The structure and unusual pH dependence of plastocyanin from the fern Dryopteris crassirhizoma - The protonation of an active site histidine is hindered by pi-pi interactions

Spectroscopic properties, amino acid sequence, electron transfer kinetics, and crystal structures of the oxidized (at 1.7 Angstrom resolution) and red uced form (at 1.8 Angstrom resolution) of a novel. plastocyanin from the fe rn Dryopteris crassirhizoma are presented. Kinetic studies show that the re duced form of Dryopteris plastocyanin remains redox-active at low pH, under conditions where the oxidation of the reduced form of other plastocyanins is inhibited by the protonation of a solvent-exposed active site residue, H is(87) (equivalent to His(90) in Dryopteris plastocyanin), The x-ray crysta l structure analysis of Dryopteris plastocyanin reveals pi-pi stacking betw een Phe(12) and His(90), suggesting that the active site is uniquely protec ted against inactivation. Like higher plant plastocyanins, Dryopteris plast ocyanin has an acidic patch, but this patch is located closer to the solven t-exposed active site His residue, and the total number of acidic residues is smaller, In the reactions of Dryopteris plastocyanin with inorganic redo x reagents, the acidic patch (the "remote" site) and the hydrophobic patch surrounding His(90) (the "adjacent" site) are equally efficient for electro n transfer. These results indicate the significance of the lack of protonat ion at the active site of Dryopteris plastocyanin, the equivalence of the t wo electron transfer sites in this protein, and a possibility of obtaining a novel insight into the photosynthetic electron transfer system of the fir st vascular plant fern, including its molecular evolutionary aspects, This is the first report on the characterization of plastocyanin and the first t hree-dimensional protein structure from fern plant.