Casaus José L, Navarro José A, Hervás Manuel, Lostao Anabel, De la Rosa Miguel A, Gómez-Moreno Carlos, Sancho Javier, Medina Milagros
Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, Zaragoza 50009, Spain.
J Biol Chem. 2002 Jun 21;277(25):22338-44. doi: 10.1074/jbc.M112258200. Epub 2002 Apr 11.
The influence of the amino acid residues sandwiching the flavin ring in flavodoxin (Fld) from the cyanobacterium Anabaena sp. PCC 7119 in complex formation and electron transfer (ET) with its natural partners, photosystem I (PSI) and ferredoxin-NADP(+) reductase (FNR), was examined in mutants of the key residues Trp(57) and Tyr(94). The mutants' ability to form complexes with either FNR or PSI is similar to that of wild-type Fld. However, some of the mutants exhibit altered kinetic properties in their ET processes that can be explained in terms of altered flavin accessibility and/or thermodynamic parameters. The most noticeable alteration is produced upon replacement of Tyr(94) by alanine. In this mutant, the processes that involve the transfer of one electron from either PSI or FNR are clearly accelerated, which might be attributable to a larger accessibility of the flavin to the reductant. However, when the opposite ET flow is analyzed with FNR, the reduced Y94A mutant transfers electrons to FNR slightly more slowly than wild type. This can be explained thermodynamically from a decrease in driving force due to the significant shift of 137 mV in the reduction potential value for the semiquinone/hydroquinone couple (E(1)) of Y94A, relative to wild type (Lostao, A., Gómez-Moreno, C., Mayhew, S. G., and Sancho, J. (1997) Biochemistry 36, 14334-14344). The behavior of the rest of the mutants can be explained in the same way. Overall, our data indicate that Trp(57) and Tyr(94) do not play any active role in flavodoxin redox reactions providing a path for the electrons but are rather involved in setting an appropriate structural and electronic environment that modulates in vivo ET from PSI to FNR while providing a tight FMN binding.
研究了蓝藻鱼腥藻7119黄素氧还蛋白（Fld）中夹着黄素环的氨基酸残基在与天然伙伴光系统I（PSI）和铁氧还蛋白-NADP（+）还原酶（FNR）形成复合物及电子转移（ET）过程中的影响，构建了关键残基色氨酸（Trp57）和酪氨酸（Tyr94）的突变体。突变体与FNR或PSI形成复合物的能力与野生型Fld相似。然而，一些突变体在其ET过程中表现出改变的动力学特性，这可以用黄素可及性和/或热力学参数的改变来解释。最显著的改变是由丙氨酸取代Tyr94引起的。在这个突变体中，涉及从PSI或FNR转移一个电子的过程明显加速，这可能归因于黄素对还原剂的更大可及性。然而，当用FNR分析相反的ET流时，还原型Y94A突变体向FNR转移电子的速度比野生型略慢。这可以从热力学角度解释，因为相对于野生型，Y94A的半醌/对苯二酚偶联（E1）的还原电位值显著偏移137 mV，导致驱动力降低（洛斯塔奥, A., 戈麦斯-莫雷诺, C., 梅休, S. G., 和桑乔, J. (1997) 《生物化学》36, 14334 - 14344）。其余突变体的行为也可以用同样的方式解释。总体而言，我们的数据表明Trp57和Tyr94在黄素氧还蛋白氧化还原反应中不发挥任何为电子提供路径 的积极作用，而是参与设定合适的结构和电子环境，在提供紧密FMN结合的同时调节体内从PSI到FNR的ET。
