Effect of pH on axial ligand coordination of cytochrome c" from Methylophilus methylotrophus and horse heart cytochrome c

The effect of protons on the axial ligand coordination and on structural aspects of the protein moiety of cytochrome c' ' from Methylophilus methylotrophus, an obligate methylotroph, has been investigated down to very low pH (i.e., 0.3). The unusual resistance of this cytochrome to very low pH values has been exploited to carry out this study in comparison with horse heart cytochrome c. The experiments were undertaken at a constant phosphate concentration to minimize the variation of ionic strength with pH. The pH-linked effects have been monitored at 23 degrees C in the oxidized forms of both cytochromes by following the variations in the electronic absorption, circular dichroism and resonance Raman spectra. This approach has enabled the conformational changes of the heme surroundings to be monitored and compared with the concomitant overall structural rearrangements of the molecule. The results indicate that horse heart cytochrome c undergoes a first conformational change at around pH 2.0. This event is possibly related to the cleavage of the Fe-Met80 bond and a likely coordination of a H(2)O molecule as a sixth axial ligand. Conversely, in cytochrome c" from M. methylotrophus, a variation of the axial ligand coordination occurs at a pH that is about 1 unit lower. Further, it appears that a concerted cleavage of both His ligands takes place, suggesting indeed that the different axial ligands present in horse heart cytochrome c (Met/His) and in cytochrome c" from M. methylotrophus (His/His) affect the heme conformational changes.     

Studies on cytochrome oxidase. Interactions of the cytochrome oxidase protein with phospholipids and cytochrome c.

1. By the application of the principle of the sequential fragmentation of the respiratory chain, a simple-method has been developed for the isolation of phospholipid-depleted and phospholipid-rich cytochrome oxidase preparations. 2. The phospholip-rich oxidase contains about 20% lipid, including mainly phosphatidylethanolamine, phosphatidylcholine, and cardiolipin. Its enzymic activity is not stimulated by an external lipid such as asolectin. 3. The phospholipid-depleted oxidase contains less than 0.1% lipid. It is enzymically inactive in catalyzing the oxidation of reduced cytochrome c by molecular oxygen. This activity can be fully restored by asolectin; and partially restored (approximately 75%) by purified phospholipids individually or in combination. The activity can be partially restored also by phospholipid mixtures isolated from mitochondria, from the oxidase itself, and from related preparations. Among the detergents tested only Emasol-1130 and Tween 80 show some stimulatory activity. 4. The phospholipid-depleted oxidase binds with cytochrome c evidently by "protein-protein" interactions as does the phospholipid-rich or the phospholipid-replenished oxidase to form a complex with the ratio of cytochrome c to heme a of unity. The complex prepared from phospholipid-depleted cytochrome oxidase exhibits a characteristic Soret absorption maximum at 415 nm in the difference spectrum of the carbon monoxide-reacted reduced form minus the reduced form. This 415-nm maximum is abolished by the replenishment of the complex with a phospholipid or by the dissociation of the complex in cholate or in a medium of high ionic strength. When ascorbate is used as an electron donor, the complex prepared from phospholipid-depleted cytochrome oxidase does not cause the reduction of cytochrome a3 which is in dramatic contrast to the complex from the phospholipid-rich or the phospholipid-replenished oxidase. However, dithionite reduces cytochrome a3 in all of the preparations of the cytochrome c-cytochrome oxidase complex. These facts suggest that the action of phospholipid on the electron transfer in cytochrome oxidase may be at the step between cytochromes a and a3. This conclusion is substantiated by preliminary kinetic results that the electron transfer from cytochrome a to a3 is much slower in the phospholipid-depleted than in phospholipid-rich or phospholipid-replenished oxidase. On the basis of the cytochrome c content, the enzymic activity has been found to be about 10 times higher in the system with the complex (in the presence of the replenishedhe external medium unless energy is provided, and that     

Effects of crystallization on the heme-carbon monoxide moiety of bovine heart cytochrome c oxidase carbonyl.

Cytochrome c oxidase isolated from bovine heart was crystallized in the fully reduced carbon monoxide (CO)-bound form. To evaluate the structure of the O2 reaction site in crystals and in solution, the bound C-O stretch infrared band in protein crystals was compared with the band for protein solution. In solution, the C-O stretch band could be deconvoluted into two extremely narrow bands, one at 1963.6 cm-1 with delta v1/2 = 3.4 cm-1 of 60% Gaussian/40% Lorentzian character represented 86% of the total band area and the other at 1960.3 cm-1 with delta v1/2 = 3.0 cm-1 of 47% Gaussian/53% Lorentzian character represented 14% of the total band area. The crystals exhibited two deconvoluted C-O infrared bands having very similar band parameters with those in solution. These findings support the presence of two structurally similar conformers in both crystals and solution. Thus crystallization of this enzyme does not affect the structure at the CO-binding site to as great extent as has been noted for myoglobin and hemoglobin carbonyls, indicating that the active (CO- or O2-binding) site of cytochrome c oxidase must be conformationally very stable and highly ordered compared to other hemoproteins such as hemoglobin.     

