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 PCuII/PCuI 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 PCuIIRuIII(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 PCuIRuIII and transient PCuIIRuII, respectively. The latter decays to PCuIRuIII 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 ACuIIRuII 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 PCuIIRuII and ACuuRun 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 ket> 5 × 103s-1 for reduction from the acidic patch (42–44) region of S. obliquus PCu11.