Alexander B. Kotlyar, Lecturer
Ph.D. Moscow State University 1985
Sherman Building, Room 626
Tel: 972-3-6407138
Fax: 972-3-6406834

Studies on the mechanism of electron and energy transfer in proteins and DNA

Electron transfer reactions are key steps in photosynthesis, respiration, drug metabolism and many other biochemical processes. In order to study long-range electron transfer one should be able to initiate the reaction by injection of oxidant or reductant within a very short time (less than 1ms). This time resolution cannot be achieved either by rapid mixing or by freeze quench techniques. The way to surmount this problem is to employ light pulse as a redox perturbant. Most mammalian proteins are photo inert and thus cannot be oxidized (or reduced) by light. Recently we have worked out a novel approach enabling us to initiate and follow up electron transfer in non-photosynthetic systems with submicrosecond time resolution. The initiation of electron transfer was achieved by photoexcitation of thiouredopyrene-trisulfonate (TUPS) molecule covalently bound to the biological molecules. Single-photon excitation of TUPS yields the low potential triplet state of the dye with high quantum efficiency. High yield, long lifetime and low redox potential of the triplet state make TUPS an efficient initiator of electron transfer processes.
Our research work concentrates on three main subjects: 1) Studies of the electron transfer mechanism in polycytochromic complexes; 2) Intramolecular electron transfer in azurin; and 3) Electron transfer activity of DNA.
1. Respiratory complexes are heme-containing proteins, specialized for efficient electron transfer and the transduction of the released energy into other forms. Functional studies of these proteins have traditionally been limited to biochemical methodologies while time resolved measurements have lagged behind due to inadequate mixing technologies. We are currently utilizing a “TUPS” redox perturbation method for delivering a rapid, reversible, step-function reductive pulse to cytochrome c-cytochrome oxidase complex. The perturbation is achieved by an intensive pulse illumination of TUPS covalently linked to cytochrome c. Rapid intramolecular electron transfer causes rapid reduction of the cytochrome c heme iron. The employment of TUPS-cytochrome c derivatives, in which reduction of the heme is completed within less than 1 m s, provides us with a direct tool for fast reduction of cytochrome c bound to cytochrome oxidase. High yield (more than 20% reduction of cytochrome c within a single pulse) and high time resolution attained by our method (t <200 ns) allow us to determine the rate constants of individual electron transfer steps in the cytochrome c-cytochrome c oxidase complex.
2. In our previous work we have studied the intramolecular electron transfer in cytochrome c. TUPS moiety was introduced to eight different sites of cytochrome c by covalent modification at specific lysine residues and the rate constants for the electron transfer reaction from the photoexcited triplet state of TUPS to the oxidized heme group and the back reaction from the ferrous heme to the oxidized dye were directly measured. Analysis of the variation of electron transfer rates with the distance separating the dye yielded a simple logarithmic dependence, supporting the homogeneous model of electron transfer in proteins. Recently we have expanded our approach for studies of electron transfer mechanism in azurin. The azurin molecule was labeled at several sites by TUPS residue and the electron transfer reactions from the photoexcited TUPS moiety to Cu+2 and back were followed by time-resolved spectroscopy. In contrast to the electron transfer in cytochrome c the dependence of the electron transfer rates on the distance separating the dye and the metal was direction-dependent. To understand the reasons for high and low conductivity along different electron-conducting pathways we intend to examine the role of particular amino acids in the conducting of electrons via the protein matrix. The amino acids will be replaced by side directed mutagenesis, and the effects of the replacements on the rates of electron transfer will be examined.
3. DNA may conduct electrons more efficiently than proteins. According to this view a double helical DNA molecule can be treated as a p stacked conductivity system which allows electrons to move effortlessly as a current through an electrical wire. The importance of the DNA controlled processes supports the need for direct measurements of electron transfer in order to determine whether or not DNA is actually a molecular wire. Such measurements are required to show the passage of electrons from an electron-donating to an electron-accepting group bridged by DNA. Our working model is based on a complementary DNA double helix labeled with electron accepting and electron donating groups at opposite ends. We have so far end-labeled and purified penta-A and penta-T oligonucleotides with TUPS and rhodamine B respectively. The electron transfer from the photoexcited TUPS to the rhodamine was followed in micro-milisecond time scale. No high electron transfer activity of the double helical DNA molecule was detected. The reason may be the nucleotide composition of DNA. In the near future we intend to measure the electron transfer parameters of the guanosine- and cytidine-rich DNA molecules. Comparison of the results from these measurements to those obtained from the adenosine-thymidine model will determine whether base composition affects the electron transfer rates. The electron conductivity may be in principle mediated by aromatic amino acid residues of proteins, intercalating into the double helical DNA in DNA-protein complexes. This may provide a mechanism of protein-mediated regulation of DNA conductivity. We intend to study the electron transfer in complexes of DNA with proteins to examine the validity of this hypothesis.

Relevant Publications

  • Kotlyar A.B. and Borovok N. (1995) Light-induced oxidation of cytochrome c. Biochim. Biophys. Acta 1228: 87-90.
  • Kotlyar A.B., Borovok N., Raviv S., Zimanyi, L. and Gutman M. (1996) Fast redox perturbation of aqueous solution by photoexcitation of pyranine. Photochem. Photobiol. 63: 448-454.
  • Kotlyar A.B. and Borovok N. (1997) Intramolecular oxidation of cytochrome c by covalently attached sulfoaromatic molecules.Biochim. Biophys. Acta 1321: 221-228.
  • Kotlyar A.B., Borovok N. and Hazani M. (1997) Use of thiouredopyrene-trisulfonate photochemistry for driving electron transfer reactions in aqueous solutions. Biochem. 36: 15823-15827.
  • Kotlyar A.B., Borovok N. and Hazani M. (1997) Photoinduced electron transfer in singly labeled thiouredopyrene-trisulfonate cytochrome c derivatives. Biochem. 36: 15828-15833.