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)     

Effect of specific lysine modification on the reduction of cytochrome c by succinate-cytochrome c reductase.

The reduction of cytochrome c by succinate-cytochrome c reductase was studied at very low cytochrome c concentrations where the reaction between cytochrome c1 and cytochrome c was rate limiting. The rate constant for the reaction was found to be independent of ionic strength up to 0.1 M chloride, and to decrease rapidly at higher ionic strength, suggesting that the interaction between cytochrome c1 and cytochrome c was primarily electrostatic. The reaction rates of cytochrome c derivatives modified at single lysine residues to form trifluoroacetylated or trifluoromethylphenylcarbamylated cytochromes c were studied to determine the role of individual lysines in the reaction. None of the modifications affected the reaction at low ionic strength, but at higher ionic strength the reaction rate was substantially decreased by modification of those lysines surrounding the heme crevice, lysine-8, -13, -27, -72, and -79. Modification of lysine-22, -25, -55, -99, and -100 had no effect on the rate. These results indicate that the binding site on cytochrome c for cytochrome c1 overlaps considerably with that for cytochrome oxidase, suggesting that cytochrome c might undergo some type of rotational diffusion during the electron-transport process.     

Electrostatic interaction of cytochrome c with cytochrome c1 and cytochrome oxidase

The reactions of horse heart cytochrome c with succinate-cytochrome c reductase and cytochrome oxidase were studied as a function of ionic strength using both spectrophotometric and oxygen electrode assay techniques. The kinetic parameter Vmax/Km for both reactions decreased very rapidly as the ionic strength was increased, indicating that electrostatic interactions were important to the reactions. A new semiempirical relationship for the electrostatic energy of interaction between cytochrome c and its oxidation-reduction partners was developed, in which specific complementary charge-pair interactions between lysine amino groups on cytochrome c and negatively charged carboxylate groups on the other protein are assumed to dominate the interaction. The contribution of individual cytochrome c lysine amino groups to the electrostatic interaction was estimated from the decrease in reaction rate caused by specific modification of the lysine amino groups by reagents that change the charge to 0 or -1. These estimates range from -0.9 kcal/mol for lysines immediately surrounding the heme crevice of cytochrome c to 0 kcal/mol for lysines well removed from the heme crevice region. The semiempirical relationship for the total electrostatic energy of interaction was in quantitative agreement with the experimental ionic strength dependence of the reaction rates when the parameters were based on the specific lysine modification results. The electrostatic energies of interaction between cytochrome c and its reductase and oxidase were nearly the same, providing additional evidence that the two reactions take place at similar sites on cytochrome c.     

Chromatium flavocytochrome c: kinetics of reduction of the heme subunit, and the flavocytochrome c-mitochondrial cytochrome c complex

The kinetics of reduction of Chromatium vinosum flavocytochrome c heme subunit by exogenous flavin neutral semiquinones generated by laser flash photolysis have been investigated. Unlike the holoprotein, the isolated heme subunit was appreciably reactive with lumiflavin neutral semiquinone. The measured rate constant for the reaction (2.7 X 10(7) M-1 S-1) was comparable to those of c-type cytochromes having similar redox potentials. The ionic strength dependence of the reaction with FMN neutral radical indicated that the heme subunit had a small negative charge at the site of reduction. Taken together, these results suggest that the active site of the heme subunit is buried on complexation with the flavin subunit in the holoprotein. Horse cytochrome c formed a strong complex with Chromatium, but not Chlorobium, flavocytochrome c. Possible physiological electron acceptors such as HiPIP, cytochrome c', and cytochrome c-555 apparently did not bind to the flavocytochromes c. The rate constant for reduction by lumiflavin radical of horse cytochrome c complexed to flavocytochrome c was about twofold smaller than for reduction of horse cytochrome c alone. Flavocytochrome c was itself unreactive with exogenous flavin semiquinones. The ionic strength dependence of the reduction of the complex by FMN radical was also smaller than for horse cytochrome c in the absence of flavocytochrome c. Sulfite, which forms an adduct with the protein-bound FAD (FAD is bound in an 8-alpha-S-cysteinyl linkage), did not affect the reduction of horse cytochrome c in its complex with flavocytochrome c. We conclude that horse cytochrome c is reduced directly by exogenous flavins in its complex with flavocytochrome c, although the kinetics are slightly modified. These results are not unlike observations made with complexes of mitochondrial cytochrome c with cytochrome oxidase or cytochrome b5.     

