and B27 threonine with lysine or arginine. These substitutions were introduced by site-specific mutagenesis in a gene coding for a single-chain insulin precursor. By tryptic transpeptidation the single-chain precursors were transformed to the double-chain insulin structure, concomitantly with incorporation of residue B30. Thus insulins combining B13 glutamine, A17 glutamine and B27 lysine or arginine with B30 threonine, threonine amide or lysine amide were synthesized. The time course of blood glucose lowering effect and the absorption were studied after subcutaneous injection in rabbits and pigs. The prolonged action of B30-substituted insulins was markedly enhanced by B27 lysine or arginine substitutions and by B13 glutamine. The B27 residue is located on the surface of the hexamer, so a basic residue in this position presumably promotes the packing of hexamers at neutral pH. The B13 residues cluster in the centre of the hexamer. When the electrostatic repulsive forces from six glutamic acid residues are abolished by substitution with glutamine, a stabilization of the hexamer can be envisaged.(ABSTRACT TRUNCATED AT 250 WORDS)     

An investigation into a cardiolipin acyl chain insertion site in cytochrome c

Mitochondrial cytochrome c associates with the phosphoplipid cardiolipin (CL) through a combination of electrostatic and hydrophobic interactions. The latter occurs by insertion into cytochrome c of an acyl chain, resulting in the dissociation of the axial Met-80 heme-iron ligand. The resulting five coordinate cytochrome c/CL complex has peroxidatic properties leading to peroxidation of CL and dissociation of the complex. These events are considered to be pre-apoptotic and culminate with release of cytochrome c from the mitochondria into the cytoplasm. Two distinct surface regions on cytochrome c have been suggested to mediate CL acyl chain insertion and this study has probed one of these regions. We have constructed a series of alanine mutants aimed at disrupting a surface cleft formed between residues 67-71 and 82-85. The physicochemical properties, peroxidase activity, CL binding, and kinetics of carbon monoxide (CO) binding to the ferrous cytochrome c/CL complex have been assessed for the individual mutants. Our findings reveal that the majority of mutants are capable of binding CL in the same apparent stoichiometry as the wild-type protein, with the extent to which the Met-80 ligand is bound in the ferrous cytochrome c/CL complex being mutant specific at neutral pH. Mutation of the species conserved Arg-91 residue, that anchors the cleft, results in the greatest changes to physicochemical properties of the protein leading to a change in the CL binding ratio required to effect structural changes and to the ligand-exchange properties of the ferrous cytochrome c/CL complex.     

Solution conformation of the His-47 to Ala-47 mutant of Pseudomonas stutzeri ZoBell ferrocytochrome c-551

In the cytochrome c-551 family, the heme 17-propionate caboxylate group is always hydrogen bonded to an invariant Trp-56 and conserved residues (His and Arg mainly, Lys occasionally) at position 47. The mutation of His-47 to Ala-47 for Pseudomas stutzeri ZoBell cytochrome c-551 removes this otherwise invariant hydrogen bond. The solution structure of ferrous H47A has been solved based on NMR-derived constraints. Results indicate that the mutant has very similar main chain folding compared to wild-type. However, less efficient packing of residues in the mutant surrounding the heme propionates leads to more solvent exposure for both propionate groups, which may account for decreased stability of the mutant. The mutant has a reduction potential different from wild-type, and furthermore, the pH dependence of this potential is not the same as for wild-type. The structure of the mutant suggests that these changes are related to the loss of the residue-47 propionate hydrogen bond and the loss of charge on the side chain of residue 47.     

Improving protein extraction yield in reversed micellar systems through surface charge engineering

The mechanism of extraction of rat cytochrome b(5) from water into a sodium dioctylsulfosuccinate (AOT) micellar organic phase was studied using protein engineering of surface charged residues. The extraction behavior of native cytochrome b(5) and modified proteins with substitutions of the type glutamic acid --> lysine at positions 44 (E44K), 56 (E56K), and 92 (E92K), was studied as a function of pH. The results indicate that an important mechanism of extraction is an electrostatic interaction of this protein with the negatively charged surfactant. We demonstrate that it is possible to improve extraction by engineering the protein surface charge, increasing the driving force responsible for the protein transfer to the micellar phase. (c) 1994 John Wiley & Sons, Inc.     

