J. Am. Chem. SOC.1988, 110, 435-439 435 Distance Dependence of Photoinduced Long-Range Electron Transfer in Zinc/Ruthenium-Modified Myoglobins Andrew W. Axup, Michael Albin, Stephen L. Mayo, Robert J. Crutchley,' and Harry B. Gray* Contribution No. 7588 from the Arthur Amos Noyes Laboratory, California Institute of Technology, Pasadena, California 91 125. Received May 8, I987 Abstract: An experimental investigation of the distance dependence of long-range electron transfer in zinc/ruthenium-modified myoglobins has been performed. The modified proteins were prepared by substitution of zinc mesoporphyrin IX diacid (ZnP) for the heme in each of four previously characterized pentaammineruthenium(II1) (a5Ru; a = NH,) derivatives of sperm whale myoglobin (Mb): a5Ru(His-48)Mb, aSRu(His-12)Mb, a5Ru(His-l 16)Mb, a5Ru(His-81)Mb. Electron transfer from the ZnP triplet excited state (,ZnP*) to Ru3+, 3ZnP*-Ru3t -+ ZnP+-Ru2+ (AEo - 0.8 V) was measured by time-resolved transient absorption spectroscopy: rate constants (kf)are 7.0 X lo4 (His-48), 1.0 X IO2 (His-12), 8.9 X 10' (His-1 16), and 8.5 X IO' (His-81) s-' at 25 OC. Activation enthalpies calculated from the temperature dependences of the electron-transfer rates over the range 5-40 OC are 1.7 i 1.6 (His-48), 4.7 i 0.9 (His-12), 5.4 i 0.4 (His-116), and 5.6 i 2.5 (His-81) kcal mol-'. Electron-transfer distances (d = closest ZnP edge to a5Ru(His) edge; angstroms) were calculated to fall in the following ranges: His-48, 11.8-16.6; His-12, 21.5-22.3; His-116, 19.8-20.4; His-81, 18.8-19.3. The rate-distance equation is kf = 7.8 X lo8 exp[-0.9l(d - 3)] 8. The data indicate that the 3ZnP*-Ru(His-12)3+ electronic coupling may be enhanced by an intervening tryptophan (Trp-14). Long-range electron transfers are important mechanistic steps it is generally assumed that the rates of these transfers are de- in many biological oxidation-reduction reactions.2-16 Although termined to a large extent by the donor-acceptor separation and the nature of the intervening medi~m,l'-~'very few experiments (1) Present address: Department of Chemistry, Carleton University, Ot- have addressed these points in a systematic manner.'^^.'' tawa, Canada KlS 5B6. One approach to the study of long-range electron transfer is (2) Hatefi, Y. Annu. Reo. Biochem. 1985, 54, 1015-1069. to attach a redox-active complex to a specific surface site of a (31 Dixit. B. P. S.N.; Vanderkooi. J. M. Curr. TOO.Bioenera. 1984. 13, 159-202. structurally characterized heme or blue copper protein, thereby (4) Michel-Beyerle, M. E.; Ed. Antennas and Reaction Centers of Pho- producing a two-site molecule with a fixed donor-acceptor dis- tosynthetic Bacteria; Springer-Verlag: Berlin, 1985. tance. The redox-active complex that has been employed suc- (5) De Vault, D. Quantum Mechanical Tunnelling in Biological Systems, 2nd ed.; Cambridge University: Cambridge, 1984. cessfully in several previous experiments is a5Ru2+/3+(a = NH,), (6) Sykes, A. G. Chem. SOC.Rev. 1985, 14, 283-315. which covalently bonds to surface histidines.8-12 This complex (7) Brunschwig, B. S.; DeLaive, P. J.; English, A. M.; Goldberg, M.; Gray, can be attached by the reaction of a5Ru(OH,)2t with native H. B.; Mayo, S. L.; Sutin, N. Inorg. Chem. 1985, 24, 3743-3749. protein under mild conditions, and the ruthenated protein can be (8) (a) Gray, H. B. Chem. SOC.Rev. 1986,15, 17-30. (b) Mayo, S. L.; Ellis, W. R., Jr.; Crutchley, R. J.; Gray, H. B. Science (Washington, D.C.) purified by ion-exchange chromatography. 