The reaction domain on Rhodospirillum rubrum cytochrome c2 and horse cytochrome c for the Rhodospirillum rubrum cytochrome bc1 complex

The interaction of the Rhodospirillum rubrum cytochrome bc1 complex with R. rubrum cytochrome c2 and horse cytochrome c was studied using specific lysine modification and ionic strength dependence methods. In order to define the reaction domain on cytochrome c2, several fractions consisting of mixtures of singly labeled carboxydintrophenyl-cytochrome c2 derivatives were employed. Fraction A consisted of a mixture of derivatives modified at lysines 58, 81, and 109 on the back of cytochrome c2, while fractions C1, C2, C3, and C4 were mixtures of singly labeled derivatives modified at lysines 9, 13, 75, 86, and 88 on the front of cytochrome c2 surrounding the heme crevice. The rate of the reaction of fraction A was found to be nearly the same as that of native cytochrome c2. However, the rate constants of fractions C1-C4 were found to be more than 20-fold smaller than that of native cytochrome c2. These results indicate that lysine residues surrounding the heme crevice of cytochrome c2 are involved in electrostatic interactions with carboxylate groups at the binding site on the cytochrome bc1 complex. Since the same domain is involved in the reaction with the photosynthetic reaction center, cytochrome c2 must undergo some type of rotational or translational diffusion during electron transport in R. rubrum. The reaction rates of horse heart cytochrome c derivatives modified at single lysine amino groups with trifluoroacetyl or trifluoromethylphenylcarbamoyl were also measured. Modification of lysines 8, 13, 25, 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 indicates that the reaction of horse cytochrome c also involves the heme crevice domain.     

Characterization of four covalently-linked yeast cytochrome c/cytochrome c peroxidase complexes: Evidence for electrostatic interaction between bound cytochrome c molecules

Four covalent complexes between recombinant yeast cytochrome c and cytochrome c peroxidase (rCcP) were synthesized via disulfide bond formation using specifically designed protein mutants (Papa, H. S., and Poulos, T. L. (1995) Biochemistry 34, 6573-6580). One of the complexes, designated V5C/K79C, has cysteine residues replacing valine-5 in rCcP and lysine-79 in cytochrome c with disulfide bond formation between these residues linking the two proteins. The V5C/K79C complex has the covalently bound cytochrome c located on the back-side of cytochrome c peroxidase, approximately 180 degrees from the primary cytochrome c-binding site as defined by the crystallographic structure of the 1:1 noncovalent complex (Pelletier, H., and Kraut J. (1992) Science 258, 1748-1755). Three other complexes have the covalently bound cytochrome c located approximately 90 degrees from the primary binding site and are designated K12C/K79C, N78C/K79C, and K264C/K79C, respectively. Steady-state kinetic studies were used to investigate the catalytic properties of the covalent complexes at both 10 and 100 mM ionic strength at pH 7.5. All four covalent complexes have catalytic activities similar to those of rCcP (within a factor of 2). A comprehensive study of the ionic strength dependence of the steady-state kinetic properties of the V5C/K79C complex provides evidence for significant electrostatic repulsion between the two cytochromes bound in the 2:1 complex at low ionic strength and shows that the electrostatic repulsion decreases as the ionic strength of the buffer increases.     

Definition of the interaction domain for cytochrome c on the cytochrome bc(1) complex. Steady-state and rapid kinetic analysis of electron transfer between cytochrome c and Rhodobacter sphaeroides cytochrome bc(1) surface mutants

