Nitroreductase: Determinants of Flavin Enzyme Activity

描述（由申请人提供）：黄素是多功能氧化还原辅因子，用于酶中，以执行从脂肪酸代谢到Hg 2+还原解毒的广泛重要化学反应。 这反映了黄素的电子灵活性和通过在多个功能位置处的各种相互作用来调谐的顺从性。 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，从这里开始与氢键和弯曲的黄素isoalloxazine环系统在简单的模型和两个互补的蛋白质。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谐的描述，其使用以下组合：（1）首次使用固态NMR来测量黄素碳和氮的化学位移张量的主值δ/ii，作为黄素电子和相互作用的敏感和直接反映，（2）电子结构的计算，能量和化学位移为了允许根据黄素电子学和相互作用对观察到的化学位移进行定量解释，（3）NMR研究直接评估氢键在硝基还原酶中氧化还原调节中的重要性，以及（4）诱变的黄素氧还蛋白和硝基还原酶操纵黄素弯曲，一个有争议的潜在机制黄素氧化还原调节。因此，我们将确定15 N和13 C的签名的黄素弯曲和不同的氢键黄素，并开始了解在一个基本的水平上的氧化还原调节蛋白质在个人的相互作用。这些工具将广泛适用于黄素蛋白。

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.