Silvestrini M C, Brunori M, Tegoni M, Gervais M, Labeyrie F
Eur J Biochem. 1986 Dec 1;161(2):465-72. doi: 10.1111/j.1432-1033.1986.tb10467.x.
Two derivatives of Hansenula anomala flavocytochrome b2 have been prepared, one deprived of the flavin prosthetic group (deflavocytochrome b2), and the other consisting of the heme-b-carrying globule (b2 core). The redox potential of the heme in the two derivatives is -5 (+/- 5) mV and -10 (+/- 5) mV respectively, fairly similar to the value of -20 (+/- 5) mV reported for the holoenzyme, indicating a minor effect of the flavin and of the flavodehydrogenase domain on heme potential. The kinetics of azurin and stellacyanin reduction by both derivatives have been investigated. At pH 7.0, I = 0.2 M and 20 degrees C the second-order rate constants are: k = 8 X 10(5) M-1 S-1 for azurin reduction by deflavocytochrome b2; k = 1.6 X 10(6) M-1 S-1 for azurin reduction by b2 core; k = 1 X 10(7) M-1 S-1 for stellacyanin reduction by deflavocytochrome b2; k = 3 X 10(7) M-1 S-1 for stellacyanin reduction by b2 core. The change in pH markedly affects the kinetics in the case of azurin, but has no effect on stellacyanin reduction. The change in ionic strength has a significant effect when deflavocytochrome b2 is the reductant, indicating that the flavodehydrogenase domain plays a role in the stabilization of the transient kinetic complex by means of electrostatic interactions. The kinetic results are discussed in the framework of the Marcus theory.
已制备出异常汉逊酵母黄素细胞色素b2的两种衍生物，一种不含黄素辅基（脱辅基黄素细胞色素b2），另一种由携带血红素b的球蛋白（b2核心）组成。两种衍生物中血红素的氧化还原电位分别为-5（±5）毫伏和-10（±5）毫伏，与报道的全酶-20（±5）毫伏的值相当接近，表明黄素和黄素脱氢酶结构域对血红素电位的影响较小。研究了两种衍生物还原天青蛋白和杨梅素的动力学。在pH 7.0、I = 0.2 M和20℃条件下，二级速率常数分别为：脱辅基黄素细胞色素b2还原天青蛋白的k = 8×10⁵ M⁻¹ S⁻¹；b2核心还原天青蛋白的k = 1.6×10⁶ M⁻¹ S⁻¹；脱辅基黄素细胞色素b2还原杨梅素的k = 1×10⁷ M⁻¹ S⁻¹；b2核心还原杨梅素的k = 3×10⁷ M⁻¹ S⁻¹。pH的变化对天青蛋白的动力学有显著影响，但对杨梅素的还原没有影响。当脱辅基黄素细胞色素b2作为还原剂时，离子强度的变化有显著影响，表明黄素脱氢酶结构域通过静电相互作用在稳定瞬态动力学复合物中起作用。在马库斯理论的框架内讨论了动力学结果。
