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 trifluoroacetylation of individual cytochrome c lysines on the reaction with cytochrome oxidase.

We have prepared three different cytochrome c derivatives, each containing a single specifically trifluoroacetylated lysine at residues 13, 55, and 99, respectively. The only modification that affected cytochrome c oxidase (EC 1.9.3.1) activity was that of lysine-13 at the top of the heme crevice. Trifluoroacetylation of lysine-13 increased the apparent Michaelis constant fivefold compared to that of native cytochrome c, but did not affect the maximum velocity. Trifluoroacetylation of lysine-55 at the left side of the cytochrome c molecule did not affect cytochrome oxidase activity in any way, nor did trifluoroacetylation of lysine-99 at the rear of the cytochrome c molecule. This indicates that the cytochrome oxidase binding site on cytochrome c involved only the front of the cytochrome c molecule and those lysines immediately surrounding the heme crevice.     

Region of cytochrome c interacting with yeast cytochrome b2: determination with singly modified carboxydinitrophenyl cytochromes c

The association and reduction reactions of ten different 4-carboxy-2,6-dinitrophenyl (CDNP) horse heart cytochromes c, singly modified at lysines 8, 13, 27, 39, 60, 72, 73, 86, 87, and 99, with Saccharomyces cerevisiae cytochrome b2 were studied to determine the region of cytochrome c interacting with cytochrome b2. In the presence of higher ratios of free cytochrome c to cytochrome b2, native cytochrome c, and the CDNP-lysine 39, 60, and 99 derivatives associated with cytochrome b2 with a binding stoichiometry close to 2:1, while CDNP-cytochromes c modified at lysines 8, 13, 27, 72, 73, 86, and 87 formed only 1:1 complexes. In the presence of lower ratios of free cytochrome c, modifications of lysines 8, 27, 86, and 87 had more inhibitory effects on the association of cytochrome c with cytochrome b2 than modifications of lysines 13, 39, 60, 72, 73, and 99. This tendency was similar to that on removal of free cytochrome c, except in the case of CDNP-lysine 13 and 73 derivatives. The rate of reduction of cytochrome c by cytochrome b2 was decreased by carboxydinitrophenylation of lysines 8, 13, 27, 72, 73, 86, and 87. In contrast, the rate of reduction of cytochrome c was not affected by modifications of lysines 39, 60, and 99. Since lysines 8, 13, 27, 72, 73, 86, and 87 are located on the front surface and lysines 39, 60, and 99 on the back side, and since different effects of modifying lysine residues located on the front surface may be interpreted in terms of effects on the complementary interaction of cytochrome c and cytochrome b2, these results indicate that the region of cytochrome c interacting with cytochrome b2 is located on the front surface of the cytochrome c molecule containing the exposed heme edge.     

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.     

Use of specific trifluoroacetylation of lysine residues in cytochrome c to study the reaction with cytochrome b5, cytochrome c1, and cytochrome oxidase

The preparation, purification, and characterization of four new derivatives of cytochrome c trifluoroacetylated at lysines 72, 79, 87, and 88 are reported. The redox reaction rates of these derivatives with cytochrome b5, cytochrome c1 and cytochrome oxidase indicated that the interaction domain on cytochrome c for all three proteins involves the lysines immediately surrounding the heme crevice. Modification of lysines 72, 79, 87 had a large effect on the rate of all three reactions, while modification of lysine 88 had a very small effect. Even though lysines 87 and 88 are adjacent to one another, lysine 87 is at the top left of the heme crevice oriented towards the front of cytochrome c, while lysine 88 is oriented more towards the back. Since the interaction sites for cytochrome c1 and cytochrome oxidase are essentially identical, cytochrome c probably undergoes some type of rotational diffusion during electron transport.     

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.     

Reaction of cytochromes c and c2 with the Rhodobacter sphaeroides reaction center involves the heme crevice domain

In order to define the interaction domain on Rhodobacter sphaeroides cytochrome c2 for the photosynthetic reaction center, positively charged lysine amino groups on cytochrome c2 were modified to form negatively charged (carboxydinitrophenyl)- (CDNP-) lysines. The reaction mixture was separated into several different fractions by ion-exchange chromatography on (carboxymethyl)cellulose. Tryptic digests of these fractions were analyzed by reverse-phase peptide mapping to determine the lysines that had been modified. Fraction A was found to consist of a mixture of singly labeled derivatives modified at lysine-35, -88, -95, -97, and -105 and several other unidentified lysines comprising 32% of the total. 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 second-order rate constant for the reaction of native cytochrome c2 with reaction centers was 2.0 X 10(8) M-1 s-1, while that for fraction A was 20-fold less, 1.0 X 10(7) M-1 s-1. This suggests that lysines surrounding the heme crevice of cytochrome c2 are involved in electrostatic interactions with carboxylate groups at the binding site of the reaction center. The reaction rates of horse heart cytochrome c derivatives modified at single lysine amino groups with trifluoroacetyl or trifluoromethylphenylcarbamoyl were also measured. Modification of lysine-8, -13, -27, -72, -79, and -87 surrounding the heme crevice significantly lowered the rate of reaction, while modification of lysines in other regions had no effect. This indicates that the reaction of horse heart cytochrome c with the reaction center also involves the heme crevice domain.     

