NMR SPECTROSCOPY OF IRON-SULFUR PROTEINS

描述（由申请人提供）：在以前的项目期间进行的工作已经证明，NMR光谱学，X射线晶体学和量子化学计算的组合作为一种高效的方法来理解铁硫蛋白的序列-结构-功能关系。核磁共振谱包含了关于电子核相互作用的极其敏感的信息。这些信息是X射线晶体结构无法获得的，它提供了对铁中心的化学性质、几何形状的细节、氢键的强度和电子离域模式的深入了解。因此，NMR作为观察控制氧化还原电位和调节电子转移途径的簇的性质的窗口。我们已经证明，高水平的量子化学方法可以成功地将实验NMR观测值与高分辨率X射线晶体学提供的几何结构相关联，从而为调整金属位点性质的因素提供详细的见解。反过来，NMR数据和量子计算可以提供结构约束的情况下，X射线晶体学还没有可行的足够的分辨率。我们建议使用这种协调的方法来研究一系列的1Fe和[2Fe-2S]蛋白质，在电子转移的研究中的模型系统，其目的是回答一些问题，关于它们的结构-功能关系。待研究的蛋白质包括：一系列具有改变的氧化还原电位的梭菌红氧还蛋白（单体1Fe蛋白）、铁氧还蛋白（二聚体1Fe蛋白）和四种具有不同光谱和功能特性的原型[2Fe-2S]蛋白（鱼腥藻营养铁氧还蛋白、人铁氧还蛋白、Rieske蛋白和风产液菌铁氧还蛋白）。我们建议，不仅收集和分析NMR数据，而且还与他人合作，以获得晶体结构和电子-核双共振（ENDOR）数据。还原的红氧还蛋白的X射线结构最近变得可用，但目前没有任何还原的[2Fe-2S]蛋白的结构可用。努力将扩大到改善1Fe蛋白质的计算和扩展的方法[2Fe-2S]蛋白质。还将努力开发顺磁NMR的新方法，特别是对于那些不适合既定方法的系统。

DESCRIPTION (provided by applicant): Work carried out in previous project periods has demonstrated that the combination of NMR spectroscopy, X-ray crystallography, and quantum chemical calculations serves as a highly productive approach to understanding sequence-structure-function relationships in iron-sulfur proteins. NMR spectra contain exquisitely sensitive information about electron nuclear interactions. This information, which is unavailable from X-ray crystal structures, provides insights into the chemical properties of the iron centers, details of their geometry, strengths of hydrogen bonds, and patterns of electron delocalization. NMR thus serves as a window for viewing the properties of the cluster that control redox potentials and regulate pathways of electron transfer. We have shown that high-level quantum chemical approaches can successfully correlate experimental NMR observables with geometric structures provided by high-resolution X-ray crystallography, and thus provide detailed insight to the factors tuning the properties of the metal site. In turn, NMR data and quantum calculations can provide structural constraints in cases where X-ray crystallography has not been feasible at sufficient resolution. We propose to use this coordinated approach to investigate a series of 1Fe and [2Fe-2S] proteins that are model systems in the study of electron transfer, with the aim of answering a number of questions regarding their structure-function relationships. The proteins to be studied include: a series of clostridial rubredoxins (monomeric 1Fe proteins) that have altered redox potentials, desulforedoxin (dimeric 1Fe protein), and four prototypical [2Fe-2S] proteins that have distinct spectral and functional properties (Anabaena vegetative ferredoxin, human ferredoxin, a Rieske protein, and Aquifex aeolicus ferredoxin). We propose, not only to collect and analyze NMR data, but also to collaborate with others to obtain crystal structures and electron-nuclear double resonance (ENDOR) data. An X-ray structure for reduced rubredoxin recently became available, but currently no structures are available for any reduced [2Fe-2S] proteins. Effort will be expended toward improving the calculations for 1Fe proteins and extending the methodology to [2Fe-2S] proteins. Effort will also be expended toward developing new methodology for paramagnetic NMR, in particular, for systems that are not amenable to established approaches.