Kinetic studies of the copper nitrite reductase from Achromobacter cycloclastes and its interaction with a blue copper protein

Transient state, burst and steady state kinetics of reactions of the blue copper nitrite reductase (NIR) and blue copper protein from Achromobacter cycloclastes are investigated. The two copper-containing species are reacted with each other and where possible with dithionite, ascorbate and nitrite. Both copper proteins are fully reduced by dithionite with both S2O4(2-) and SO2-. species active. NIR is only partially reduced by ascorbate in an unusual biphasic reaction consistent with complete reduction of type-one copper followed by partial reduction of type-two copper. The rate of reduction of the type-one copper is accelerated using phenazine methosulfate as mediator. Nitrite can oxidize dithionite-reduced NIR but cannot reduce oxidized NIR. Rate constants were determined for all observed reactions.     

Kinetics of dithionite reduction of the heme nonapeptide of cytochrome c

The kinetics of dithionite reduction of the oxidized heme nonapeptide fragment of horse heart cytochrome c have been measured as a function of ionic strength at pH 7 and pH 9 by the stopped-flow technique. Dithionite concentration dependences indicate that the radical anion monomer, SO2-., is the active reductant. The pH 7 ionic strength dependence suggests that the heme peptide is reacting as a negatively charged molecule (its overall charge is calculated to be -1). Comparison of these results with the known rate of dithionite reduction of cytochrome c indicates that the heme nonapeptide has substantially greater inherent reactivity than cytochrome c, perhaps due to the greater accessibility of the heme.     

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].     

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.     

Cyanide reactivity of cytochrome c derivatives.

The kinetic rates and equilibrium association constants for cyanide binding have been measured for a series of cytochrome c derivatives as a probe of heme accessibility. The series included horse and yeast cytochromes iodinated at Tyr 67 and 74, horse cytochrome formylated at Trp 59 in both a low and high redox potential form, the Met 80 sulfoxide derivative of horse cytochrome and the N-acylisourea heme propionate derivative of tuna cytochrome. Native cytochromes c are well known to bind cyanide slowly in a reaction simply first order both in cytochrome and cyanide up to at least 100 mM in cyanide. The derivative demonstrate markedly different kinetics which indicate the following conclusions. (1) In spite of chemical modification at different loci, all the derivatives have highly similar reactivity, suggesting common ligation structures and mechanisms for reaction. (2) Compared to native cytochromes, reaction rates are 10-20 fold greater. This is in accord with a more accessible heme crevice, but not a completely opened crevice. For the completely opened case, rate increases are expected to be between three and five orders of magnitude. (3) Reaction rates are either independent of cyanide concentration (zero order) or show only slight variation. A mechanism which accounts for the data over four orders of magnitude in concentration postulates a protein conformation step, opening of the heme crevice, as the rate determining step. This conformation change has a limiting rate of 6 . 10(-2) s-1.     

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.     

Cytochrome c: ion binding and redox properties. Studies on ferri and ferro forms of horse, bovine, and tuna cytochrome c

The ion binding properties of horse, bovine, and tuna cytochrome c (both oxidized and reduced) have been measured using a combination of ultrafiltration, neutron activation, and ion chromatography. The ions investigated were chloride, phosphate, and Tris-cacodylate. Ion chromatography and neutron activation analysis techniques were employed to determine the concentration of free anions. Binding constants are obtained from modified Scatchard plots (in the range of 10-2000 M-1). The redox potentials for cytochrome c at different ionic strengths, pH 7.0, have been determined. In this paper we report the ionic strength and ion binding effects on the redox properties of horse, bovine, and tuna cytochrome c. Potential versus ionic strength dependence for horse, bovine, and tuna cytochrome c from the experimental data were compared with a theoretical model.     

Kinetics and mechanism of electron transfer from dithionite to microsomal cytochrome b5 and to forms of the protein associated with charged and neutral vesicles.

The kinetics of the dithionite reduction of calf liver microsomal cytochrome b5, both free in solution and bound to dimyristoyl phosphatidylcholine vesicles, are consistent with electron transfer between SO2- and the exposed haem edge of the protein. The vesicle membrane does not hinder the approach of SO2- to the site of electron transfer on the protein. In 0.01 M-Tris/HCl buffer, pH 8.1, ket (25 degrees C), delta H et and delta S et are estimated to be 1.44 x 10(6) M-1.s-1, 7.8 kJ.mol-1 and -92.3 J.K-1.mol-1 respectively. The cytochrome exhibits an acid dissociation, pKa 9.3 +/- 0.3, and the rate of electron transfer from dithionite to the high-pH form is about one-third of that to the neutral-pH form. The effect of ionic strength on the kinetics is consistent with a reaction between like-charged species and is discussed in terms of a number of theoretical models. In systems comprising cytochrome b5 and negatively charged vesicles, the effect of increasing the charge density of mixed dimyristoyl phosphatidylcholine/dicetyl phosphate vesicles and of increasing the concentration of dicetyl phosphate vesicles is to lower the rate of electron transfer from dithionite to the haem moiety of the cytochrome. With vesicles of high charge density, however, the kinetics are complicated by vesicle-induced conformation changes of the cytochrome.     

