Nitroreductase: Determinants of Flavin Enzyme Activity

说明(申请人提供)：黄素是一种多功能的氧化还原辅因子，用于酶中执行从脂肪酸代谢到Hg2+的还原解毒等广泛的重要化学作用。这反映了Flavin的电子灵活性和顺应性，可以通过多种功能位置的各种交互进行调整。我们试图了解蛋白质指定黄素的氧化还原活性的个别机制，从这里开始，在简单的模型和两个互补蛋白质中，黄素异氧嘧啶环系统的氢键和弯曲。我们提出了一种在电子结构水平上的酶促黄素氧化还原调节的描述，它结合以下两种方法：(1)第一次使用固体核磁共振测量黄素碳和氮的化学位移张量的主值，增量/II，作为黄素电子和相互作用的灵敏和直接的反映；(2)电子结构、能量和化学位移的计算，以便能够根据黄素电子和相互作用来定量解释观察到的化学位移；(3)核磁共振研究直接评估氢键在硝基还原酶氧化还原调节中的重要性；以及(4)黄毒素和硝基还原酶的诱变来操纵黄素弯曲，黄素氧化还原调节的一个有争议的潜在机制。因此，我们将识别黄素弯曲的15N和13C特征以及黄素产生的不同氢键，并开始在基础水平上了解蛋白质在个别相互作用方面应用的氧化还原调节。这些工具将对黄素蛋白具有广泛的适用性。

DESCRIPTION (provided by applicant): Flavins are versatile redox cofactors which are used in enzymes to perform a wide range of vital chemistry ranging from fatty acid metabolism to reductive detoxification of Hg 2+. This is a reflection of the flavin's electronic flexibility and amenability to tuning via a variety of interactions at multiple functional positions. We seek to understand individual mechanisms by which proteins specify the redox activity of flavins, beginning here with hydrogen bonds and bending of the flavin isoalloxazine ring system in simple models and two complementary proteins. We propose a description of enzymatic flavin redox tuning at the level of electronic structure using a combination of (1) the first use of solid state NMR to measure the principal values,delta/ii, of the chemical shift tensors of flavin carbons and nitrogens as sensitive and direct reflections of flavin electronics and interactions, (2) computation of electronic structure, energies and chemical shifts in order to permit quantitative interpretation of the observed chemical shifts in terms of flavin electronics and interactions, (3) NMR studies directly evaluating the importance of hydrogen bonding in redox tuning in nitroreductase and (4) mutagenesis of flavodoxin and nitroreductase to manipulate flavin bending, a controversial potential mechanism of flavin redox tuning. Thus, we will identify 15N and 13C signatures of flavin bending and the different hydrogen bonds the flavin makes, and begin to understand at a fundamental level the redox tuning applied by proteins in terms of individual interactions. These tools will have widespread applicability to flavoproteins.