The interaction domain for cytochrome c on the cytochrome bc(1) complex was studied using a series of Rhodobacter sphaeroides cytochrome bc(1) mutants in which acidic residues on the surface of cytochrome c(1) were substituted with neutral or basic residues. Intracomplex electron transfer was studied using a cytochrome c derivative labeled with ruthenium trisbipyridine at lysine 72 (Ru-72-Cc). Flash photolysis of a 1:1 complex between Ru-72-Cc and cytochrome bc(1) at low ionic strength resulted in electron transfer from photoreduced heme c to cytochrome c(1) with a rate constant of k(et) = 6 x 10(4) s(-1). Compared with the wild-type enzyme, the mutants substituted at Glu-74, Glu-101, Asp-102, Glu-104, Asp-109, Glu-162, Glu-163, and Glu-168 have significantly lower k(et) values as well as significantly higher equilibrium dissociation constants and steady-state K(m) values. Mutations at acidic residues 56, 79, 82, 83, 97, 98, 213, 214, 217, 220, and 223 have no significant effect on either rapid kinetics or steady-state kinetics. These studies indicate that acidic residues on opposite sides of the heme crevice of cytochrome c(1) are involved in binding positively charged cytochrome c. These acidic residues on the intramembrane surface of cytochrome c(1) direct the diffusion and binding of cytochrome c from the intramembrane space.     

Reaction of cytochrome c2 with photosynthetic reaction centers from Rhodopseudomonas viridis.

The reactions of Rhodopseudomonas viridis cytochrome c2 and horse cytochrome c with Rps. viridis photosynthetic reaction centers were studied by using both single- and double-flash excitation. Single-flash excitation of the reaction centers resulted in rapid photooxidation of cytochrome c-556 in the cytochrome subunit of the reaction center. The photooxidized cytochrome c-556 was subsequently reduced by electron transfer from ferrocytochrome c2 present in the solution. The rate constant for this reaction had a hyperbolic dependence on the concentration of cytochrome c2, consistent with the formation of a complex between cytochrome c2 and the reaction center. The dissociation constant of the complex was estimated to be 30 microM, and the rate of electron transfer within the 1:1 complex was 270 s-1. Double-flash experiments revealed that ferricytochrome c2 dissociated from the reaction center with a rate constant of greater than 100 s-1 and allowed another molecule of ferrocytochrome c2 to react. When both cytochrome c-556 and cytochrome c-559 were photooxidized with a double flash, the rate constant for reduction of both components was the same as that observed for cytochrome c-556 alone. The observed rate constant decreased by a factor of 14 as the ionic strength was increased from 5 mM to 1 M, indicating that electrostatic interactions contributed to binding. Molecular modeling studies revealed a possible cytochrome c2 binding site on the cytochrome subunit of the reaction center involving the negatively charged residues Glu-93, Glu-85, Glu-79, and Glu-67 which surround the heme crevice of cytochrome c-554.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure of an electron transfer complex. I. Covalent cross-linking of cytochrome c peroxidase and cytochrome c

Cytochrome c peroxidase and cytochrome c form a noncovalent electron transfer complex in the course of the peroxidase-catalyzed reduction of H2O2. The two hemoproteins were cross-linked in 40% yield to a covalent 1:1 complex with the aid of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The covalent complex was found to be a valid model of the noncovalent electron transfer complex for the following reasons. The covalent complex had only 5% residual peroxidase activity toward exogeneous ferrocytochrome c indicating that the cross-linked cytochrome c covers the electron-accepting site of cytochrome c peroxidase. The residual peroxidase activity was almost independent of ionic strength indicating that the electron-accepting site is much less accessible even when ionic bonds between the two cross-linked hemoproteins are severed. The rate of reduction of heme c by ascorbate is 15 times slower in the covalent complex than in free cytochrome c and is independent of ionic strength. Although the covalent complex may not have been entirely pure with respect to the number and location of the cross-links, two major cross-links could be localized to within a few residues. One is from Lys 13 of cytochrome c to an acidic residue in positions 32, 33, 34, 35, or 37 of cytochrome c peroxidase, the other from Lys 86 of cytochrome c to a carboxyl group in the same cluster of acidic residues. The result stresses the importance of a peculiar stretch of acidic residues of cytochrome c peroxidase and of Lys 13 and 86 of cytochrome c.     

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.     

