MOLECULAR BASIS FOR CARBON MONOXIDE SENSING AND RESPONSE

红色红螺菌通过表达一系列基因来响应环境中一氧化碳(CO)的存在，这些基因的产物将CO氧化为CO2并在此过程中产生能量。CooA是这种一氧化碳依赖的转录所必需的蛋白质，是CRP的同源物。在上一个授权期，我们已经将CooA纯化到均一性，并证明它是一个约54kD的二聚体，包含原血红素作为CO结合位点。由于CooA在所有条件下都是二聚体，但只有在CO结合时才与DNA结合，所以CooA可能会经历与cAMP结合时CRP类似的构象变化。CooA的血红素在CO结合前后是6配位的，这表明CO取代配体可能是这种构象变化的触发因素。CooA必须被还原才能与CO结合，还原时会发生不寻常的配体转换。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反应的分子基础。在此过程中，我们将确定剩余的未知配体的血红素和其他残基的反应CO的关键。这些包括参与氧化还原依赖的配体交换的残基，以及参与构象变化本身的残基。在CooA的许多有趣的特性中，有一点是它对CO的专一性，以及它不能对通常结合血红素的其他小分子做出功能反应。我们提出了多种方法来理解这种特异性，包括突变方法，它很有可能识别新特性的CooA变体。随后对这些变体的生化和光谱分析不仅应该揭示CooA活性的重要特征，还应该为小分子与血红素的结合提出一般规则。与许多其他血红素蛋白相比，通过使用报告系统选择CooA的新变体的能力是一个巨大的技术优势。这种自我报告的特性将被用来深入了解血红素在具有医学意义的蛋白质中的反应和功能的分子基础，如血红蛋白和可溶性鸟苷酸环化酶。

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.