ATP induces conformational changes in mitochondrial cytochrome c oxidase. Effect on the cytochrome c binding site

ATP influences the kinetics of electron transfer from cytochrome c to mitochondrial oxidase both in the membrane-embedded and detergent-solubilized forms of the enzyme. The most relevant effect is on the so-called "high affinity" binding site for cytochrome c which can be converted to "low affinity" by millimolar concentrations of ATP (Ferguson-Miller, S., Brautigan, D. L., and Margoliash, E. (1976) J. Biol. Chem. 251, 1104-1115). This phenomenon is characterized at the molecular level by the following features. ATP triggers a conformational change on the water-exposed surface of cytochrome c oxidase; in this process, carboxyl groups forming the cluster of negative charges responsible for binding cytochrome c change their accessibility to water-soluble protein modifier reagents; as a consequence the electrostatic field that controls the enzyme-substrate interaction is altered and cytochrome c appears to bind differently to oxidase; photolabeling experiments with the enzyme from bovine heart and other eukaryotic sources show that ATP cross-links specifically to the cytoplasmic subunits IV and VIII. Taken together, these data indicate that ATP can, at physiological concentration, bind to cytochrome c oxidase and induce an allosteric conformational change, thus affecting the interaction of the enzyme with cytochrome c. These findings raise the possibility that the oxidase activity may be influenced by the cell environment via cytoplasmic subunit-mediated interactions.     

Effect of the hinge protein on the heme iron site of cytochrome c1

X-ray absorption spectroscopic (XAS) studies on cytochrome C1 from beef heart mitochondria were conducted to identify the effect of the hinge protein [Kim, C.H., & King, T.E. (1983) J. Biol. Chem. 258, 13543-13551] on the structure of the heme site in cytochrome c1. A comparison of XAS data of highly purified "one-band" and "two-band" cytochrome c1 [Kim, C.H., & King, T.E. (1987) Biochemistry 26, 1955-1961] demonstrates that the hinge protein exerts a rather pronounced effect on the heme environment of the cytochrome c1: a conformational change occurs within a radius of approximately 5 A from the heme iron in cytochrome c1 when the hinge protein is bound to cytochrome c1. This result may be correlated with the previous observations that the structure and reactivity of cytochrome c1 are affected by the hinge protein [Kim, C.H., & King, T.E. (1987) Biochemistry 26, 1955-1961; Kim, C.H., Balny, C., & King, T.E. (1987) J. Biol. Chem. 262, 8103-8108].     

An investigation of protein conformation of cytochrome c by using cytochrome c insolubilized on to agarose gel.

Cytochrome c insolubilized on to agarose gel was shown to be more resistant to denaturation and carboxymethylation than the soluble protein. These differences are discussed both with respect to the conformational changes that take place during denaturation of cytochrome c and with respect to the pH-dependent forms of carboxymethyl-cytochrome c.     

The cytochrome c binding site on cytochrome c oxidase.

Cytochrome $\text{c}$ was chemically coupled to cytochrome $\text{c}$ oxidase using the reagent 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) which couples amine groups to carboxyl residues. The products of this reaction were analyzed on 2.5–27% polyacrylamide gradient gels electrophoretically. Since cytochrome $\text{c}$ binds to cytochrome oxidase electrostatically in an attraction between certain of its lysine residues and carboxyl residues on the oxidase surface, EDC is an especially appropriate reagent probe for binding-subunit studies. Coupling of polylysine to cytochrome oxidase using EDC was also performed, and the products of this reaction indicate that polylysine, an inhibitor of the cytochrome $\text{c}$ reaction with oxidase, binds to the same oxidase subunit as does cytochrome $\text{c}$, subunit IV in the gel system used.     

Reduction of oxidized cytochrome c by ascorbate ion

The kinetics and mechanism of the reduction of oxidized cytochrome c by ascorbate has been investigated in potassium nitrate, potassium 4-morpholineethanesulfonate (KMes), potassium sulfate and potassium ascorbate media. The results are consistent with simple second order electron transfer from ascorbate dianion to cytochrome c and do not support electron transfer from an ascorbate dianion bound to the protein of the cytochrome as recently proposed by Myer and Kumar. A rate constant of 8 X 10(5) M-1 X s-1 (25 degrees C, ionic strength, 0.1) was found for the electron-transfer step. This rate constant is essentially independent of the specific ions used in controlling ionic strength.     

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.     

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.     

Amino acid substitutions at tryptophan-51 of cytochrome c peroxidase: effects on coordination, species preference for cytochrome c, and electron transfer.

