Horseradish peroxidase-catalyzed oxidation of cis-[Ru-II(LL)(2)XY] complexes by hydrogen peroxide (LL=2,2 '-bipyridine and 1,10-phenanthroline): Equilibria, kinetics, mechanism, and active site reassembly

HRP-catalyzed oxidation of a series of Ru-II complexes cis-[Ru(LL)(2)XY] (B F4)(n) (LL = bpy, 4,4 ' -Me(2)bpy, phen; X,Y = Cl-, Br-, SCN-, NO, CO32-, H 2O, dmso; n = 0-2) by H2O2 into the corresponding Ru-III species follows a 2:1 Ru-II to H2O2 Stoichiometry and obeys first-order kinetics both in a Ru II complex and the enzyme, the reaction rate being weakly dependent on the H2O2 concentration in the range (1-4)x10(-4) M. The second-order rate const ant k(3), which is believed to refer to the rate-limiting electron transfer from Ru-II at the HRP Compound II, are measured for a series of Ru complex es. The largest rate constant of the nine species tested is observed for ci s-[Ru(4,4 ' -Me(2)bpy)(2)(H2O)(2)](2+) and equals 7.2x10(5) M-1 s(-1) at 25 degreesC and pH 6.7. In general, the rate constants k(3) are influenced by the redox potentials of the complexes E degrees ' and low oxidation rates are observed when E degrees ' becomes close to 500 mV versus SCE. This reac tivity trend is confirmed by the example of the equilibrium cis-[Ru(bpy)(2) (H2O)(2)](2+) + py reversible arrow cis-[Ru(bpy)(2)(H2O) (py)](2+) giving r ise to the species with E degrees ' = 465 mV (vs SCE), which are significan tly more resistant to the enzymatic oxidation than the parent complex. Two procedures have been used to reassemble apo-HRP employing the hemin-1-(3-am inopropyl)imidazole conjugate (3). The first included the complexation of c is-[Ru(phen)(2)(H2O)(2)](2+) with the imidazole residue of 3 followed by th e loading of the complex formed into the apo enzyme. The second procedure i nvolved the reconstitution of apo peroxidase with 3 followed by the complex ation with cis-[Ru(phen)(2)(H2O)(2)](2+) Both approaches gave similar Ru-co ntaining catalytically active (with respect to ABTS) preparations, capable of the intramolecular oxidation by H2O2 of ruthenium(II) coordinated with a n imidazole arm of 3.