Intramolecular electron transfer in cytochrome c and complex of cytochrome c with cytochrome oxidase

Información del proyecto

Identificador del acuerdo de subvención: INTAS-2001-00002

Proyecto cerrado

Fecha de inicio 1 Junio 2002

Fecha de finalización 31 Mayo 2005

Financiado con arreglo a

• International Association for the promotion of cooperation with scientists from the independent states of the former Soviet Union (INTAS), 1993-

Coste total

Sin datos

Aportación de la UE

€ 90 000,00

Coordinado por

Tel Aviv University
Israel

Objetivo

The proposal is aimed at studying the mechanism of intramolecular electron transfer in cytochrome c and in the complex of cytochrome c with cytochrome oxidase.
In the first phase of the work we are going to investigate the mechanism of intramolecular electron transfer in cytochrome c. The electron transfer will be initiated by an intensive pulse illumination of thiouredopyrene-trisulfonate (TUPS), a reaction that generates a strong reductant. The SH-modifying form of TUPS will be covalently attached to desired sites of cytochrome c, e.g. to SH-groups introduced into the protein by site directed mutagenesis. Intramolecular reduction of the heme iron by the excited form of the dye will be followed by transient absorption spectroscopy looking at heme reduction. The high yield of heme reduction and the ability to follow the reaction on time scales from hundreds of picoseconds to milliseconds will allow us to determine the rate constants of electron transfer with high level of accuracy and reliability. The dependence of the electron transfer rates on the distance between the heme and different sites on the protein surface will be analysed to estimate possible electron transfer pathways in cytochrome c.

With this knowledge at hand we will directionally replace by site-directed mutagenesis "non-conductive" amino acids with "conductive" ones and vice versa along calculated pathways, looking at the effect of replacement on the rate of electron transfer. The data we are going to accumulate will serve as experimental basis for understanding the role of protein structural elements in electron conductivity.
The second phase of the work will be devoted to the investigation of the electron transfer mechanism of cytochrome oxidase. To achieve this goal we will produce TUPS-cytochrome c derivatives, which are capable of undergoing fast (in less than 1 ms) self-reduction upon illumination, and also capable of forming functionally active complexes with cytochrome oxidase. We will use the knowledge accumulated in the first phase of the research for selecting "fast" derivatives. The derivatives will be prepared by the covalent attachment of SH-specific TUPS label to a singular cysteine residue introduced at desired sites of cytochrome c by site-directed mutagenesis. The time resolved kinetics of electron redistribution within the complex of TUPS-cytochrome c with cytochrome oxidase will be measured by recording transient difference spectra in a wide range of wavelengths on time scales from hundreds of picoseconds to milliseconds. The analysis of the spectral data by singular value decomposition with self-modeling, combined with a global exponential fitting procedure will yield the spectra of the intermediates involved in the electron transfer in cytochrome oxidase and the individual rate constants associated with the electron transfer steps.

The effect of cytochrome oxidase inhibitors (CN-, N3-) as well as the effect of pH on the individual rate constants will be studied.
The third phase of the work will be devoted to the investigation of the electrogenic mechanism of COX. We will employ TUPS-cytochrome c derivatives as efficient photoinduced electron donors and will utilize the unique electrometric approach developed by Professor achieve in A.N.Belozersky Institute of Moscow State University, for studying charge translocation in COX.

The main points to be addressed in these studies, regarding the electrogenic mechanism of the enzyme, are:
1. Electrogenic nature of CuA reduction by bound cytochrome c;
2. Coupling of heme a reduction with vectorial proton movement;
3. Kinetics of the electrogenic phase associated with electron transfer from CuA to heme a.

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Programa(s)

• IC-INTAS - International Association for the promotion of cooperation with scientists from the independent states of the former Soviet Union (INTAS), 1993-

Tema(s)

• 4 - Life Sciences
• OPEN - OPEN Call

Convocatoria de propuestas

Datos no disponibles

Régimen de financiación

Datos no disponibles

Coordinador

Tel Aviv University

• Aportación de la UE: Sin datos
• Dirección: Ramat Aviv ; 69978 Tel Aviv ; Israel
• Coste total: Sin datos

Participantes (4)

Hungarian Academy of Sciences

Hungría

• Aportación de la UE: Sin datos
• Dirección: Temesvari krt. 62 ; 6701 Szeged
• Coste total: Sin datos

M.V. Lomonosov Moscow State University

Rusia

• Aportación de la UE: Sin datos
• Dirección: Vorobjevy Gory, Khokhlova 6 ; 119899 Moscow
• Coste total: Sin datos

National Academy of Sciences of Belarus

Bielorrusia

• Aportación de la UE: Sin datos
• Dirección: F. Skaryna Ave., 70 ; 220072 Minsk
• Coste total: Sin datos

Russian Academy of Sciences

Rusia

• Aportación de la UE: Sin datos
• Dirección: 16/10 Miklukho-Maklaya Street ; 117997 Moscow
• Coste total: Sin datos