1986, 233, 948-952. In order to probe distance effects on long-range electron (9) (a) Yo", K. M.; Shelton, J. B.; Shelton, J. R.; Schroeder, W. A,; transfer, we have replaced the heme in four ruthenated myoglobins Worosila, G.; Isied, S. S.; Bordignon, E.; Gray, H. B. Proc. Natl. Acad. Sci. (a5Ru(His-48)Mb; a5Ru(His-12)Mb; a5Ru(His-l16)Mb; a5Ru- U.S.A. 1982,79,7052-7055. (b) Winkler, J. R.; Nocera, D. G.; Yocom, K. whale myoglobin)'O M.; Bordignon, E.; Gray, H. B. J. Am. Chem. SOC.1982, 104, 5798-5800. (His-81)Mb: Mb = sperm by zinc meso- (c) Yocom, K. M.; Winkler, J. R.; Nocera, D. G.; Bordignon, E.; Gray, H. porphyrin IX diacid (ZnP). The ZnP excited triplet state (3ZnP*) B. Chem. Scr. 1983, 21, 29-33. (d) Kcstic, N. M.; Margalit, R.; Che, C.-M.; is a much more powerful electron donor than a reduced heme Gray, H. B. J. Am. Chem. SOC.1983, 105, 7765-7767. (e) Margalit, R.; (hE0(3ZnP*-Ru3+- ZnP+-Ru2+) - 0.8 V)," thereby allowing Kostic, N. M.; Che, C.-M.; Blair, D. F.; Chiang, H.-J.; Pecht, I.; Shelton, J. different electron-transfer distances to B.; Shelton, J. R.; Schroeder, W. A.; Gray, H. B. Proc. Natl. Acari. Sci. U.S.A. four be examined in a single 1984, 81, 6554-6558. (f) Nocera, D. G.; Winkler, J. R.; Yocom, K. M.; protein molecule. Bordimon. E.: Grav. H. B. J. Am. Chem. SOC.1984. 106. 5145-5150. (E\ Section Crutciicy,'R. J.; Elk, W. R., Jr.; Gray, H. B. J. Am.Chem. Soc. 1985, IO?, Experimental 5002-5004. (h) Lieber, C. M.; Karas, J. L.; Gray, H. B. J. Am. Chem. SOC. Materials and Apparatus. Distilled water, filtered through a Barnstead -.19117.- . , -109. - ., -3778-3779. - - . Nanopure water purification system (No. 2794, specific resistance >18 (10) Crutchley, R. J.; Ellis, W. R., Jr.; Gray, H. B. Frontiers in Bioinor- Ma-cm), was used in the preparation of all aqueous solutions. 2-Buta- ganic Chemistry; Xavier, A. V., Ed.; VCH: Weinheim, FRG, 1986; pp none (MCB) was stored over aluminum oxide (Woelm neutral, Waters 679-693. Crutchley, R. J.; Ellis, W. R., Jr.; Shelton, J. B.; Shelton, J. R.; Associates) at 4 OC to prevent the accumulation of peroxides. All other Schroeder, W. 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R.; Mauk, A. G.; Gray, H. B. J. Am. Chem. (17) Marcus, R. A,; Sutin, N. Biochim. Biophys. Acta 1985,811,265-322. Soc. 1985,107,739-740. (c) McLendon, G.; Miller, S. R. J. Am. Chem. SOC. (18) Hopfield, J. J. Proc. Natl. Acad. Sci. U.S.A. 1974, 71, 3640-3644. 1985, 107, 781 1-7816. (d) Cheung, E.; Taylor, K.; Komblatt, J. A.; English, (19) Hush, N. S. Coord. Chem. Reu. 1985, 64, 135-157. A. M.; McLendon, G. L.; Miller, J. R. Proc. Natl. Acad. Sci. U.S.A. 1986, (20) Larsson, S. J. Chem. Soc., Faraday Trans. 1983, 79, 1375-1388. 83, 1330-1333. (e) Conklin, K. T.; McLendon, G. L. Inorg. Chem. 1986,25, (21) Scott, R. A,; Mauk, A. G.; Gray, H. B. J. Chem. Educ. 1985, 62, 4804-4806. 932-938. 0002-7863/88/15 10-0435$0 1 .50/0 0 1988 American Chemical Society 436 J. Am. Chem. SOC.,Vol. 110, No. 2, 1988 Axup et al. Samples were degassed and purged with purified argon (passed 3 mL of water. The reaction mixture was heated and refluxed for 2 h. through a manganese oxide column) on a dual-manifold vacuum argon Hydrochloric acid (6 N) was added to precipitate the porphyrin diacid. line. At least five vacuum/purge cycles were used to deoxygenate sam- The reaction mixture was refrigerated overnight.