Amino acid replacements of an aromatic residue, Trp-51, which is in contact with the heme of yeast cytochrome c peroxidase have a number of significant effects on the kinetics and coordination state of the enzyme. Six mutants at this site (W51F, W51M, W51T, W51C, W51A, and W51G) were examined. Optical and EPR spectra show that each of these mutations introduces a shift from the 5-coordinate to 6-coordinate form, and slightly increases the asymmetry of the heme ligand field. Conversion from a 6-coordinate high-spin form at pH 5 to a 6-coordinate low-spin form at pH 7 is observed for several of the variants (W51F, W51T, and W51A), while W51G and W51C appear as predominantly low-spin species between pH 5 and 7. Addition of 50% glycerol prevents the facile conversion to the low-spin conformation for W51F, W51T, and W51A, and only W51F can be stabilized in a 5-coordinate configuration by glycerol. For the oxidation of cytochrome c by H2O2, three of the variants (W51F, W51M, and W51T) exhibit values of kcat(app) that are greater than for the wild-type enzyme, while the other mutations give decreased rates of enzyme turnover. Unlike the wild-type enzyme, which functions more efficiently with cytochrome c from yeast than with the horse heart protein, the mutant W51F does not show a preference for substrate from its native organism. The three mutants which exhibit increased values of kcat(app) show a pH optimum at 6.8 compared with that of 5.25 for the wild-type enzyme when measured with horse heart cytochrome c. This shift in pH optimum is not observed with yeast cytochrome c. Construction of single and multiple mutations at Trp-51, Ile-53, and Gly-152 shows that these kinetic properties are not due to natural amino acid variations observed at these sites. Pre-steady-state kinetics show that the bimolecular rate constant for the fast phase of the reaction of the enzyme with H2O2 is only slightly decreased from 3.03 (0.09) X 10(7) to 2.2 (0.1) X 10(7) M-1 s-1 for W51F and to 1.5 (0.1) X 10(7) M-1 s-1 for W51A. The slow phase of the reaction (4.9 s-1) which contributes approximately 30% to the amplitude of the change for the wild-type enzyme is not observed for W51F or W51A.(ABSTRACT TRUNCATED AT 400 WORDS)     

Interaction of dimeric horse cytochrome c with cyanide ion

We have previously shown that methionine–heme iron coordination is perturbed in domain-swapped dimeric horse cytochrome c. To gain insight into the effect of methionine dissociation in dimeric cytochrome c, we investigated its interaction with cyanide ion. We found that the Soret and Q bands of oxidized dimeric cytochrome c at 406.5 and 529 nm redshift to 413 and 536 nm, respectively, on addition of 1 mM cyanide ion. The binding constant of dimeric cytochrome c and cyanide ion was obtained as 2.5 × 10⁴ M^?1. The Fe–CN and C–N stretching (ν _Fe–CN and ν _CN) resonance Raman bands of CN^?-bound dimeric cytochrome c were observed at 443 and 2,126 cm^?1, respectively. The ν _Fe–CN frequency of dimeric cytochrome c was relatively low compared with that of other CN^?-bound heme proteins, and a relatively strong coupling between the Fe–C–N bending and porphyrin vibrations was observed in the 350–450-cm^?1 region. The low ν _Fe–CN frequency suggests weaker binding of the cyanide ion to dimeric cytochrome c compared with other heme proteins possessing a distal heme cavity. Although the secondary structure of dimeric cytochrome c did not change on addition of cyanide ion according to circular dichroism measurements, the dimer dissociation rate at 45 °C increased from (8.9 ± 0.7) × 10^?6 to (3.8 ± 0.2) × 10^?5 s^?1, with a decrease of about 2 °C in its dissociation temperature obtained with differential scanning calorimetry. The results show that diatomic ligands may bind to the heme iron of dimeric cytochrome c and affect its stability.     

Interaction of cytochrome c with cytochrome c oxidase studied by monoclonal antibodies and a protein modifying reagent.

C/57 black mice were immunized with beef heart cytochrome c oxidase, generating 48 hybrid cell lines that secrete antibodies against the different subunits of the enzyme. Immunoblot analysis showed reactions with 7 of the 13 subunits. Among the monoclonal antibodies produced, only those to subunit II gave significant inhibition; these inhibited the enzyme activity completely and prevented cytochrome c binding to the enzyme. Epitope mapping studies indicate that a peptide including residues 200-227 reacts with the antibody, suggesting that the C-terminus of the protein is essential for the binding of this antibody. The carboxyl modifying reagent 1-ethyl-3-[3-(trimethylammonio)propyl]carbodiimide (ETC) was chosen to investigate further the relationship between antibody and cytochrome c binding domains. ETC caused 50% inhibition of the enzyme activity with a first-order time during the first 20 min; a slower reaction over 3 h resulted in 90% inhibition. Cytochrome c binding to the oxidase was inhibited to a similar extent as cytochrome c oxidation, and protection against both effects was afforded by the presence of cytochrome c during ETC modification. Anion-exchange of FPLC of the modified forms of cytochrome oxidase revealed extensive inhomogeneity, indicating random derivatization of a number of different carboxyls even during the first-order reaction, and precluding identification of carboxyl residues related to a specific phase of the reaction. Cytochrome c and the subunit II-specific antibody protected against radioactive labeling of subunit II by ETC in the presence of [14C]glycine ethyl ester, demonstrating that the antibody and cytochrome c occupy significant and overlapping areas on the subunit II surface.(ABSTRACT TRUNCATED AT 250 WORDS)