Lysine Residues Of Cytochrome Research Articles

Factors Affecting Protein Transfer into Surfactant-Isooctane Solution: A Case Study of Extraction Behavior of Chemically Modified Cytochrome c

The extraction mechanism of proteins by surfactant molecules in an organic solvent has been investigated using a chemically modified protein. We conducted guanidylation on lysine residues of cytochrome c by replacing their amino groups with homoarginine to enhance the protein-surfactant interaction. Results have shown that guanidylated cytochrome c readily forms a hydrophobic complex with dioleyl phosphoric acid (DOLPA) through hydrogen bonding between the phosphate moiety and the guanidinium groups. Although improved protein-surfactant interaction activated the formation of a hydrophobic complex at the interface, it could not improve the protein transfer in isooctane. It has been established that the protein extraction mechanism using surfactant molecules is mainly governed by two processes: formation of an interfacial complex at the oil-water interface and the subsequent solubilization of the complex into the organic phase. In addition, a kinetic study demonstrated that guanidylation of lysine accelerated the initial extraction rate of cytochrome c. This fact implies that the protein transferability from aqueous phase into organic phase depends on the protein-surfactant interaction which can be modified by protein surface engineering.

Application of Promoter Modified Electrodes to Bioelectrochemical Measurements on the Effects of Origin and Modification of Lysine Residues of Cytochrome c

Using promoter-modified electrodes, electron-transfer reactions of cytochrome c at an electrode and with cytochrome c oxidase were examined. The electrochemical behavior of cytochromes c from horse, bovine, chicken and tuna hearts at an electrode was similar to each other. Little difference was observed for the reactions between cytochromes c of various origins and cytochrome c oxidase from bovine in a phosphate buffer solution. Also, when a few (less than three) lysine residues of cytochrome c were treated with 4-chloro-3,5-dinitrobenzoic acid (CDNP) or 2,4-dinitrofluorobenzene (DNP), the electrode reaction of the modified (especially, mono-substituted) cytochrome c still showed no significant difference from that of the native one. On the other hand, the electron-transfer reaction between CDNP- or DNP-substituted cytochrome c at lysine 13 and/or 72 and cytochrome c oxidase was greatly affected, even when only one or two lysine residues of cytochrome c were modified. Since the cytochrome c molecules of different origins examined have about 3–19 differences in their amino acid sequences, but most lysine residues remain invariant, the present results indicate that lysine residues of cytochrome c play an important role in the interaction with cytochrome c oxidase; however, the promoter modified electrode surface does not recognize cytochrome c molecule very rigorously. The present study also shows that electrochemical techniques with the aid of functional electrodes are applicable as useful, convenient methods to analyze the biological reactions of proteins.

Electrical communication between horse heart cytochrome c and electrodes in the presence of DNA or RNA

Electrical communication between electrodes and enzymes has received active interest from the viewpoints of the clarification of electron transport mechanisms in biological systems, and of applications to biochemical sensors and biocatalyst electrodes. As has been reviewed by Armstrong et al. [l], direct electron exchange between electrodes and cytochrome c, one of the electron transport enzymes in mitochondria, has been successfully investigated by using metal oxide electrodes like In,O, [2], and promotors such as 4,4’-bipyridyl [3], dithiobis(ethanoic acid) [4], and bis(4-pyridyl)disulfide [5]. A feature common to the above promotors is that electron-rich nitrogen atoms or anions, which are able to interact with positively charged lysine residues in cytochrome c, are present at several tenths of a nm apart from electrode surfaces at the adsorbed state. We chose DNA or RNA and studied their suitability as a promotor, because they have negative charges and are actually present in mitochondria. As a result, we discovered that DNA or RNA works as an effective promotor by interacting electrostatically with the positively charged patch of cytochrome c.

Lipid and subunit III depleted cytochrome c oxidase purified by horse cytochrome c affinity chromatography in lauryl maltoside.

Cytochrome oxidase is purified from rat liver and beef heart by affinity chromatography on a matrix of horse cytochrome c-Sepharose 4B. The success of this procedure, which employs a matrix previously found ineffective with beef or yeast oxidase, is attributed to thorough dispersion of the enzyme with nonionic detergent and a low density of cross-linking between the lysine residues of cytochrome c and the cyanogen bromide activated Sepharose. Beef heart oxidase is purified in one step from mitochondrial membranes solubilized with lauryl maltoside, yielding an enzyme of purity comparable to that obtained on a yeast cytochrome c matrix [Azzi, A., Bill, K., & Broger, C. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 2447-2450]. Rat liver oxidase is prepared by hydroxyapatite and horse cytochrome c affinity chromatography in lauryl maltoside, yielding enzyme of high purity (12.5-13.5 nmol of heme a/mg of protein), high activity (TN = 270-400 s-1), and very low lipid content (1 mol of DPG and 1 mol of PI per mol of aa3). The activity of the enzyme is characterized by two kinetic phases, and electron transfer can be stimulated to maximal rates as high as 650 s-1 when supplemented with asolectin vesicles. The rat liver oxidase purified by this method does not contain the polypeptide designated as subunit III. Comparisons of the kinetic behavior of the enzyme in intact membranes, solubilized membranes, and the purified delipidated form reveal complex changes in kinetic parameters accompanying the changes in state and assay conditions, but do not support previous suggestions that subunit III is a critical factor in the binding of cytochrome c at the high-affinity site on oxidase or that cardiolipin is essential for the low-affinity interaction of cytochrome c. The purified rat liver oxidase retains the ability to exhibit respiratory control when reconstituted into phospholipid vesicles, providing definitive evidence that subunit III is not solely responsible for the ability of cytochrome oxidase to produce or respond to a membrane potential or proton gradient.

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 b 5, cytochrome c 1 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 c 1 and cytochrome oxidase are essentially identical, cytochrome c probably undergoes some type of rotational diffusion during electron transport.

Binding of cytochrome [formula omitted] to the cytochrome [formula omitted]1 complex (complex III) and its subunits cytochrome [formula omitted]1 and [formula omitted

Cytochrome c 1, the electron donor for cytochrome c , is a subunit of the mitochondrial cytochrome bc 1 complex (complex III, cytochrome c reductase). To test if cytochrome c 1 is the cytochrome c -binding subunit of the bc 1 complex, binding of cytochrome c to the complex and to isolated cytochrome c 1 was compared by a gel-filtration method under non-equilibrium conditions (a bc 1 complex lacking the Rieske ironsulfur protein was used; von Jagow et al. (1977) Biochim. Biophys. Acta 462 , 549–558). The approximate stoichiometries and binding affinities were found to be very similar. Binding of cytochrome c to isolated cytochrome b which is another subunit of the reductase was not detectable by the gel-filtration method. Further, the same lysine residues of cytochrome c were shielded towards chemical acetylation in the complexes c: c 1 and c: bc 1. From this we conclude that the same surface area of cytochrome c is in direct contact with cytochrome bc 1 and with cytochrome c 1 in the respective complexes and that therefore cytochrome c is most probably the structural ligand for cytochrome c in mitochondrial cytochrome c reductase.