Photoinduced electron transfer in singly labeled thiouredopyrenetrisulfonate azurin derivatives

A novel method for the initiation of intramolecular electron transfer react ions in azurin is reported. The method is based on laser photoexcitation of covalently attached thiouredopyrenetrisulfonate (TUPS), the reaction that generates the low potential triplet state of the dye with high quantum effi ciency. TUPS derivatives of azurin, singly labeled at specific lysine resid ues, were prepared and purified to homogeneity by ion exchange HPLC. Transi ent absorption spectroscopy was used to directly monitor the rates of the e lectron transfer reaction from the photoexcited triplet state of TUPS to Cu (II) and the back reaction from Cu(I) to the oxidized dye. For all singly l abeled derivatives, the rate constants of copper ion reduction were one or two orders of magnitude larger than for its reoxidation, consistent with th e larger thermodynamic driving force for the former process. Using 3-D coor dinates of the crystal structure of Pseudomonas aeruginosa azurin and molec ular structure calculation of the TUPS modified proteins, electron transfer pathways were calculated. Analysis of the results revealed a good correlat ion between separation distance from donor to Cu ligating atom (His-N or Cy s-S) and the observed rate constants of Cu(II) reduction. (C) 1999 Federati on of European Biochemical Societies.