Effect of surface charges on the rates of intermolecular electron-transfer between de novo designed metalloproteins.

A de novo designed coiled-coil metalloprotein was prepared that uses electrostatic interactions to control both its conformational and bimolecular electron-transfer properties. The title protein exists as a coiled-coil heterodimer of the [Ru(trpy)(bpy)-KK(37-mer)] and [Ru(NH(3))(5)-EE(37-mer)] polypeptides which is formed by interhelix electrostatic attractions. Circular dichroism studies show that the electrostatic heterodimer has K(d) = 0.19 +/- 0.03 microM and is 96% helical at high concentrations. Intercomplex electron-transfer reactions were studied that involve the [Ru(NH(3))(5)-H21](2+) electron-donor and the [Ru(trpy)(bpy)-H21](3+) electron-acceptor belonging to different 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)(NH(3))(5)-H21 site was placed on the surface of 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 chain which was negatively charged. The rates of intermolecular electron-transfer increased from (1.9 +/- 0.4) x 10(7) M(-1) s(-1) to (3.7 +/- 0.5) x 10(7) M(-1) s(-1) as the ionic strength was increased from 0.01 to 0.20 M. This indicates that the electrostatic repulsion between the ruthenium centers dominates the kinetics of these reactions. However, the presence of the oppositely charged protein surfaces in the coiled-coils creates an electrostatic recognition domain that substantially ameliorates the effects of this repulsion.