Proton-NMR studies of the effects of ionic strength and pH on the hyperfine-shifted resonances and phenylalanine-82 environment of three species of mitochondrial ferricytochrome c.

Ferricytochromes c from three species (horse, tuna, yeast) display sensitivity to variations in solution ionic strength or pH that is manifested in significant changes in the proton NMR spectra of these proteins. Irradiation of the heme 3-CH3 resonances in the proton NMR spectra of tuna, horse and yeast iso-1 ferricytochromes c is shown to give NOE connectivities to the phenyl ring protons of Phe82 as well as to the beta-CH2 protons of this residue. This method was used to probe selectively the Phe82 spin systems of the three cytochromes c under a variety of solution conditions. This phenylalanine residue has previously been shown to be invariant in all mitochondrial cytochromes c, located near the exposed heme edge in proximity to the heme 3-CH3, and may function as a mediator in electron transfer reactions [Louie, G. V., Pielak, G. J., Smith, M. & Brayer, G. D. (1988) Biochemistry 27, 7870-7876]. Ferricytochromes c from all three species undergo a small but specific structural rearrangement in the environment around the heme 3-CH3 group upon changing the solution conditions from low to high ionic strength. This structural change involves a decrease in the distance between the Phe82 beta-CH2 group and the heme 3-CH3 substituent. In addition, studies of the effect of pH on the 1H-NMR spectrum of yeast iso-1 ferricytochrome c show that the heme 3-CH3 proton resonance exhibits a pH-dependent shift with an apparent pK in the range of 6.0-7.0. The chemical shift change of the yeast iso-1 ferricytochrome c heme 3-CH3 resonance is not accompanied by an increase in the linewidth as previously described for horse ferricytochrome c [Burns, P. D. & La Mar, G. N. (1981) J. Biol. Chem. 256, 4934-4939]. These spectral changes are interpreted as arising from an ionization of His33 near the C-terminus. In general, the larger spectral changes observed for the resonances in the vicinity of the heme 3-CH3 group in yeast iso-1 ferricytochrome c with changes in solution conditions, relative to the tuna and horse proteins, suggest that the region around Phe82 is more open and that movement of the Phe82 residue is less constrained in yeast ferricytochrome c. Finally, it is demonstrated here that both the heme 8-CH3 and the 7 alpha-CH resonances of yeast ferricytochrome c titrate with p2H and exhibit apparent pK values of approximately 7.0. The titrating group responsible for these spectral changes is proposed to be His39.     

Kinetics of reduction by free flavin semiquinones of the components of the cytochrome c-cytochrome c peroxidase complex and intracomplex electron transfer

The kinetics of reduction by free flavin semiquinones of the individual components of 1:1 complexes of yeast ferric and ferryl cytochrome c peroxidase and the cytochromes c of horse, tuna, and yeast (iso-2) have been studied. Complex formation decreases the rate constant for reduction of ferric peroxidase by 44%. On the basis of a computer model of the complex structure [Poulos, T.L., & Finzel, B.C. (1984) Pept. Protein Rev. 4, 115-171], this decrease cannot be accounted for by steric effects and suggests a decrease in the dynamic motions of the peroxidase at the peroxide access channel caused by complexation. The orientations of the three cytochromes within the complex are not equivalent. This is shown by differential decreases in the rate constants for reduction by neutral flavin semiquinones upon complexation, which are in the order tuna much greater than horse greater than yeast iso-2. Further support for differences in orientation is provided by the observation that, with the negatively charged reductant FMNH., the electrostatic environments near the horse and tuna cytochrome c electron-transfer sites within their respective complexes with peroxidase are of opposite sign. For the horse and tuna cytochrome c complexes, we have also observed nonlinear concentration dependencies of the reduction rate constants with FMNH.. This is interpreted in terms of dynamic motion at the protein-protein interface. We have directly measured the physiologically significant intra-complex one electron transfer rate constants from the three ferrous cytochromes c to the peroxide-oxidized species of the peroxidase. At low ionic strength these rate constants are 920, 730, and 150 s-1 for tuna, horse, and yeast cytochromes c, respectively. These results are also consistent with the contention that the orientations of the three cytochromes within the complex with CcP are not the same. The effect on the intracomplex electron-transfer rate constant of the peroxidase amino acid side chain(s) that is (are) oxidized by the reduction of peroxide was determined to be relatively small. Thus, the rate constant for reduction by horse cytochrome c of the peroxidase species in which only the heme iron atom is oxidized was decreased by only 38%, indicating that this oxidized side-chain group is not tightly coupled to the ferryl peroxidase heme iron. Finally, it was found that, in the absence of cytochrome c, neither of the ferryl peroxidase species could be rapidly reduced by flavin semiquinones.(ABSTRACT TRUNCATED AT 400 WORDS)     

Active-site structural comparison of streptococcal NADH peroxidase and NADH oxidase. Reconstitution with artificial flavins.

