4925
J. Phys. Chem. 1991,95,4925-4928
Electron Transfer In Azurln and the Role of Aromatic Side Groups of the Protein A. Broo and S . Lamson* Departments of Physical Chemistry, Gothenburg University and Chalmers University of Technology, S-412 96 Gothenburg, Sweden (Received: January 4, 1991; In Final Form: April 1. 1991)
Intramolecular electron-transfer reaction in azurin, studied experimentally by Farver and Pecht, is examined with respect to the electronic coupling between the redox centers. The electronic factor is calculated by using a tight-binding method for two possible pathways, one with aromatic groups and one without such groups. Although the former pathway has a larger electronic factor, closer inspection of the results shows that this is not due to electron-transfer-promoting properties of the aromatic groups but can be explained as differences in the peptide backbone interactions.
Introduction Electron transfer (ET) is one of the most important processes in living systems and occurs in many different reaction steps in respiration and photosynthesis and in other biochemical p r o " . It is commonly accepted that the physical mechanism for ET in proteins is vibration-induced electron tunneling. Biological ET reactions then resemble inorganic redox reactions and the relevant theory is derived mainly from the latter field.'+ Electronic tunneling leads to an exponential decrease of the ET rate with distance. The tunneling barrier is due to the protein but as such it is much lower than the corresponding barrier in water solvent or in vacuum.5-' Thus the parts of the protein which are connected by electronic overlap are in fact promoting ET and define Ypathways*. There are now several experiments which confirm the physical principles behind the model.*-'* Due to the complicated nature (1) Marcus, R. A. J. Chrm. Phys. 1956, 24, 966; 1957, 26, 867, 872; Trans. N.Y. Acad. Sci. 1957, 19,423; Discuss. Faraday Soc. 1960,29,21, 129; Annu. Rev. Phys. Chrm. 1964, 15, 155. (2) Hopfield, J. J. Proc. Natl. Acad. Sci. U.S.A. 1974,71, 3640; Biophys. J . 1977, 18,311. Potasek, M.; Hopfield, J. J. Proc. Natl. Acad. Sci. U.S.A. 1977, 74, 3817. (3) Jortner. J. J. Chem. Phys. 1976,64,4860. Jortner, J. Biochcm. Biophys. Acta 1980, 594, 193. Kestner, N. R.;Logan, J.; Jortner, J. J. Phys. Chcm. 1974, 78,2148. Ulstrup, J.; Jortner, J. J. Chcm. Phys. 1975,63,4358. (4) Marcus, R. A.; Sutin, N. Biochim. Biophys. Acta 1985, 811, 265. (5) Larsson, S.J. Am. Chem. Soc. 1981, 103,4034. (6) Larsson, S.J. Chem. Soc., Faraday Trans. 1 1983, 79, 1375; In?. J . Quantum Chem. 1982, 9, 385. (7) Beratan, D. N.; Hopfield, J. J. J. Am. Chcm. Soc. 1984, 106, 1584. Beratan, D. N.; Onuchi. J. N.; Hopfield, J. J. J. Chcm. Phys. 1985,83, 5325; 1987,86,4488. Beratan, D. N. J. Am. Chem. Soc. 1986, 108,4321. (8) Farver, 0.;Pecht, I. Isr. J. Chcm. 1981, 21, 13; Prof. Natl. Acad. Sci. US.A. 1981, 78,4190. Farver, 0.;Shahak, Y.;Pecht, I. Biochemistry 1982, 21, 1885, 3556. (9) McGourty, J. L.; Blough, N. V.; Hoffman, B. M. J. Am. Chem. Soc. 1982, 105, 4470. (IO) Isied, S.S.;Worosila, G.; Atherton, S . J. J. Am. Chem. Soc. 1982, 104, 7659. hied, S.S.;Kuehn, C.; Worosila, G . J. Am. Chrm. Soc. 1984, 106. 1722. Bechtold, R.;Kuehn, C.; Lepre, C.; Isied, S.S.Nature 1986,322, 286. (1 1) Winkler, J. R.;Nocera, D. G.; Yocom, K. M.; Bordignon, E.;Gray, H. B. J. Am. Chrm. Soc. 1982,101,5798. Lieber, C. M.; Karas. J. L.; Gray, H. B. J. Am. Chem. Soc. 1987, 109, 3778. Mayo, S.L.; Ellis, Jr., W. R.; Crutchley, R. J.; Gray, H. B. Science 1986, 233, 948. (12) Bcchtold, R.;Gardiner, M. B.; Kazmi, A.; van Hemelryck, B.; Isied, S.S.J . Phys. Chem. 1986, 90, 3800. Isied, S.S.; Vassilian, A.; Wishart. J.; Creutz, C.; Schwarz, H.; Sutin, N. J. Am. Chem. Soc. 1988, 110, 635. (13) Margalit, R.; Pecht, I.; Gray, H. B. J. Am. Chem. SOC.1983, 105, 301. Kostic, N. M.; Margalit. R.;Che. C.