Protonation of the copper(I) form of the blue copper proteins plastocyaninand amicyanin - A molecular dynamics study

The conformational space of the copper(I) forms of the cupredoxines amicyan in (ami) (Paracoccus versutus) and plastocyanin (pla) (Populus nigra) with a protonated histidine donor close to the C-terminus ("C-terminal histidine " ami: His96; pla: His87) was investigated with force field calculations th at involve the experimentally determined structures of the protonated or un protonated copper(I/II) forms: a setup of 36 conformers with a constrained torsional angle that involves the C-imidazole-C-methylene (C-gamma-C-beta) bond (chi2; 10 degrees increments); strain energy minimization of all const rained conformers; a 10 ps molecular dynamics search around each conformer; constrained, followed by unconstrained optimization of each of the resulti ng low energy structures; cluster analysis of the resulting conformations. For both cupredoxines there are two major conformers which differ in their solvent accessibility. The plastocyanin and amicyanin structures are in exc ellent agreement with the experimentally observed crystallographic data; th e two analyses lead to the proposal for a general mechanism for the protona tion of reduced blue copper proteins. This is also in agreement with the re sults of a conformational analysis of a complex of the protonated copper(I) form of amicyanin with phosphate.