MOLECULAR BASIS FOR CARBON MONOXIDE SENSING AND RESPONSE

红红螺旋菌对环境中一氧化碳（CO）的存在作出反应，表达一组基因，其产物将CO氧化为CO2并在此过程中产生能量。CooA是这种共依赖转录所必需的蛋白质，是CRP的同源物。在之前的授权期内，我们纯化了CooA至均匀性，并表明它是一个大约54 kD的二聚体，含有原血红素作为CO结合位点。由于CooA在所有条件下都是二聚体，但仅在CO结合时与DNA结合，因此CooA很可能发生类似于CRP与cAMP结合时的构象变化。CooA的血红素在CO结合前后为6位，表明CO的配体位移可能是引起构象变化的原因。CooA必须被还原才能与CO结合，并且在还原过程中会发生不寻常的配体开关。在本提案中，我们将通过各种突变，光谱和结构分析来确定CooA对CO响应的分子基础。在此过程中，我们将识别血红素的剩余未知配体和其他对CO反应至关重要的残基。这些残基包括参与氧化还原依赖的配体开关的残基，以及参与构象变化本身的残基。CooA有许多有趣的特性，其中之一是它对CO的特异性，以及它对通常结合血红素的其他小分子不起功能反应。我们提出了多种方法来理解这种特异性，包括一种极有可能识别新特性的CooA变体的诱变方法。随后对这些变异进行生化和光谱分析，不仅可以揭示CooA活性的重要特征，还可以揭示小分子与血红素结合的一般规律。与许多其他血红素蛋白相比，通过使用报告系统选择CooA新变体的能力是一项巨大的技术优势。这种自我报告性质将被利用来深入了解血红素反应性的分子基础和血红素蛋白的功能，如血红蛋白和可溶性胍基环化酶。

Rhodospirillum rubrum responds to the presence of carbon monoxide (CO) in its environment by expressing a set of genes whose products oxidize CO to CO2 and generate energy in the process. CooA is the protein that is necessary for this CO-dependent transcription, and is a homolog of CRP. In the previous grant period, we have purified CooA to homogeneity and shown it to be a dimer of approximately 54 kD that contains protoheme as the CO- binding site. Because CooA is a dimer under all conditions, but only binds DNA upon CO binding, it is likely that CooA undergoes a conformational change analogous to that of CRP upon cAMP binding. The heme of CooA is 6-coordinate before and after CO binding, suggesting that ligand displacement by CO is the likely trigger for that conformational change. CooA must be reduced in order to bind CO and an unusual ligand switch occurs upon reduction. In the present proposal, we will determine the molecular basis for the response of CooA to CO by a variety of mutational, spectroscopic and structural analyses. In the course of this, we will identify the remaining unknown ligand to the heme and the other residues critical for the response to CO. These include the residues that are involved in the redox-dependent ligand switch, as well as those involved in the conformational change itself. Among the many interesting properties of CooA is its specificity for CO and its failure to respond functionally to other small molecules that typically bind hemes. We propose a variety of approaches to understand this specificity including a mutagenesis approach which is highly likely to identify CooA variants of novel properties. Subsequent biochemical and spectral analysis of these variants should not only reveal the important features of CooA activity, but suggest general rules for the binding of small molecules to hemes. The ability to select novel variants of CooA through the use of reporter systems is an enormous technical advantage compared to the case with many other heme proteins. This self-reporting property will be exploited to gain an insight into the molecular basis for heme reactivity and function in heme proteins of medical interest, such as hemoglobins and soluble guanylyl cyclase.