Aromatic Residues May Enhance Intramolecular Electron Transfer in Azurin Ole Farver,* ,† Lars K. Skov, ‡ Simon Young, § Nicklas Bonander, § B. Go ¨ran Karlsson, § Tore Va ¨nngård, § and Israel Pecht | Institute of Analytical and Pharmaceutical Chemistry The Royal Danish School of Pharmacy DK-2100 Copenhagen, Denmark Department of Chemistry, UniVersity of Copenhagen DK-2100 Copenhagen, Denmark Department of Biochemistry and Biophysics UniVersity of Go ¨ teborg and Chalmers Institute of Technology, S-412 96 Go ¨ teborg, Sweden Department of Immunology, The Weizmann Institute of Science, RehoVot 76100, Israel ReceiVed December 23, 1996 Electron transfer (ET) plays an important role in many biological systems, and a central question is whether specific amino acid residues may promote ET. 1 The semiclassical Marcus theory for nonadiabatic processes predicts that intramo- lecular ET in proteins is governed by the standard free energy of reaction (ΔG°), the nuclear reorganization energy (λ), and the electronic coupling (H DA ) between electron donor (D) and acceptor (A) at the transition state: 2 The electronic coupling energy, H DA , is expected to decay exponentially with the distance between D and A as For protein ET the distance between D and A may be considerable (g1.0 nm), leading to a very small electronic coupling. Still, intramolecular ET over distances of 2.0 nm or more has been observed. 3 The blue single-copper protein azurin is engaged in biological ET and serves as an ideal system for examination of intramo- lecular long range ET (LRET) in proteins. 4 It consists almost exclusively of a rigid -sheet polypeptide, and three-dimensional structures have been determined for a large number of wild- type (WT) and single site mutated azurins. 5 Furthermore, no attachment of external redox group is needed, since it contains two potential redox centers, the copper ion coordinated directly to amino acid residues and a disulfide bridge (RSSR) in the opposite end of the molecule. We have previously demonstrated that intramolecular LRET between the two centers can be induced by pulse radiolysis. 4 Using both WT and single site mutated azurins, we have studied the effect of specific amino acid substitutions on the rate of intramolecular ET. In order to understand better the effect of the polypeptide matrix between D and A, we have used the structure-dependent pathway model developed by Beratan and Onuchic for identifying the relevant ET routes. 6 In this model, the total coupling of a pathway is given as a repeated product of the couplings of the individual links. The optimum pathway between the two redox sites, Πǫ, is then identified. Pathway calculations for the above intramolecular ET were performed using the high-resolution three-dimensional structures of Pseudomonas aeruginosa azurin and of its mutants, when available. 5 For other mutants, structures based on 2D NMR studies and energy minimization calculations were employed. The calculations predict two major electron transfer routes in all of the azurins 4 listed in Table 1: one longer path through the peptide chain to the copper-ligating imidazole of His46 and one shorter path through the buried residue 48 (usually a tryptophan), necessitating a through-space jump from Val31 to this side chain, and further to the copper ligand, Cys112. The electronic coupling factors were found to be Πǫ ) 2.5 × 10 -7 and 3.0 × 10 -8 , respectively. 4 However, in this analysis the electronic interaction between the Cu(II) ion and its ligands was not included. It has been demonstrated that the high degree of anisotropic covalency in the blue single-copper protein, plas- tocyanin, would enhance ET through the Cys ligand. 7 By similar arguments, from the ligand coefficients of Ψ HOMO in azurin obtained by Larsson et al., 8 it can be estimated that ET through Cys would be enhanced by a factor of ∼150 over ET via one of the His ligands. This means that the two pathways would be about equally important. † The Royal Danish School of Pharmacy. ‡ University of Copenhagen. § University of Go ¨teborg and Chalmers Institute of Technology. | The Weizmann Institute of Science. (1) (a) Broo, A.; Larsson, S. J. Phys. Chem. 1991, 95, 4925. (b) Beratan, D. N.; Onuchic, J. N.; Winkler, J. R.; Gray, H. B. Science 1992, 258, 1740. (c) Moser, C. C.; Keske, J. M.; Warncke, K.; Farid, R. S.; Dutton, P. L. Nature 1992, 355, 796. (d) Farid, R. S.; Moser, C. C.; Dutton, P. L. Curr. Opin. Struct. Biol. 1993, 3, 225. (e) Siddarth, P.; Marcus, R. A. J. Phys. Chem. 1993, 97, 13078. (f) Gray, H. B.; Winkler, J. R. Ann. ReV. Biochem. 1996, 65, 537. (2) Marcus, R. A.; Sutin, N. Biochim. Biophys. Acta 1985, 811, 265. (3) Struct. Bond.(Long Range Electron Transfer in Biology) 1991, 75. (4) (a) Farver, O.; Pecht, I. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 6968. (b) Farver, O.; Pecht, I. J. Am. Chem. Soc. 1992, 114, 5764. (c) Farver, O.; Skov, L. 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N.; Onuchic, J. N. J. Phys. Chem. 1993, 97, 13083. (b) Skourtis, S. S.; Regan, J. J.; Onuchic, J. N. J. Phys. Chem. 1994, 98, 3379. (7) (a) Penfield, K. W.; Gewirth, A. A.; Solomon, E. I. J. Am. Chem. Soc. 1985, 107, 4519. (b) Christensen, H. E. M.; Conrad, L. S.; Mikkelsen, K. V.; Nielsen, M. K.; Ulstrup, J. Inorg. Chem. 1990, 29, 2808. (c) Lowery, M. D.; Guckert, J. A.; Gebhard, M. S.; Solomon, E. I. J. Am. Chem. Soc. 1993, 115, 3012. (8) Larsson, S.; Broo, A.; Sjo ¨lin, L. J. Phys. Chem. 1995, 99, 4860. k ) 2π p H DA 2 (4πλRT) 1/2 e -(∆G°+λ) 2 /4λRT (1) H DA ) H° DA e -/2(r - r 0 ) (2) Figure 1. Calculated pathways for ET from the sulfur of Cys3 to the copper center in WT P. aeruginosa azurin. Some interconnecting distances (three H-bonds and one van der Waals contact) are given (Å). In the V31W mutant, the closest distance between the two tryptophans (3.5 Å) occurs between W48 C 3 and W31 C ǫ3 . The coordinates were obtained from ref 5b. 5453 J. Am. Chem. Soc. 1997, 119, 5453-5454 S0002-7863(96)04386-7 CCC: $14.00 © 1997 American Chemical Society