Effects of amino acid replacements in yeast iso-1 cytochrome c on heme accessibility and intracomplex electron transfer in complexes with cytochrome c peroxidase

The kinetics of reduction of wild type and several site-specific mutants of yeast iso-1 cytochrome c (Arg-13----Ile, Gln-16----Ser, Gln-16----Lys, Lys-27----Gln, Lys-72----Asp), both free and in 1:1 complexes with yeast cytochrome c peroxidase, by free flavin semiquinones have been studied. Intramolecular one-electron transfer from the ferrous cytochromes c to the H2O2-oxidized peroxidase at both low (8 mM) and high (275 mM) ionic strengths was also studied. The accessibility of the cytochrome c heme within the electrostatically stabilized complex and the rate constants for intramolecular electron transfer at both low and high ionic strength are highly dependent on the specific amino acids present at the protein-protein interface. Importantly, replacement by uncharged amino acids of Arg or Lys residues thought to be important in orientation and/or stabilization of the electron-transfer complex resulted in increased rates of electron transfer. In all cases, an increase in ionic strengths from 8 to 275 mM also produced increased intramolecular electron-transfer rate constants. The results suggest that the electrostatically stabilized 1:1 complex is not optimized for electron transfer and that by neutralization of key positively charged residues, or by an increase in the ionic strength thereby masking the ionic interactions, the two proteins can orient themselves to allow the formation of a more efficient electron-transfer complex.     

Oxidative titrations of reduced cytochrome aa3: influence of cytochrome c and carbon monoxide on the midpoint potential values.

Oxidative titrations were performed on the electrostatic complex formed between cytochrome c and cytochrome aa3 at low ionic strength. Midpoint potentials of the redox centers in the proteins in 1:1 and 2:1 complexes were compared with those in mixtures of the cytochromes at high ionic strength. Computer simulations of all titrations yielded midpoint potentials for the components of cytochrome aa3 which were consistent with literature values for isolated cytochrome aa3 or mixture of cytochromes c and aa3. However, the unequal heme extinction coefficients observed previously (Schroedl, N.A., and Hartzell, C.R. (1977), Biochemistry 16, 1327) during oxidative titrations of cytochrome aa3 became equal in magnitude under these experimental conditions. The binding of cytochrome c to cytochrome aa3 changed the midpoint potentials of cytochrome aa3 by 15-20 mV, while the midpoint potentials for cytochrome c were altered by 50-60 mV. Careful analysis of these titrations including computer simulation revealed that cytochrome c was able to bind to cytochrome aa3 only after cytochrome aL2+ had become oxidized. When bound to cytochrome aa3, the midpoint potential of cytochrome c was 210 7V. Titrations performed under a carbon monoxide atmosphere revealed cytochrome aa3 midpoint potentials unchanged from reported values. Cytochrome c again exhibited a midpoint potential of 210 mV after binding to cytochrome aa3.     

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)     

Effect of pyridoxal phosphate on the formation of cytochrome c1-c and cytochrome c-cytochrome oxidase complexes.

The cytochrome c-cytochrome oxidase complex is formed when c reacts with cytochrome oxidase (Kuboyama et al. (1962) Biochem. Biophys. Res. Commun. 9, 534) and the cytochrome c1-cytochrome c complex is formed when c reacts with cytochrome c1 in the presence of the hinge protein (Kim, C.H. and King, T.E. (1981) Biochem. Biophys. Res. Commun. 101, 607). Both complexes are considered to be possible intermediates in electron transfer reaction between these cytochromes. Triply substituted modified cytochrome c by pyridoxal phosphate at lysine residues (Lys-79, 86 and one to be identified) abolishes both complex formations and electron transfer activity with succinate cytochrome c reductase or cytochrome oxidase.     

Vectorially oriented monolayers of the cytochrome c/cytochrome oxidase bimolecular complex.

Vectorially oriented monolayers of yeast cytochrome c and its bimolecular complex with bovine heart cytochrome c oxidase have been formed by self-assembly from solution. Both quartz and Ge/Si multilayer substrates were chemical vapor deposited with an amine-terminated alkylsiloxane monolayer that was then reacted with a hetero-bifunctional cross-linking reagent, and the resulting maleimide endgroup surface then provided for covalent interactions with the naturally occurring single surface cysteine 102 of the yeast cytochrome c. The bimolecular complex was formed by further incubating these cytochrome c monolayers in detergent-solubilized cytochrome oxidase. The sequential formation of such monolayers and the vectorially oriented nature of the cytochrome oxidase was studied via meridional x-ray diffraction, which directly provided electron density profiles of the protein(s) along the axis normal to the substrate plane. The nature of these profiles is consistent with previous work performed on vectorially oriented monolayers of either cytochrome c or cytochrome oxidase alone. Furthermore, optical spectroscopy has indicated that the rate of binding of cytochrome oxidase to the cytochrome c monolayer is an order of magnitude faster than the binding of cytochrome oxidase to an amine-terminated surface that was meant to mimic the ring of lysine residues around the heme edge of cytochrome c, which are known to be involved in the binding of this protein to cytochrome oxidase.     