Use of specific trifluoroacetylation of lysine residues in cytochrome c to study the reaction with cytochrome b5, cytochrome c1, and cytochrome oxidase

The preparation, purification, and characterization of four new derivatives of cytochrome c trifluoroacetylated at lysines 72, 79, 87, and 88 are reported. The redox reaction rates of these derivatives with cytochrome b₅, cytochrome c₁ and cytochrome oxidase indicated that the interaction domain on cytochrome c for all three proteins involves the lysines immediately surrounding the heme crevice. Modification of lysines 72, 79, and 87 had a large effect on the rate of all three reactions, while modification of lysine 88 had a very small effect. Even though lysines 87 and 88 are adjacent to one another, lysine 87 is at the top left of the heme crevice oriented towards the front of cytochrome c, while lysine 88 is oriented more towards the back. Since the interaction sites for cytochrome c₁ and cytochrome oxidase are essentially identical, cytochrome c probably undergoes some type of rotational diffusion during electron transport.     

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.     

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.     

Binding of arylazidocytochrome c derivatives to beef heart cytochrome c oxidase: cross-linking in the high- and low-affinity binding sites

Two arylazidocytochrome c derivatives, one modified at lysine-13 and the second modified at lysine-22, were reacted with beef heart cytochrome c oxidase. The lysine-13 modified arylazidocytochrome c was found to cross-link both to the enzyme and with lipid bound to the cytochrome c oxidase complex. The lysine-22 derivative reacted only with lipids. Cross-linking to protein was through subunit II of the cytochrome c oxidase complex, as first reported by Bisson et al. [Bisson, R., Azzi, A., Gutweniger, H., Colonna, R., Monteccuco, C., & Zanotti, A. (1978) J. Biol. Chem. 253, 1874]. Binding studies show that the cytochrome c derivative covalently bound to subunit II was in the high-affinity binding site for the substrate. Evidence is also presented to suggest that cytochrome c bound to the lipid was in the low-affinity binding site [as defined by Ferguson-Miller et al. [Ferguson-Miller, S., Brautigan, D. L., & Margoliash, E. (1976) J. Biol. Chem. 251, 1104]]. Covalent binding of the cytochrome c derivative into the high-affinity binding site was found to inhibit electron transfer even when native cytochrome c was added as a substrate. Inhibition was almost complete when 1 mol of the Lys-13 modified arylazidocytochrome c was covalently bound to the enzyme per cytochrome c oxidase dimer (i.e., congruent to 280 000 daltons). Covalent binding of either derivative with lipid (low-affinity site) had very little effect on the overall electron transfer activity of cytochrome c oxidase. These results are discussed in terms of current theories of cytochrome c-cytochrome c oxidase interactions.     

Pyridoxalphosphate-modified derivatives of cytochrome c. Mono- and disubstituted derivatives: characteristics and effect on electron transport in cytochrome c-depleted mitochondria

Cytochrome c is modified by covalent binding of pyridoxal phosphate (PLP) to lysine residues. One di-substituted [(PLP)2--C] and two mono-substituted derivatives [(PLP)--c and (PLP)'--c] were obtained and precisely purified. The peak at 695 nm and CD-spectra in 190--600 nm region show that all derivatives have native conformation. The differential UV-spectra of the derivatives against native protein show that in (PLP)2--c there is a contact dipole-dipole interaction between PLP chromophores. It is calculated that the N-atoms of the two PLP-substituted lysines must be at a distance less than or equal to 12 A. Analysing our and literature data, one may suppose that Lys-13 and Lys-87 are the most probable candidates for modification with PLP. (PLP)---c and (PLP)'--c behave differently during ion-exchange chromatography and when added to cytochrom c-depleted mitochondria. (PLP)'--c restores electron transfer at higher concentrations than (PLP)'--c. Both they restore fully succinate and ascorbate oxidation but at considerably higher concentrations than the native protein, i. e. modification of any one of the reactive towards PLP lysines descreases but does not exclude the interaction with its reductase and oxidase. The effective equilibrium constants of binding of modified derivatives to cytochrome c-depleted mitochondria are lower than the constant for native protein. Together with decrease in binding activity, Hill coefficients increase. From our results it may be supposed that probably the binding sites of cytochrome c for its reductase and oxidase partially overlap.     

Ion binding to lysine-modified derivatives of cytochrome c.