The apoproteins of the streptococcal NADH peroxidase (H2O2----2H2O) and NADH oxidase (O2----2H2O) stabilize the neutral forms of 6-hydroxy- and 6-mercapto-FAD, respectively. The redox behavior of the 6-hydroxy-FAD peroxidase closely mimics that of the native enzyme with both dithionite and NADH. Both oxidase and peroxidase preferentially stabilize the N(1)-protonated p-quinonoid species of 8-mercapto-FAD, and the 8-position of the bound flavin is accessible to solvent in both proteins. The 8-mercapto-FAD peroxidase yields an EH2 spectrum on reduction virtually identical to that seen with 8-mercapto-FAD glutathione reductase, but no distinct EH2.NADH form appears. The dramatic decreases in reactivity at the flavin 2- and 4-positions for both the peroxidase and the oxidase, assessed with the reconstituted 2- and 4-thio-FAD enzymes, suggest that these positions are buried by elements of both protein structures. Furthermore, reconstitution of the peroxidase with the higher potential 2- and 4-thioflavins yields enzyme forms which are fully reducible with 1.4 eq of NADH/FAD, giving rise to stable thio-FADH2.NAD+ complexes. This behavior closely mimics that of the native NADH oxidase and provides further evidence supporting the hypothesis that a major functional distinction between the two structurally related proteins is determined by the redox potential and/or NADH reactivity of the bound flavin coenzyme.     

The reduction of methemerythrin by e-aq and CO2- from pulse radiolysis studies.

The rate constants for reduction of methemerythrin from Phascolopsis gouldii and Themiste pyroides by hydrated electrons are 2.0 and 3.9 x 10(9) M(-1)s(-1), respectively, at pH 8.2, I = 0.03 M, and 25 degrees C. There is only a small increase in rate when the pH is lowered to 6.3 and a very small decrease when the ionic strength is raised to 0.1 M. Adding azide ion (to form the met-azide adduct) has little effect on the reactivity towards e-aq. For the monomer form, metmyohemerythrin from T. pyroides, the reaction rate constant is 4.5 x 10(9) M(-1)s(-1). Methemerythrin from T. pyroides reacts with CO2- with a rate constant 6.8 x 10(7) M(-1)s(-1). The reactivity sequence e-aq greater than CO2- greater than SO2- (from dithionite reduction) towards methemerythrin is the same as that observed with reduction of heme proteins but the rate constants are some 10 to 100 times smaller for the former. Only 10 to 20% of the e-aq or CO2- radicals generated effect reduction of the iron centers in methemerythrin.     

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)     

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.     

Site-directed mutagenesis of Azotobacter vinelandii ferredoxin I. Changes in [4Fe-4S] cluster reduction potential and reactivity.

We have used site-directed mutagenesis to obtain two variants of Azotobacter vinelandii ferredoxin I (AvFdI), whose x-ray structures are now available. In the C20A protein, a ligand to the [4Fe-4S] cluster was removed whereas in the C24A mutant a free cysteine next to that cluster was removed. Like native FdI, both mutants contain one [4Fe-4S] cluster and one [3Fe-4S] cluster. The structure of C24A is very similar to that of native FdI, while the structure of C20A is rearranged in the region of the [4Fe-4S] cluster to allow it to use the free Cys-24 as a replacement ligand. Here we compare the properties of the native, C20A, and C24A proteins. Although all three proteins are O2 stable in vitro, the C20A protein is much less stable toward proteolysis than the other two in vivo. Spectroscopic results show that all three proteins exhibit the same general redox behavior during O2-oxidation and dithionite reduction. Electrochemical data show that the [3Fe-4S] clusters in all three proteins have the same pH-dependent reduction potentials (-425 mV versus SHE, pH 7.8), whereas the [4Fe-4S] cluster potentials vary over a approximately 150 mV range from -600 mV (C24A) to -647 mV (native) to -746 mV (C20A). Despite this variation in potential both the C20A and C24A proteins appear to be functional in vivo. Native FdI reacts with three equivalents of Fe(CN)3-(6) to form a paramagnetic species previously proposed to be a cysteinyl-disulfide radical. Neither the C20A nor the C24A variant undergoes this reaction, strongly suggesting that it involves the free Cys-24.     

Partitioning of electrostatic and conformational contributions in the redox reactions of modified cytochromes c.