Identification of the binding site on cytochrome c1 for cytochrome c

The reagent 1-ethyl-3-(3-[14C]trimethylaminopropyl)carbodiimide (ETC) was used to identify specific carboxyl groups on the cytochrome bc1 complex (ubiquinol-cytochrome c reductase, EC 1.10.2.2) involved in binding cytochrome c. Treatment of the cytochrome bc1 complex with 2 mM ETC led to inhibition of the electron transfer activity with cytochrome c. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that both the cytochrome c1 heme peptide and the Mr = 9175 "hinge" peptide were radiolabeled by ETC. In addition, a new band appeared at a position consistent with a 1:1 cross-linked cytochrome c1-hinge peptide species. Treatment of a 1:1 cytochrome bc1-cytochrome c complex with ETC led to the same inhibition of electron transfer activity observed with the uncomplexed cytochrome bc1, but to decreased radiolabeling of the cytochrome c1 heme peptide. Two new cross-linked species corresponding to cytochrome c-hinge peptide and cytochrome c-cytochrome c1 were formed in place of the cytochrome c1-hinge peptide species. In order to identify the specific carboxyl groups labeled by ETC, a purified cytochrome c1 preparation containing both the heme peptide and the hinge peptide was dimethylated at all the lysines to prevent internal cross-linking. The methylated cytochrome c1 preparation was treated with ETC and digested with trypsin and chymotrypsin, and the resulting peptides were separated by high pressure liquid chromatography. ETC was found to label the cytochrome c1 peptides 63-81, 121-128, and 153-179 and the hinge peptides 1-17 and 48-65. All of these peptides are highly acidic and contain one or more regions of adjacent carboxyl groups. The only peptide consistently protected from labeling by cytochrome c binding was 63-81, demonstrating that the carboxyl groups at residues 66, 67, 76, and 77 are involved in binding cytochrome c. These residues are relatively close to the heme-binding cysteine residues 37 and 40 and indicate a possible site for electron transfer from cytochrome c1 to cytochrome c.     

A pivotal heme-transfer reaction intermediate in cytochrome c biogenesis

c-Type cytochromes are widespread proteins, fundamental for respiration or photosynthesis in most cells. They contain heme covalently bound to protein in a highly conserved, highly stereospecific post-translational modification. In many bacteria, mitochondria, and archaea this heme attachment is catalyzed by the cytochrome c maturation (Ccm) proteins. Here we identify and characterize a covalent, ternary complex between the heme chaperone CcmE, heme, and cytochrome c. Formation of the complex from holo-CcmE occurs in vivo and in vitro and involves the specific heme-binding residues of both CcmE and apocytochrome c. The enhancement and attenuation of the amounts of this complex correlates completely with known consequences of mutations in genes for other Ccm proteins. We propose the complex is a trapped catalytic intermediate in the cytochrome c biogenesis process, at the point of heme transfer from CcmE to the cytochrome, the key step in the maturation pathway.     

