KINETIC STUDIES ON 1-1 ELECTRON-TRANSFER REACTIONS INVOLVING BLUE COPPER PROTEINS .3. PROTONATION EFFECTS, PROTEIN-COMPLEX ASSOCIATION, AND BINDING-SITES IN REACTIONS OF PSEUDOMONAS-AERUGINOSA AZURIN WITH CO(PHEN)33+, CO(4,7-DPSPHEN)33-, AND FE(CN)63-(OXIDANTS) AND FE(CN)64-(REDUCTANT)

Whereas the reaction of reduced Pseudomonas aeruginosa azurin. ACu1, with Co(4, 7-DPSphen)33-is independent of pH in the range 6.3-9.0, the reactions with other oxidants Co(phen)33+and Fe(CN)63~ are pH dependent, exhibiting behavior consistent with protonation at or near to the site on the protein at which the complex binds, i.e., associates. The oxidant Co(phen>33+binds at a site on ACu1with a pKaof 7.6, whereas Fe(CN)63-binds at a site with a pKaof 7.1, which is shifted to pKa= 6.1 for the reaction of ACu11with Fe(CN)64-. The sites are probably close to the two histidine groups of the azurin which are not bound to the copper. Protein-complex association (K) prior to electron transfer (ket) has been observed in the reactions of Co(phen)33+, Co(4, 7-DPSphen)33-, and Fe(CN)63-with ACu1and in the reaction of ACu11with Fe(CN)64-, the latter two reactions being less extensively studied here because of the existence of previous data. For Co(phen)33+, pH 9.1, l = 0.10 M (NaCl), the association constant K=(25 °C) = 457 M-1, πH° = -1.2 kcal mol-1, πS° = 8.1 cal K-1mol-1, and Ket(25 °C) = 21.3 πH° = 14.3 kcal molπS° = -4.5 cal K-lmol-1. For Co(4, 7-DPSphen)33-pH 7.0, l = 0.10 M (NaCl), K (25 °C) = 2750 M-1, πH° = -3.7 kcal mol-1, π5° = 3.3 cal mol-1, and ket(25 °C) = 0.21 s-1, πH° = 10.7 kcal mol-1, πS° = -25.9 cal K-1mol-1. The reaction of Fe(CN)3-with ACu1is unaffected by the presence of Co(4, 7-DPSphen)33bound to the protein, confirming that these two reactants use different binding sites. The various data obtained are compared with previous results for similar reactions of parsley plastocyanin. © 1979, American Chemical Society. All rights reserved.