Effect of surface charges on the rates of intermolecular electron-transferbetween de novo designed metalloproteins

A de novo designed coiled-coil metalloprotein was prepared that uses electr ostatic interactions to control both its conformational and bimolecular ele ctron-transfer properties. The title protein exists as a coiled-coil hetero dimer of the [Ru(trpy)(bpy)-KK(37-mer)] and [Ru(NH3)(5)-EE(37-mer)] polypep tides which is formed by interhelix electrostatic attractions. Circular dic hroism studies show that the electrostatic heterodimer has Kd 0.19 +/- 0.03 muM and is 96% helical at high concentrations. Intercomplex electron-trans fer reactions were studied that involve the [Ru(NH3)(5)-H21](2+) electron-d onor and the [Ru(trpy)(bpy)H21](3+) electron-acceptor belonging to differen t electrostatic dimers. An important feature of the designed metalloprotein is its two cationic redox centers embedded within protein surfaces having opposite charge. Thus, the Ru-II(NH3)5-H21 site was placed on the surface o f one chain of the coiled-coil which was made to be positively charged, and the Ru-III(trpy)(bpy)-H21 site was placed on the surface of the other chai n which was negatively charged. The rates of intermolecular electron-transf er increased from (1.9 +/- 0.4) x 10(7) M-1 s(-1) to (3.7 +/- 0.5) x 107 M- 1 s(-1) as the ionic strength was increased from 0.01 to 0.20 M. This indic ates that the electrostatic repulsion between the ruthenium centers dominat es the kinetics of these reactions. However, the presence of the oppositely charged protein surfaces in the coiled-coils creates an electrostatic reco gnition domain that substantially ameliorates the effects of this repulsion .