Dissociation of bovine cytochrome c1 subcomplex and the status of cysteine residues in the subunits

Purified bovine heart two-band cytochrome c1 subcomplex was dissociated by treatment with p-chloromercuribenzoic acid (pCMB) into its heme subunit and a colorless subunit called hinge protein, which is essential for the formation of cytochrome c1-c complex. The subcomplex was found by titration to react with 4 mol of pCMB per mol of cytochrome c1. The contents of mercury of the dissociated heme subunit and the hinge protein were 3 and 1 mol per mol of polypeptide, respectively. These results, together with the sequence analysis, indicated that the three cysteine residues in cytochrome c1 heme subunit not involved in heme-binding existed in free thiol form. One of the five cysteine residues in the hinge protein was in free form and four in two disulfide bonds. The dissociated hinge protein was digested with staphylococcal protease and the cysteine-containing peptides were separated by reversed-phase high-performance liquid chromatography (HPLC). The content of mercury and the result of performic acid oxidation of cystine peptides revealed that Cys-30 existed in free thiol form and two disulfide bridges were formed between Cys-24 and Cys-68 and between Cys-40 and Cys-54. The conformation of the hinge protein was predicted to be composed largely of either two-alpha-helical or four-alpha-helical conformation with the amino (N)-terminal 20 residues being in a random structure.     

Electronic and vibrational spectroscopy of the cytochrome c:cytochrome c oxidase complexes from bovine and Paracoccus denitrificans.

The 1:1 complex between horse heart cytochrome c and bovine cytochrome c oxidase, and between yeast cytochrome c and Paracoccus denitrificans cytochrome c oxidase have been studied by a combination of second derivative absorption, circular dichroism (CD), and resonance Raman spectroscopy. The second derivative absorption and CD spectra reveal changes in the electronic transitions of cytochrome a upon complex formation. These results could reflect changes in ground state heme structure or changes in the protein environment surrounding the chromophore that affect either the ground or excited electronic states. The resonance Raman spectrum, on the other hand, reflects the heme structure in the ground electronic state only and shows no significant difference between cytochrome a vibrations in the complex or free enzyme. The only major difference between the Raman spectra of the free enzyme and complex is a broadening of the cytochrome a3 formyl band of the complex that is relieved upon complex dissociation at high ionic strength. These data suggest that the differences observed in the second derivative and CD spectra are the result of changes in the protein environment around cytochrome a that affect the electronic excited state. By analogy to other protein-chromophore systems, we suggest that the energy of the Soret pi* state of cytochrome a may be affected by (1) changes in the local dielectric, possibly brought about by movement of a charged amino acid side chain in proximity to the heme group, or (2) pi-pi interactions between the heme and aromatic amino acid residues.     

Steady state kinetics and binding of eukaryotic cytochromes c with yeast cytochrome c peroxidase.

1. The steady state kinetics for the oxidation of ferrocytochrome c by yeast cytochrome c peroxidase are biphasic under most conditions. The same biphasic kinetics were observed for yeast iso-1, yeast iso-2, horse, tuna, and cicada cytochromes c. On changing ionic strength, buffer anions, and pH, the apparent Km values for the initial phase (Km1) varied relatively little while the corresponding apparent maximal velocities varied over a much larger range. 2. The highest apparent Vmax1 for horse cytochrome c is attained at relatively low pH (congruent to 6.0) and low ionic strength (congruent to 0.05), while maximal activity for the yeast protein is at higher pH (congruent to 7.0) and higher ionic strength (congruent to 0.2), with some variations depending on the nature of the buffering ions. 3. Direct binding studies showed that cytochrome c binds to two sites on the peroxidase, under conditions that give biphasic kinetics. Under those ionic conditions that yield monophasic kinetics, binding occurred at only one site. At the optimal buffer concentrations for both yeast and horse cytochromes c, the KD1 and KD2 values approximate the Km1 and Km2 values. At ionic strengths below optimal, binding becomes too strong and above optimal, too weak. 4. Under ionic conditions that are optimal and give monophasic kinetics with horse cytochrome c but are suboptimal for the yeast protein, yeast cytochrome c strongly inhibits the reaction of horse cytochrome c with peroxidase, uncompetitively at one site and competitively at a second site. The appearance of the second site under monophasic conditions is interpreted as an allosteric effect of the inhibitor binding to the first site. 5. The simplest model accounting for these observations postulates two kinetically active sites on each molecule of peroxidase, a high affinity and a low affinity site, that may correspond to the free radical and the heme iron (IV) of the oxidized enzyme, respectively. Both oxidizing equivalents may be discharged at either site. Furthermore, the enzyme appears to exist as an equilibrium mixture of a high ionic strength form, EH and a low ionic strength form, EL, the former reacting optimally with yeast cytochrome c, and the latter with horse cytochrome c.