Tegoni M, Silvestrini M C, Guigliarelli B, Asso M, Brunori M, Bertrand P
Architecture et Fonction de Macromolécules Biologiques, IFR1, UPR 9039-CNRS, Marseille, France.
Biochemistry. 1998 Sep 15;37(37):12761-71. doi: 10.1021/bi980192z.
The kinetics of intramolecular electron transfer between flavin and heme in Saccharomyces cerevisiae flavocytochrome b2 were investigated by performing potentiometric titrations and temperature-jump experiments on the recombinant wild type and Y143F and Y254F mutants. The midpoint potential of heme was determined by monitoring redox titrations spectrophotometrically, and that of semiquinone flavin/reduced flavin (Fsq/Fred) and oxidized flavin (Fox)/Fsq couples by electron paramagnetic resonance experiments at room temperature. The effects of pyruvate on the kinetic and thermodynamic parameters were also investigated. At room temperature, pH 7.0 and I = 0.1 M, the redox potential of the Fsq/Fred, Fox/Fsq, and oxidized heme/reduced heme (Hox/Hred) couples were -135, -45, and -3 mV, respectively, in the wild-type form. Although neither the mutations nor excess pyruvate did appreciably modify the potential of the heme or that of the Fsq/Fred couple, they led to variable positive shifts in the potential of the Fox/Fsq couple, thus modulating the driving force that characterizes the reduction of heme by the semiquinone in the -42 to +88 mV range. The relaxation rates measured at 16 degreesC in temperature-jump experiments were independent of the protein concentrations, with absorbance changes corresponding to the reduction of the heme. Two relaxation processes were clearly resolved in wild-type flavocytochrome b2 (1/tau1 = 1500 s-1, 1/tau2 = 200 +/- 50 s-1) and were assigned to the reactions whereby the heme is reduced by Fred and Fsq, respectively. The rate of the latter reaction was determined in the whole series of proteins. Its variation as a function of the driving force is well described by the expression obtained from electron-transfer theories, which provides evidence that the intramolecular electron transfer is not controlled by the dynamics of the protein.
通过对重组野生型、Y143F和Y254F突变体进行电位滴定和温度跃升实验，研究了酿酒酵母黄素细胞色素b2中黄素和血红素之间的分子内电子转移动力学。通过分光光度法监测氧化还原滴定来测定血红素的中点电位，通过室温下的电子顺磁共振实验测定半醌黄素/还原黄素（Fsq/Fred）和氧化黄素（Fox）/Fsq偶联的中点电位。还研究了丙酮酸对动力学和热力学参数的影响。在室温、pH 7.0和I = 0.1 M条件下，野生型形式中Fsq/Fred、Fox/Fsq和氧化血红素/还原血红素（Hox/Hred）偶联的氧化还原电位分别为-135、-45和-3 mV。尽管突变和过量的丙酮酸均未明显改变血红素或Fsq/Fred偶联的电位，但它们导致Fox/Fsq偶联的电位发生不同程度的正向偏移，从而在-42至+88 mV范围内调节了半醌还原血红素的驱动力。在温度跃升实验中于16℃测得的弛豫速率与蛋白质浓度无关，吸光度变化对应于血红素的还原。在野生型黄素细胞色素b2中清晰分辨出两个弛豫过程（1/tau1 = 1500 s-1，1/tau2 = 200 +/- 50 s-1），分别归因于血红素被Fred和Fsq还原的反应。在整个蛋白质系列中测定了后一个反应的速率。其作为驱动力函数的变化可以通过电子转移理论得到的表达式很好地描述，这表明分子内电子转移不受蛋白质动力学的控制。
