J. Am. Chem. SOC. zyxwvu 1993, 115, zyxwvut 1485-1489 1485 Electron Transfer in Ruthenium/Zinc Porphyrin Derivatives of Recombinant Human Myoglobins. Analysis of Tunneling Pathways in Myoglobin and Cytochrome c Danilo R. Casimiro, Luet-L. Wong,'. Jorge L. Co16n,lb Thomas E. Zewert, John H. Richards, I-Jy Chang, Jay R. W i d e r , * and Harry B. Gray* Contribution No. 8716 from the Beckman Institute, California Institute of Technology, Pasadena, California 91 125. Received August 28, 1992 Abstract: Site-directed mutants of human myoglobin have been prepared and characterized; each protein has a single surface-modifiable histidine (at position 48, 70, or 83). The proteins were .modified by covalent attachment of penta- ammineruthenium (a5Ru) to the surface histidine and substitution of zinc mesoporphyrin zyxw IX diacid (ZnP) for the heme. Donor-acceptor separations (edge-edge distances d) in the modified proteins are 9.5 A, His70; 12.7 A, His48; and 15.5 A, His83. Rates of photoinduced electron transfer in these ruthenium-modified myoglobins were measured by transient absorption spectroscopy. The 3ZnP* zyxwvutsrq - Ru3+ rate constants are 1.6 X IO' (His'lO), 7.2 X lo4 (His48), and 4.0 X lo2 s-I (His83) (-AGO = 0.82 eV); charge-recombination (Ru2+ - ZnP') rates are 1.1 X lo5 (His48) and 7.3 X lo2 s-I (His83) (-AGO = 0.96 eV). Activationless (maximum) rates assuming h = 1.3 eV are 7.2 X lo7 (His70), 3.3 X lo5 (His48). and 1.8 X lo3 s-l (His83). Distant electronic couplings, which limit the maximum rates in the modified myoglobins, have been analyzed along with data from Ru-modified cytochromes c in terms of a tunneling pathway model. Single dominant pathways adequately describe. the electronic couplings in cytochrome zyxwvutsr c but do not satisfactorily account for the myoglobin couplings. The correlation of electronic coupling with tunneling length for myoglobin is improved significantly by the inclusion of multiple pathways. Introduction Both theoretical and experimental investigations have shown that variations in distant donor-acceptor (D-A) electronic cou- plings can control the rates of many protein electron-transfer (ET) reaction^.^-'^ If a simple homogeneous medium separates donor (1) (a) Present address: Inorganic Chemistry Laboratory, University of Oxford, South Parks Road, Oxford OX1 3QR, U.K. (b) Present address: Department of Chemistry, University of Puerto Rico, Rio Piedras, Puerto Rim 00931. (2) Marcus, R. A.; Sutin, N. Biochim. Biophys. Acra 1985, 811, 265. (3) McLendon, G. Acc. Chem. Res. 1988, 21, 160. (4) Bertrand, P. Sfrucr. Bonding 1991, 75, 1. (5) Kuki, A. Sfrucr. Bonding 1991, 75, 49. (6) Beratan, D. N.; Betts, J. N.; Onuchic, J. N. Science 1991, 252, 1285. (7) (a) Beratan, D. N.; Onuchic, J. N.; Hopfield, J. J. J. Chem. Phys. 1987,86,4488. (b) Beratan, D. N.; Onuchic, J. N. Phorosynrh. Res. 1989, 22, 173. (c) Beratan, D. N.; Onuchic, J. N.; Betts, J. N.; Bowler, B. E.; Gray, H. B. J. 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