The effect of Cl- and K+ ions on the apparent equilibrium constant of the reaction between horse ferricytochrome c and potassium ferrocyanide was studied. Unmodified cytochrome was compared with two lysine-modified derivatives. One, guanidinated, had all lysyl groups converted to homoarginine (but retained the same positive charge); the other was trinitrophenylated at one lysine (measured spectrophotometrically). Both modified derivatives had a somewhat larger equilibrium constant in the reaction of the reduced protein with ferricyanide, but, unlike trifluoroacetylated cytochrome c (which has a negative charge), the redox properties were not dramatically different. The native protein and the lysine-modified cytochromes showed differential K+ binding in Tris-cacodylate buffer at constant ionic strength (0.003-0.005 M). More K+ was bound to ferrocytochrome c. This redox-linked binding, however, was unaffected by modification of lysine. All three derivatives also showed redox-linked differential Cl- ion binding (more Cl- ion was bount to ferricytochrome); however, in this case, the binding was reduced in the lysine-modified molecules. This was interpreted as loss of a single anion site. This anion site critically depends on one or a few lysines which are more reactive with trinitrobenzene sulfonate.     

The reaction of cytochromes c and c2 with the Rhodospirillum rubrum reaction center involves the heme crevice domain

In order to define the interaction domain on Rhodospirillum rubrum cytochrome c2 for the photosynthetic reaction center, positively charged lysine amino groups on cytochrome c2 were modified to form negatively charged carboxydinitrophenyl lysines. The reaction mixture was separated into six different fractions by ion exchange chromatography on carboxymethylcellulose and sulfopropyl-Sepharose. Peptide mapping studies indicated that fraction A consisted of a mixture of singly labeled derivatives modified at lysines 58, 81, and 109 on the back of cytochrome c2. Fractions C1, C2, C3, and C4 were found to be 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 photooxidation of the carboxydinitrophenyl-cytochrome c2 derivatives by reaction centers purified from R. rubrum was measured following excitation with a laser pulse. The second-order rate constant of fraction A modified at backside lysines was found to be 2.3 X 10(7) M-1 s-1, nearly the same as that of native cytochrome c2, 2.6 X 10(7) M-1 s-1. However, the rate constants of fractions C1-C4 were found to be 6 to 12-fold smaller than that of native cytochrome c2. These results indicate that lysines surrounding the heme crevice of cytochrome c2 are involved in electrostatic interactions with carboxylate groups at the binding site of the reaction center. 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, or 87 surrounding the heme crevice was found to significantly lower the rate of reaction, while modification of lysines in other regions had no effect. This indicates that the reaction of horse heart cytochrome c with the reaction center also involves the heme crevice domain.     

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.     

Use of specific lysine modifications to identify the site of reaction between cytochrome c and ferricyanide

The site of the reaction between horse heart ferrocytochrome c and ferricyanide was investigated by measuring the reaction rate of cytochrome c derivatives specifically modified at single lysine residues to form trifluoroacetyl or trifluoromethylphenylcarbamyl amino groups. Cytochrome c derivatives singly modified at lysines 8, 13, 25, 27, 72, 79, and 87 surrounding the heme crevice had rate constants decreased from that of native cytochrome c by factors of 1.29, 2.03, 1.12, 1.35, 1.46, 1.29, and 1.19, respectively. Modification of a given lysine with the bulky trifluoromethylphenylcarbamyl group caused nearly the same decrease in reaction rate as modification with the trifluoroacetyl group, indicating that the effect was due to removal of an electrostatic interaction between the protonated lysine amino group and ferricyanide. Modification of lysines 22, 55, 99, and 100 at the right side, bottom, and back of cytochrome c had no effect on the reaction rate. These results indicate that the reaction site is located at the exposed edge of the heme and that the electrostatic interaction between ferricyanide and cytochrome c is dominated by the lysine amino groups surrounding the heme crevice, which include lysine 86, in addition to the ones listed above. We have used the specific lysine modification results to estimate the contribution of each lysine amino group to the electrostatic interaction and have developed a semiempirical relation for the total electrostatic interaction.     

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.     

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)     

An enzyme kinetics and 19F nuclear magnetic resonance study of selectively trifluoroacetylated cytochrome c derivatives.

The reaction of cytochrome c with ethyl thioltrifluoroacetate was carried out under conditions which led to the selective trifluoroacetylation of a small number of the 19 lysines. The mixture of derivatives was separated by ion-exchange chromatography and four different derivatives with well-resolved 19F nuclear magnetic resonance (NMR) spectra were obtained. Peptide mapping techniques indicated that one of these derivatives contained a single trifluoroacetyl group at lysine 22, and another derivative was singly labeled at lysine 25. The trifluoroacetylated lysine 22 derivative was fully active toward both succinate-cytochrome c reductase (EC 1.3.99.1) and cytochrome oxidase (EC 1.9.3.1) white the trifluoroacetylated lysine 25 derivative was fully active toward the reductase, but had a threefold greater Michaelis constant in the cytochrome oxidase reactin. This supports the hypothesis that the cytochrome oxidase binding site is located in the heme cervice region, and that Lys-25 is important in the binding. 19FNMR spectra of the cytochrome c derivatives bound to phospholipid vesicles were obtained. The reasonably narrow line widths (35-65 Hz) and good sensitivity of the trifluoroacetyl resonances indicated that they might be useful probes for the interaction of cytochrome c with intact mitochondria.