Binding Site For Cytochrome Research Articles

Topology and neighbor analysis of the photosynthetic reaction center from Rhodopseudomonas sphaeroides.

The subunit arrangement of the reaction center complex (RC) of Rhodopseudomonas sphaeroides was studied by chemical modification with four different cross-linking reagents using purified RC in lauryldimethylamine oxide, RC incorporated into liposomes, and intact chromatophore membranes, from which RCs are isolated. The RC of R. sphaeroides is composed of three polypeptide subunits, H, M, and L, apparent molecular mass as determined in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, of 28,000, 24,000, and 21,000, respectively. The intra-complex products produced, were found to contain the polypeptides H-M-L, H-M, H-L, and M-L linked together. In addition, the cross-linking of cytochrome c to solubilized and membrane-bound RCs was observed with all four reagents. The products were found to be only a cytochrome c linked to either the M or L polypeptide. These results indicate that a portion of the L and M subunits of the RC must be exposed in situ on the periplasmic surface of the membrane near a binding site for cytochrome c on the RC, and all three subunits must be in close proximity to one another.

Structure of an electron transfer complex. II. Chemical modification of carboxyl groups of cytochrome c peroxidase in presence and absence of cytochrome c.

Cytochrome c peroxidase forms an electron transfer complex with cytochrome c. The complex is governed by ionic bonds between side chain amino groups of cytochrome c and carboxyl groups of peroxidase. To localize the binding site for cytochrome c on the peroxidase, we have used the method of differential chemical modification. By this method the chemical reactivity of carboxyl groups (toward carbodiimide/aminoethane sulfonate) was compared in free and in complexed peroxidase. When ferricytochrome c was bound to cytochrome c peroxidase, acidic residues 33, 34, 35, 37, 221, 224, and 1 to 3 carboxyls at the C terminus became less reactive by a factor of approximately 4, relative to the remaining 39 carboxylates of peroxidase. Of the less reactive residues those in the 30-40 region and the 221/224 pair are on opposite sides of the surface area which contains the heme propionates. We, therefore, propose that the binding site for cytochrome c on cytochrome c peroxidase spans the area where one heme edge comes close to the molecular surface. The results are in very good agreement with chemical cross-linking studies (Waldmeyer, B., and Bosshard, H.R. (1985) J. Biol. Chem. 260, 5184-5190); they also support a hypothetical model predicted on the basis of the known crystal structures of cytochrome c and peroxidase (Poulos, T.L., and Kraut, J. (1980) J. Biol. Chem. 255, 10322-10330).

Spectroscopic analysis of the interaction between cytochrome c and cytochrome c oxidase.

Complex formation between cytochrome c oxidase and cytochrome c perturbs the optical absorption spectrum of heme c and heme a in the region of the alpha-, beta, and gamma-bands. The perturbations have been used to titrate cytochrome c oxidase with cytochrome c. A stoichiometry of one molecule of cytochrome c bound per molecule of cytochrome c oxidase is obtained (1 heme c per heme aa3). In contrast, a stoichiometry of 2:1 was found earlier using a gel-filtration method (Rieder, R., and Bosshard, H.R. (1978) J. Biol. Chem. 253, 6045-6053). From the result of the spectrophotometric titration and from the wavelength position of the perturbation signals it is concluded that cytochrome c oxidase contains only a single binding site for cytochrome c which is close enough to heme a to function as an electron transfer site. The second site detected earlier by the gel-filtration method must be remote from this electron transfer site. Scatchard plots of the titration data are curvilinear, possibly indicating interactions between cytochrome c-binding sites on adjacent monomers of dimeric cytochrome c oxidase. The relationship between cytochrome c binding and the reaction of cytochrome c oxidase with ferrocytochrome c is discussed.

Reaction of thionitrobenzoate-modified yeast cytochrome c with monomeric and dimeric forms of beef heart cytochrome c oxidase

Thionitrobenzoate-modified yeast cytochrome c was shown to react with both monomeric and dimeric forms of beef heart cytochrome c oxidase through subunit III. This cytochrome c derivative was found to inhibit electron transfer in the dimer but not in the monomer. These results are interpreted to show that the high affinity binding site for cytochrome c is a cleft at the interface between monomers in the cytochrome c oxidase dimer.

Study of the Hansenula anomala yeast flavocytochrome-b2-cytochrome-c complex 2. Localization of the main association area.

The reversible association of the Zn2+-substituted Hansenula anomala cytochrome c dimer (Thomas et al., preceding paper in this issue) to flavocytochrome b2 in oxidized or lactate-reduced state has been investigated by fluorimetry. The same method has been used for the determination of Zn-cytochrome c complexing to defined proteolytic fragments of flavocytochrome b2, either heme-b2-containing monomers or a flavin-linked tetramer. All these fragments but the isolated cytochrome b2 core showed binding stoichiometries, Kd values and ionic strength dependences quite similar to those found for native flavocytochrome b2. These data allowed localization of the single high-affinity binding site of cytochrome c on a particular globule in the dehydrogenase domain of the flavocytochrome b2 protomers. Quenching of the Zn-porphyrin c fluorescence in the various complexes occurred with only minor changes of the fluorescence lifetime and did not show any direct relationship to the presence or the redox state of the heme b2 group.

Photosystem I reaction centers from Chlamydomonas and higher plant chloroplasts.