Modification of a lysine residue of adrenodoxin reductase, essential for complex formation with adrenodoxin

The NADPH-cytochrome c reductase activity of NADPH-adrenodoxin reductase from NADPH to cytochrome c via adrenodoxin was inhibited by pyridoxal 5'-phosphate and other reagents that modified the lysine residues. However, the NADPH-ferricyanide reductase activity was not affected. Loss of the cytochrome c reductase activity could be prevented by adrenodoxin, but not by NADP+. One lysine residue of the adrenodoxin reductase could be protected from the modification with pyridoxal 5'-phosphate by complex formation with adrenodoxin. Loss of the NADPH-cytochrome c reductase activity was not due to the conformational change of the modified adrenodoxin reductase, judging from circular dichroism spectrometric studies.     

The binding domain on horse cytochrome c and Rhodobacter sphaeroides cytochrome c2 for the Rhodobacter sphaeroides cytochrome bc1 complex

The interaction of the Rhodobacter sphaeroides cytochrome bc1 complex with Rb. sphaeroides cytochrome c2 and horse cytochrome c was studied by using specific lysine modification and ionic strength dependence methods. The rate of the reactions with both cytochrome c and cytochrome c2 decreased rapidly with increasing ionic strength above 0.2 M NaCl. The ionic strength dependence suggested that electrostatic interactions were equally important to the reactions of the two cytochromes, even though they have opposite net charges at pH 7.0. In order to define the interaction domain on horse cytochrome c, the reaction rates of derivatives modified at single lysine amino groups with trifluoroacetyl or trifluoromethylphenylcarbamoyl were measured. Modification of lysine-8, -13, -27, -72, -79, and -87 surrounding the heme crevice was found to significantly lower the rate of the reaction, while modification of lysines in other regions had no effect. This result indicates that lysines surrounding the heme crevice of horse cytochrome c are involved in electrostatic interactions with carboxylate groups at the binding site on the cytochrome bc1 complex. In order to define the reaction domain on cytochrome c2, a fraction consisting of a mixture of singly labeled 4-carboxy-2,6-dinitrophenylcytochrome c2 derivatives modified at lysine-35, -88, -95, -97, and -105 and several unidentified lysines was prepared. Although it was not possible to resolve these derivatives, all of the identified lysines are located on the front surface of cytochrome c2 near the heme crevice. The rate of reaction of this fraction was significantly smaller than that of native cytochrome c2, suggesting that the binding domain on cytochrome c2 is also located at the heme crevice.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modification by homocysteine thiolactone affects redox status of cytochrome C

Homocysteine (Hcy)-thiolactone mediates a post-translational incorporation of Hcy into protein in humans. Protein N-homocysteinylation is detrimental to protein structure and function and is linked to pathophysiology of hyperhomocysteinemia observed in humans and experimental animals. The modification by Hcy-thiolactone can be detrimental directly by affecting the function of an essential lysine residue or indirectly by interfering with the function of other essential residues or cofactors. Previous work has shown that cytochrome c is very sensitive to Hcy-thiolactone, which causes formation of N-Hcy-cytochrome c multimers. However, it was unclear what sites in cytochrome c were prone to Hcy attachment and whether N-linked Hcy can affect the structure and redox function of cytochrome c. Here we show that 4 lysine residues (Lys8 or -13, Lys86 or -87, Lys99, and Lys100) of cytochrome c are susceptible to N-homocysteinylation. We also show that N-homocysteinylation of 1 mol of lysine/mol of protein affects the redox state of the heme ligand of cytochrome c by rendering it reduced. The modification causes subtle structural changes, manifested as increased resistance of the N-Hcy-cytochrome c to proteolysis by trypsin, chymotrypsin, and Pronase. However, no major secondary structure perturbations were observed as shown by circular dichroism spectroscopy. Our data illustrate how N-homocysteinylation can interfere with the function of heme-containing proteins.