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首页> 外文学位 >Fast kinetic studies of electron transfer reactions for cytochrome bc1 and cytochrome b5.
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Fast kinetic studies of electron transfer reactions for cytochrome bc1 and cytochrome b5.

机译：细胞色素bc1和细胞色素b5电子转移反应的快速动力学研究。

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摘要

Fast kinetic studies of biological electron transfer (ET) reactions have been carried out using laser flash photolysis techniques in our laboratory. A high quantum yield ruthenium complex Ru(bpz) was labeled at the cysteine residue of mutated electron transfer proteins. Upon laser flash, photo-excited Ru(bpz) complex is able to donate or accept an electron depending on the redox state of the environment. Photo-excited Ru(II*) is a strong reductant and can reduce the labeled protein. In the presence of the sacrificial electron acceptor, pre-reduced cytochrome (cyt) can also be photo-oxidized by the covalently attached Ru(bpz) complex. Four binding residues of cyt b5 were mutated (E44Q, E48Q, E56Q, D60N) and the intra-protein, intercomparable and inter-protein electron transfer reactions in the classic cyt b5--cyt c system were studied using laser photolysis. Intra-protein ET from Ru(II) to oxidized heme b5 is found with a rate constant of 1.2x10 6 s-1. In the photo-oxidation process, the rate constant of Ru(II*)-Fe(II)→Ru(I)-Fe(III) is above 107 s -1. Intra-complex ET reactions for wild type cyt b5 and cyt c were found as two phases with a fast phase of 3∼4x10 5 s-1 and slow phase of 3∼6x104 s-1. Four binding residue mutants showed significantly slower 2nd order rate constants than wild type cyt b5 in the whole range of ionic strengths. However, the differences between each mutant at mid to high ionic strength (more than 20 mM) are not significant. It suggests that the effect of diffusion is more significant than the effect of different single mutations at the binding surface.;The ET reactions from cyt c1 to photo-oxidized cyt c 552 or cyt c were studied using the same technique. For the reaction between Paracoccus denitrificans (P.D.) cyt c1 and cyt c552, the observed rate constant is 15,000 s-1 under 10 mM ionic strength, pH 8.0 conditions. Increasing the ratio of cyt c1 to cyt c552 results the linear relationship of increased observed rate constant, suggesting ET between cyt c1 and Ru(bpz)-N23C cyt c552 is a second order interprotein reaction and no intercomparable ET is observed at 10 mM ionic strength. From 0.2 to 0.6 of square root of ionic strength, the logarithms of 2nd order rate constants are in good linear relationship, demonstrating a diffusion controlled electrostatic interaction process between cyt c1 and cyt c552. For the reaction between P.D. cyt c1 to yeast cyt c, the rate constant is 50,000 s-1 under low ionic strength conditions. As ionic strength increased, two different phases of transient of ET were observed. The fast phase represents the intercomparable ET and the rate constants are almost independent of ionic strength. The slow phase rate constants are highly dependent on the ionic strength, suggesting second order ET between the protein molecules that are surrounded with ions. Kinetic data of cyt c1 mutants in the binding region are compared and discussed.;Studies of cyt bc1 were carried out as experiments of cyt bc1 mutants with JG144 inhibition, stigmatellin titration and heme bH redox titration. Kinetic properties of the cd1 helix and of loop mutants to JG144 inhibitor were characterized. Half-of-the-site inhibition of stigmatellin to cyt bc1 was observed in fast kinetic experiments. Mutants lacking heme bL (H198N) or heme bH (H111N) were also studied with Qo site inhibitors stigamtellin, famoxadone and JG144. Mutants of disulfide-linked ISP and cyt c1 S141C(ISP)/G180C(cyt c1) and super-reductase S287R(cyt b)/V135S(ISP) were also studied with different reduction states and Qo site inhibitors. The results and possible explanation are discussed.
机译：在我们的实验室中，已经使用激光闪光光解技术对生物电子转移（ET）反应进行了快速动力学研究。在突变的电子转移蛋白的半胱氨酸残基处标记了高量子产率的钌络合物Ru（bpz）。激光闪光后，受光激发的Ru（bpz）络合物能够根据环境的氧化还原状态捐赠或接受电子。光激发的Ru（II *）是一种强还原剂，可以还原标记的蛋白质。在牺牲电子受体的存在下，预先还原的细胞色素（cyt）也可以被共价连接的Ru（bpz）复合物光氧化。 cyt b5的四个结合残基被突变（E44Q，E48Q，E56Q，D60N），并使用激光光解研究了经典cyt b5--cyt c系统中的蛋白内，可比和蛋白间电子转移反应。发现从Ru（II）到氧化血红素b5的蛋白内ET的速率常数为1.2x10 6 s-1。在光氧化过程中，Ru（II *）-Fe（II）→Ru（I）-Fe（III）的速率常数大于107 s -1。发现野生型cyt b5和cyt c的复杂ET反应为两个阶段，快速阶段为3〜4x10 5 s-1，慢阶段为3〜6x104 s-1。在整个离子强度范围内，四个结合残基突变体显示出比野生型cyt b5慢得多的二阶速率常数。但是，每个突变体在中等或高离子强度（大于20 mM）之间的差异并不明显。提示扩散作用远大于结合表面不同单突变的影响。用相同的方法研究了从cyt c1到光氧化cyt c 552或cyt c的ET反应。对于反硝化副球菌cyt c1和cyt c552之间的反应，在10 mM离子强度，pH 8.0条件下，观察到的速率常数为15,000 s-1。 cyt c1与cyt c552的比例增加导致观察到的速率常数呈线性关系，这表明cyt c1和Ru（bpz）-N23C之间的ET是二级蛋白间反应，在10 mM离子强度下未观察到可比的ET 。从离子强度的平方根的0.2到0.6，二阶速率常数的对数具有良好的线性关系，表明cyt c1和cyt c552之间存在扩散控制的静电相互作用过程。对于P.D. cyt c1到酵母cyt c的速率常数在低离子强度条件下为50,000 s-1。随着离子强度的增加，观察到ET瞬变的两个不同阶段。快相代表可比的ET，速率常数几乎与离子强度无关。缓慢的相速率常数高度依赖于离子强度，这表明被离子包围的蛋白质分子之间的二阶ET。对cyt c1突变体在结合区的动力学数据进行了比较和讨论。以cyt bc1突变体的JG144抑制，柱头蛋白滴定和血红素bH氧化还原滴定为实验对象，对cyt bc1进行了研究。表征了cd1螺旋和JG144抑制剂的环状突变体的动力学特性。在快速动力学实验中观察到了柱头蛋白对cyt bc1的半数抑制作用。缺乏血红素bL（H198N）或血红素bH（H111N）的突变体也用Qo部位抑制剂stigamtellin，famoxadone和JG144进行了研究。还研究了具有不同还原状态和Qo位点抑制剂的二硫键连接的ISP和cyt c1 S141C（ISP）/ G180C（cyt c1）和超还原酶S287R（cyt b）/ V135S（ISP）的突变体。讨论了结果和可能的解释。

著录项

  • 作者

    Yuan, Quan.;

  • 作者单位

    University of Arkansas.;

  • 授予单位 University of Arkansas.;
  • 学科 Chemistry Biochemistry.
  • 学位 Ph.D.
  • 年度 2009
  • 页码 177 p.
  • 总页数 177
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 生物化学;
  • 关键词

  • 入库时间 2022-08-17 11:38:28

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