Kurzeev Sergey A, Vilesov Alexander S, Fedorova Tatyana V, Stepanova Elena V, Koroleva Olga V, Bukh Christian, Bjerrum Morten J, Kurnikov Igor V, Ryabov Alexander D
A. N. Bach Institute of Biochemistry Russian Academy of Science, Leninskii pr. 33, Moscow, 117071 Russia.
Biochemistry. 2009 Jun 2;48(21):4519-27. doi: 10.1021/bi8020058.
The reactivity of the acido Ru(II) complexes cis-[RuCl(2)(LL)(2)], [RuCO(3)(LL)(2)], cis-[RuCO(3)-(bquin)(2)] (LL = 2,2'-bipyridine (bpy) and 1,10-phenanthroline (phen); bquin = 2,2'-biquinoline) and cyclometalated Ru(II) derivatives of 2-phenylpyridine and 4-(2-tolyl)pyridine [Ru(o-C(6)H(4)-2-py)(phen)(2)]PF(6) (1), [Ru(o-C(6)H(3)-p-R-2-py)(bpy)(MeCN)(2)]PF(6) (2), and [Ru(o-C(6)H(3)-p-R-2-py)(phen)(MeCN)(2)]PF(6) (3) (R = H (a), Me (b)) toward laccase from Coriolus hirsutus has been investigated by conventional UV-vis spectroscopy at pH 3-7 and 25 degrees C. The acido and cyclometalated complexes are readily oxidized into the corresponding Ru(III) species, but the two types of complexes differ substantially in reactivity and obey different rate laws. The acido complexes are oxidized more slowly and the second-order kinetics, first-order in laccase and Ru(II), holds with the rate constants around 5 x 10(4) M(-1) s(-1) at pH 4.5 and 25 degrees C. The cyclometalated complexes 1-3 react much faster and the hyperbolic Michaelis-Menten kinetics holds. However, it is not due to formation of an enzyme-substrate complex but rather because of the ping-pong mechanism of catalysis, viz. E(ox) + Ru(II) --> E(red) + Ru(III) (k(1)); E(red) + 1/4O(2) --> E(ox) (k(2)), with the rate constants k(1) in the range (2-9) x 10(7) M(-1) s(-1) under the same conditions. The huge values of k(1) move the enzymatic oxidation toward a kinetic regime when the dioxygen half-reaction becomes the rate-limiting step. Cyclometalated compounds 1-3 can therefore be used for routine estimation of k(2), that is, the rate constant for reoxidation for laccases by dioxygen. The mechanism proposed was confirmed by the direct stopped-flow measurements of the k(2) rate constant (8.1 x 10(5) M(-1) s(-1) at 26 degrees C) and supported by the theoretical modeling of interaction between the bpy analogue of 1 and Coriolus hirsutes laccase using Monte Carlo simulations.
通过常规紫外可见光谱法，在pH值为3 - 7和25℃的条件下，研究了酸性Ru(II)配合物顺式-[RuCl₂(LL)₂]、[RuCO₃(LL)₂]、顺式-[RuCO₃(bquin)₂]（LL = 2,2'-联吡啶（bpy）和1,10-菲咯啉（phen）；bquin = 2,2'-联喹啉）以及2-苯基吡啶和4-(2-甲苯基)吡啶的环金属化Ru(II)衍生物[Ru(o-C₆H₄-2-py)(phen)₂]PF₆ (1)、[Ru(o-C₆H₃-p-R-2-py)(bpy)(MeCN)₂]PF₆ (2)和[Ru(o-C₆H₃-p-R-2-py)(phen)(MeCN)₂]PF₆ (3)（R = H (a)，Me (b)）对毛糙革盖菌漆酶的反应活性。酸性和环金属化配合物很容易被氧化成相应的Ru(III)物种，但这两种配合物在反应活性上有很大差异，并且遵循不同的速率定律。酸性配合物被氧化得更慢，其二级动力学，即对漆酶和Ru(II)均为一级动力学，在pH 4.5和25℃时速率常数约为5×10⁴ M⁻¹ s⁻¹。环金属化配合物1 - 3反应快得多，遵循双曲线米氏动力学。然而，这不是由于形成了酶 - 底物复合物，而是由于催化的乒乓机制，即E(ox) + Ru(II) → E(red) + Ru(III) (k₁)；E(red) + 1/4O₂ → E(ox) (k₂)，在相同条件下速率常数k₁在(2 - 9)×10⁷ M⁻¹ s⁻¹范围内。k₁的巨大值使酶促氧化进入动力学区域，此时双氧半反应成为限速步骤。因此，环金属化化合物1 - 3可用于常规测定k₂，即漆酶被双氧再氧化的速率常数。所提出的机制通过对k₂速率常数的直接停流测量（26℃时为8.1×10⁵ M⁻¹ s⁻¹）得到证实，并通过使用蒙特卡罗模拟对1的bpy类似物与毛糙革盖菌漆酶之间相互作用的理论建模得到支持。
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