-M.; Gray, H. B. J. Am. Chem. Soc. 1983, 105, 7765. Margalit, R.;Kostic, N. M.; Che, C.-M.; Blair, D. F.; Chiang, H.-J.; Pacht, 1.; Shelton, J. B.; Shelton, J. R.;Schroedcr, W. A.; Gray, H. B. Proc. Natl. Acad. Sci. U.S.A. 1984,81,6554. Cowan, J. A.; Gray, H. B. Chrm. Scr. 1987. 2 8 4 21. Axup, A. W.; Albin, M.; Mayo, S. L.; Crutchley, R. J.; Gray, H. B. J . Am. Chem. Soc. 1988, 110,435. Karas, J. L.; Lieber, C. M.; Gray, H.B. J. Am. Chcm. Soc. 1988, 110, 599. Cowan, J. A.; Upmacis, R.K.; Beratan, D. N.; Onuchic, J. N.; Gray, H. B.Annu. Rev. N.Y. Acad. Sei. 1988, 550, 68. Beratan, D. N.; Onuchic, J. N.; Betts, J. Bowler, B. E.;Gray, H. B. J. Am. Chem. Soc. 1990, 112, 7915.
0022-365419 1 /2095-4925$02.50/0
TABLE I: Calculated and Experimental Spectrum at the Cu Center in Azurin calcd energy,
oscill
cm-l
strength
4900 16100
0.001 0.04
U
20400
0.012
uHu
U
exptl' energy,
-
character M + ~ *cy ~ CUT + *cy M + ~ TC,, CUx + *cy +
+ *cy
+
CU x
+ *cy
cm-'
oscill strength
12 830 15 850 17650 20790
0.009 0.044 0.005 0.007
"270 K, ref 31.
of the barrier it is hardly surprising that there is no uniform exponential decrease with distance as in the textbook example of tunneling. To determine the nature of the structure dependence it is necessary to involve the protein in an electron structure calculation.6 An interesting issye is whether aromatic side groups of the protein are promoting ET as has been suggested on many occasions.*J9 It is frequently found that aromatic groups are conserved in similar proteins with identical ET steps.20 On the other hand, there is experimental2' and theoreticalU evidence that aromatic side groups have no special ET properties and even may serve to insulate the active centers from ET in undesired directions.6*21q22One way to answer this important question is to measure the ET rate in as many different systems as possible to get experience for different types of systems. A more direct way is to compare different pathways in quantum mechanical calculations. Progress in the latter field has been achieved in recent year^.^*^^-^' In the present report we examine possible ET (14) Liang, N.; Kang, C. H.; Ho, P. S.;Margoliash, E.; Hoffman, B. M. J . Am. Chem. Soc. 1986,108,4665. Liadg, N.; Pielak, G. L.; Mauk, A. G.; Smith, M.; Hoffman, B. M. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 1249. Liang, N.; Pielak, G. J.; Johnson, J. A.; Smith, M.; Hoffman, B. M. Science 1988, 240, 311. See also: Zemel, H.; Hoffman, B. M. J. Am. Chem. Soc.
1981, 103, 1192. (15) Jackman, M. P.;Sykes, A. G.; Salmon, G. A. J . Chem. Soc., Chem. Commun. 1987, 65. Jackman, M. P.; McGinnis. J.; Powls, R.;Salmoln, G. A.; Sykes, A. G. J. Am. Chem. Soc. 1988,110, 5880. Osvath, P.; Salmon, G. A.; Sykes, A. G. J. Am. Chem. Soc. 1988, 110, 7114. Jackman, M. P.; Lim, M. C.; Salmon, G. A.; Sykes, A. G. J. Chem. Soc., Dalton Trans. 1988, 11; J . Chem. Soc., Chem. Commun. 1988, 179. (16) Farver, 0.;Licht, A.; Pecht, 1. Biochemistry 1987, 26, 7317. (17) Farver, 0.;Pecht, I. Proc. Natl. Acad. Sci. U.S.A. 1989,86, 6968. (18) McLendon, G.; Pardue, K.; Bak, P. J. Am. Chem. Soc. 1987, 109, 7541. (19) Poulus, T. L.; Kraut, J. J. Biol. Chem. 1980. 255, 10322. (20) Wendoloski, J. J.; Matthew, J. B.; Weber, P.C.; Salemme, F. R. Science 1987, 238, 794. (21) Williams, R. J. P. In Electron Transfer in Biology and rhe Solid State, Advances in Chemistry Series No. 226; Johnson, M. K., et al., Eds.;
American Chemical Society: Washington, DC. (22) Onuchic, J. N.; Beratan, D. N. J. Am. Chem. Soc. 1987, 109,6771. Beratan. D. N.; Onuchic, J. N.; Hopfield. J. J. J. Chcm. Phys. 1987.86,4488. Onuchic, J. N.; Beratan, D. N. J . Chem. Phys. 1990, 92, 722. (23) Larsson, S.;Broo, A.; KHllebring, B.; Volosov, A. In?. J. Quantum Chem., Quantum Biol. Symp. 1988, 15, I .