The reduction of acetylated, fully succinylated and dicarboxymethyl horse cytochromes c by the radicals CH3CH(OH), CO2.-, O2.-, and e-aq' and the oxidation of the reduced cytochrome c derivatives by Fe(CN)3-6 were studied using the pulse radiolysis technique. Many of the reactions were also examined as a function of ionic strength. By obtaining rate constants for the reactions of differently charged small molecules redox agents with the differently charged cytochrome c derivatives at both zero ionic strength and infinite ionic strength, electrostatic and conformational contributions to the electron transfer mechanism were effectively partioned from each other in some cases. In regard to cytochrome c electron transfer mechanism, the results, especially those for which conformational influences predominate, are supportive of the electron being transferred in the heme edge region.     

Proton hyperfine resonance assignments using the nuclear Overhauser effect for ferric forms of horse and tuna cytochrome c.

Proton hyperfine resonance assignments for cytochromes c from several species are currently being successfully pursued by several laboratories. These efforts focus mostly on the ferrous forms. In contrast to that work, we have pursued assignments of the proton hyperfine shifted resonances for horse and tuna ferricytochromes c. Our results indicate that assignments are nearly identical in those two proteins. Using the pre-steady state nuclear Overhauser effect, several additional assignments have been made for the tuna protein, whereas for the horse protein, the following protons have been assigned: heme 7, alpha CH2; heme 7, beta CH2; histidine 18, beta CH2 and alpha CH; and the methionine 80, beta CH2.     

Probing local thermal stabilities of bovine,horse, and tuna ferricytochromes<Emphasis Type="Italic"> c</Emphasis> at pH 7

Correlation between the flexibility of the Met80 loop (residues 75-86) and the local stabilities of native ferricytochromes c from horse, bovine, and tuna was examined. By monitoring the heme bands versus temperature, absorption changes associated with altered ligation in the alkaline isomers were observed. In addition, the intensity of the 695-nm absorption band, which is associated with the heme-crevice stability, decreased with increasing temperature and exhibited biphasic temperature dependence, with transition temperatures (Tc) at 35 degrees C in tuna c, 55 degrees C in horse c, and 58 C in bovine c. Since the heme crevice plays a key role in the thermal stabilities of cytochromes c, their susceptibility to proteolytic attack was examined as a function of temperature. Proteolytic digestion, which requires local conformational instability, revealed that the local stabilities of the cytochromes follow the order: bovine > horse > tuna, and increased digestion occurred at temperatures close to the 695-nm Tc for each protein. This is consistent with the actual substitution of the Met80 ligand above the 695-nm Tc, which is reflected in the thermodynamic parameters for the two phases. Also, tuna c, unlike horse and bovine c, exhibits different 695-nm (35 degrees C) and Soret (approximately 46 degrees C) Tc values, but its local stability is controlled by the transition detected at 695 nm. The combined spectroscopic and proteolysis results clearly indicate that the flexibility of the Met80 loop determines the local stability of cytochromes c.     

Probing stability and dynamics of proteins by protease digestion. I: Comparison of protease susceptibility and thermal stability of cytochromes c

Protease susceptibility of homologous proteins in their native conformations was studied. This work aims to establish a broad and quantitative basis for the utilization of protease digestion to analyze the local stability of native proteins. Using high-performance liquid chromatography (HPLC) the time course of the proteolytic degradation of intact proteins was quantitatively traced. Rapid separation of peptide fragments with HPLC made possible the elucidation of sequential digestion originating from the cleavage at a very few sites which are locally unstable in the protein structure. Using four serine proteases, chymotrypsin, trypsin, elastase and subtilisin BPN', we found some common trends in proteolysis for a group of proteins of the cytochrome c family. By comparing of the proteolysis and thermal denaturation with ten homologous cytochromes c extracted from horse, beef, Candida krusei, Saccharomyces cerevisiae, chicken, tuna, pigeon, rabbit, dog and rat, protease susceptibility was related to locally unfolding states intrinsic to the native conformation.     

Surface potential changes of mitoplasts in the presence of pyridoxal phosphate modified cytochromes c.

1. The addition of native cytochrome c to mitoplasts leads to a decrease of surface potential of the mitoplast membrane. However the surface potential is slightly decreased (approximately 3 mV) when PLP(Lys 86)-cytochrome c and PLP(Lys 79)-cytochrome c were added. 2. The native and PLP-modified cytochromes c do not influence the order parameters S and isotropic constant a when both spin probe I and probe II were used. It is shown that cytochrome c binding to the membrane does not affect the hydrophobic intermembrane area as well as the lipid arrangements of the mitoplast membrane. 3. At low ionic strength there was observed a significant difference in the membrane potential when PLP-cytochromes c were added to the mitoplasts. 4. At high ionic strength the addition of native or PLP-modified cytochromes c does not change the membrane potential.