A photosystem I reaction center has been isolated from Chlamydomonas chloroplasts and compared with the photosystem I reaction center from higher plants. While the higher plant reaction center is active in cytochrome 552 photooxidation, the Chlamydomonas preparation was not active unless salts were included in the assay medium or the pH was lowered to 5. Subunit III-depleted photosystem I reaction center from higher plants is also inactive in cytochrome 552 photooxidation in the absence of salts. As with the Chlamydomonas reaction center, salts induced its activity. Subunit I of the photosystem I reaction center has tentatively been identified as the binding site of cytochrome 552.

Interactions of cytochrome c with mitochondrial membranes. Binding to succinate-cytochrome c reductase.

Methyl-4-azidobenzoimidate was reacted with horse heart cytochrome c to give a photoaffinity-labeled derivative of this heme protein. The modified cytochrome c bound to cytochrome c-depleted mitochondria with the same Kd as native cytochrome c and restored oxygen uptake to the same extent. Irradiation of cytochrome c-depleted mitochondrial membranes with 3- to 4-fold excess of photoaffinity-labeled cytochrome c over cytochrome c oxidase resulted in covalent binding of the derivative to the membranes. Fractionation of the irradiated mitochondria in the presence of detergents and salts followed by chromatography on an agarose Bio-Gel-A-5m showed that the labeled cytochrome c was bound covalently to succinate-cytochrome c reductase. The covalently bound cytochrome c was active in mediating electron transfer between its reductase and oxidase. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the succinate-cytochrome c reductase containing photoaffinity-labeled 125I-cytochrome c showed that the reductase contained a protein binding site for cytochrome c. It is suggested that cytochrome c1 is the most likely site for the cytochrome c binding in mitochondria in situ.

Photoaffinity labeling of yeast cytochrome oxidase with arylazido cytochrome c derivatives

The interaction of cytochrome c with cytochrome c oxidase, and the electron transfer occurring in the complex, are important events in the mitochondrial respiratory chain, leading to oxygen reduction, asymmetric proton release and conservation of the free energy of the redox reaction as an electrochemical gradient of hydrogen ions [l-3] . Among the approaches utilized to elucidate the structure of the cytochrome oxidase-cytochrome c complex, crosslinking of the components of the system has been recently employed by a number of investigators. Using yeast cytochrome c, modified at its free sulihydryl group by the reaction with 5,5’-dithiobis (2-nitrobenzoate), subunit III of yeast cytochrome c oxidase was indicated [4] to be close to the binding site for cytochrome c. Using horse heart cytochrome c labeled with a 3nitrophenylazido group at lysine 13, evidence was provided [5,6] that subunit II was the subunit nearest to cytochrome c in the complex with the beef-heart oxidase. This conclusion was also supported by [7] in which dithiobissuccininidylpropionate was used as crosslinking reagent. Using an azidocytochrome c derivative, the binding site of cytochrome c was found [S] to occur in the two smallest

Formation of a complex between yeast l-lactate dehydrogenase (cytochrome b2) and cytochrome c: Ultracentrifugal and gel chromatographic analyses

Yeast l-lactate dehydrogenase formed a stable complex with cytochrome c in weakly alkaline solution of low ionic strength. The binding ratio of cytochrome c to the enzyme depended on whether free cytochrome c was present: In the presence of a micromolar concentration of cytochrome c the enzyme formed a complex with about two molecules of cytochrome c, whereas the enzyme was in a 1:1 molecular complex after removal of free cytochrome c. This suggests that the binding of one molecule of cytochrome c changes the affinity of the other binding site on the enzyme for cytochrome c. The enzyme consists of four presumably identical subunits, each containing a binding site for cytochrome c. Thus, present data confirm the concept of negative cooperativity between the subunits of the enzyme molecule in their interaction with cytochrome c.

Biochemical and biophysical studies on cytochrome c oxidase. XIX. An EPR study of the photodissociation of carboxycytochrome c oxidase in the presence of azide

1. The photodissociation reaction of the cytochrome c oxidase-CO compound in the presence of azide was studied by EPR at 15°K. Addition of CO in the dark to cytochrome c oxidase, partially reduced (2 electrons per 4 metal ions) in the presence of azide brings about a decrease in intensity of the azide-induced low-spin heme signal at g = 2.9, 2.2 and 1.67 and an increase in intensity of both the low-spin heme signal at g = 3 and the copper signal at g = 2. Subsequent illumination with white light at room temperature of this sample causes an enhancement of the azide-induced signal at g = 2.9, and a decrease in intensity of both signals at g = 3 and g = 2. It is shown that these changes in the EPR spectrum are reversible. 2. These results demonstrate that upon photodissociation, CO is replaced by azide wheras upon incubation in the dark CO expels azide from its binding site in cytochrome c oxidase. 3. Concomitantly with the binding of CO and dissociation of the azide molecule, and vice versa, electron redistributions occur as inferred from the changes in the intensity of the copper signal at g = 2. 4. The results are explained in a model of cytochrome c oxidase with either a common binding site (cytochrome a 3) ∗ ∗ For practical reasons we maintain the cytochrome a and cytochrome a and cytochrome a 3 terminology. Cytochrome a 3 then is that part of the protein in which the heme group is less readily reduced (cyanide and azide) or oxidized (CO and NO) in the presence of ligands. for CO and azide or in a model with anti-cooperative interaction between two different sites of binding. 5. Similar types of experiments with cyanide instead of azide show that cyanide is more firmly bound to partially reduced cytochrome c oxidase than CO and azide. The affinity of ligands for partially reduced enzyme decreases in the sequence: cyanide, CO (dark), azide and CO (illuminated).