0 1991 American Chemical Society
Letters
4926 The Journal of Physical Chemistry, Vol. 95, No. 13, 1991
12
d
Figure 1. Heavy atoms included in the calculation for the 'backbone pathway".
pathways in an intramolecular ET reaction in azurin which has been invented and studied by Farver et aI.l6J7 In the mentioned experiment" a disulfide bridge between Cys-3 and Cys-26 of the blue protein azurin is reduced by using ET from a COT radical ions produced by pulse radiolysis. In a much slower step (k = 44 s-l) the Cu(I1) ion is reduced through ET from the disulfide bridge. Inspection of the azurin structure26reveals at least two independent ET pathways: one along a peptide backbone (Figure 1) without aromatic side groups and another where three aromatic groups bridge the distance between the cystine disulfur group and the Cu ion (Figure 2). The former pathway runs along the residues Cys-3, Ser-4, Val-5, Asp6, Ile-7, Gln-8, and Gly-9. Gly-9 is in close contact with His-46 which binds to the Cu ion. The aromatic pathway, on the other hand, runs along Cys-26, Lys-27, Gln-28, Phe-29, Thr-30, Val-3 1 with a well-placed phenyl group at Phe-29. The distance from this phenyl group to an indole group at Trp48 is 5 A between the closest C atoms. The indole group of Trp-48 is in contact with the phenyl group of Phe-15 which in its turn is located close to the -H2C-S ligands of Cu belonging to Cys- 121 and Cys-1 12. The aromatic side groups of Phe- 15 and Trp-48 are almost parallel. There is another pure backbone pathway along Cys-26 to His-35 but it turned out to have a much smaller coupling than the backbone pathway described above. Calculations For the case of small electronic factor IAI, compared to nuclear frequencies, the rate constant may be 0btained~3~~ from h (47rXk
where X is the reorganization energy and AG' the activation energy. A will here be obtained by using tight-binding "extended Huckel" (EH) type An avoided crossing is found by modifying nuclear coordinates in a way which corresponds to activation. The activated conformation is characterized by an avoided crossing where the orbitals of interest have the form N(% + cpA + 6a)and N'(% - cpA + 6dB)where N and "are normalization factors. c p and ~ cpA are the main components of the orbital, localized to the donor and acceptor, respectively, whereas 6 a and &dB are the small bridge components. (24) Broo, A.; Larsson, S. Inr. J . Quantum Chem. Quantum Biol. Symp. 1989, 16, 185.
(25) Christensen, H. E. M.; Conrad, L. C.; Mikkelsen, K. V.; Nielsen, M. K.; Ulstrup, J. Inorg. Chem. 1990, 29, 2808. (26) Adman, E. T.; Jensen, L. H. Isr. J . Chem. 1981,21,8. Adman, E. T.; Sieker, L. C.; Jensen, L. H. Brookhaven Data Bank, Sept 80-Sept 83. (27) Sutin, N. Ann. Rev. Nucl. Sei. 1%2,12,285; Ace. Chem. Res. 1968, I , 225; in Inorganic Biochemistry; Eichorn, G . L., Eds.; American Elsevier: New York, 1973; Vol. 2, p 61 1.
TABLE 11: Parameters Used in the Calculations"
B
a S
CU C N 0
S H
-10.47 -21.85 -26.37 -27.75 -25.12 -15.8
P -5.31 -13.35 -15.04 -13.08 -14.24
d -1499
S
-5 -17 -26 -31 -25 -12
P -5 -17 -26 -31 -25
d -20
"The a values and B(Cu,3d) are obtained in an iterative EH calculation for the Cu site (see text). @ for H, C, N, 0, and S are standard values.29 The unimportant @(Cu,4s,4p) are chosen small in agreement with ref 23.
TABLE 111: Results for Different Pathways B = backbone (see text) A = aromatic (see text) phenyl of Phe- 15 turned 90' in A most of backbone removed in A indole removed from A phenyl group of Phe-29 removed Phe- 15 removed Asn-47 removed from A all three aromatic groups removed only Phe-29 and Phe-15 removed
IAI X lo8. eV 2.9 10.3 5.9
