Preparation and characterization of two His-59 ruthenium-modified algal plastocyanins and an unusually small rate constant for ruthenium(II) → copper(II) intramolecular electron transfer over ~12 Å

Plastocyanins from the algae Anabaena variabilis and Scenedesmus obliquus possess a single uncoordinated surface histidine at position 59. Procedures for Ru modification of this residue using [Ru(NH₃)₅H₂O]²⁺ are described. The modification time required is strongly dependent on the net charges on the proteins, estimated as 1+ and 9- respectively for PCu¹ at pH 7. The major product in each case has been characterized by ICP atomic emission spectroscopy (1:1 ratios of Cu to Ru). The His-59 residue of the Ru-modified products no longer reacts with diethyl pyrocarbonate (DEPC). Also the sharp ^lH NMR His-59 C₂H resonance at 8.2 ppm is lost due to paramagnetic line broadening by the adjacent Ru(III). The PCu^II/PCu^I reduction potentials remain essentially unchanged, and the PCu¹¹ UV-vis spectrum is unperturbed by Ru modification, except for the additional shoulder at 300 nm due to the [Ru(NH₃)₅His]³⁺ moiety. On pulse radiolysis using CO₂^•- to reduce PCu^IIRu^III(pH 7, 20 °C) the behavior observed in both cases is very similar. Reduction is partitioned between the Cu(II) (72%) and Ru(III) (28%), rate constant 6.7 × 10⁸M^-1 s^-1, yielding stable PCu^IRu^III and transient PCu^IIRu^II, respectively. The latter decays to PCu^IRu^III by intra- and/or intermolecular processes (k₁ k₂), which together constitute the second stage. For A. variabilis, k₁= 0.024 ± 0.058 s^-1 and k₂= 1.2 × 10⁵M^-1 s^-1; and for S. obliquus, k₁= 0.04 ± 0.22 s^-1 and k₂ = 3.3 × 10⁵M^-1s^-1. Therefore k₁ values are <0.082 and <0.26 s^-1, respectively, with zero values not excluded from this study. Modification of Pseudomonas aeruginosa azurin was also carried out by a procedure already described. From four pulse radiolysis runs, transient ACu^IIRu^II gives k₁ = 2.5 ± 0.8 s^-1(pH 7, 17 °C), with no significant competition from k₂, in satisfactory agreement with the flash photolysis value of 1.9 ± 0.4 s^-1 from the Gray group. Donor—acceptor distances (~ 12 A) and driving forces are similar for the PCu^IIRu^II and ACu^uRuⁿ systems. Of particular interest is the very small k₁ for both Ru-modified plastocyanins, indicating that electron transfer from the His-59 site through to the Cu is not a favorable route. On the other hand when unattached [Ru(NH₃)₅Im]²⁺ is the reductant, stopped-flow studies indicate k_et> 5 × 10³s^-1 for reduction from the acidic patch (42–44) region of S. obliquus PCu¹¹.