哺乳类动物中血红素蛋白调控的硫化物新陈代谢机理

Sulfur is a critical element for human health, and is present in the amino acids cysteine, methionine, homocysteine, and in a variety of iron-sulfur electron transfer proteins. Interestingly, it is now known that the terminal product of sulfite reduction, namely hydrogen sulfide (H2S), is also biosynthesized in humans! This pungent smelly gas has been proposed to be a physiological signaling molecule for vasodilation in humans, in addition to being implicated in neurotransmission, inflammation, and in diabetes...There is growing evidence of reactions of various sulfur-containing compounds with human and mammalian heme proteins. However, to the best of our knowledge, there have been no studies on the iron-mediated sulfur metabolism by human and mammalian heme proteins. This represents a significant gap in knowledge, and the lack of understanding negatively impacts our ability to accurately determine the causes of various ailments associated with the physiology and pathology of sulfur...Our long-term goal is to delineate the role that the iron (Fe) center in heme proteins play in the metabolism of biologically important small molecules, and our recent work has focused on the heme-mediated biological chemistry of various NOx species associated with vasodilation, nitriheme formation, and nitrite-induced heme loss from intact human Hb. The goal of this proposal is to determine the factors that lead to the interconversion of sulfur species mediated by heme Fe in the muscle protein myoglobin (Mb) and the blood protein hemoglobin (Hb). Our proposed work is based on our recent discoveries that these mammalian proteins do engage in similar reactivities (e.g, in nitrite reduction) to those previously assigned exclusively to the plant and bacterial sulfite/nitrite reductase enzymes. ..We hypothesize that the heme sites of the Mb and Hb actively participate in the metabolism of sulfur-containing species such as H2S, sulfite, and sulfur-containing drugs. Our proposal challenges existing paradigms in the field that are based on the non-participation of these mammalian proteins in sulfur metabolic reactions. Our success with the related nitrogen-metabolism systems has uniquely prepared us to research this unchartered territory. We propose the following Specific Aims:.. Specific Aim 1: We will prepare and isolate adducts of wild-type, mutant, and reconstituted Mbs with sulfur compounds such as sulfite and sulfide that represent likely intermediates along the sulfur metabolism pathway.. Specific Aim 2: We will determine the conditions that promote the formation of the green-pigmented sulfheme Mbs for the wild-type, mutant, and reconstituted derivatives, and examine the related reactions with human Hb.. We will employ mutagenesis, spectroscopy, electrochemistry, macromolecular X-ray crystallography, and correlated single-crystal spectroscopy/crystallography to achieve these aims.

虽然研究表明不同的硫化物可以与哺乳动物体内的血红素蛋白发生反应，然而至今还没有任何有关人体内血红素蛋白通过铁活性中心介导的含硫化合物新陈代谢（硫循环）过程机理的研究报道，这一知识空白极大阻碍了人们对若干与硫的生理或病理相关疾病起因的深入理解。本项目的目标是确定肌球蛋白（肌肉中）和血红蛋白（血液内）铁活性中心对硫化物之间转化反应的控制因素，并研究过量硫化物对该类血红素蛋白结构与功能的影响。本项目拟表达、分离和纯化在肌球蛋白控制的硫代谢过程中可能产生的一系列中间产物；拟采用野生型、再造型及突变型肌球蛋白，通过改变活性中心氢键数目与位置以及改变血红素大环结构，精确分析蛋白活性中心对硫化物结构的影响；并研究体内过量硫化物对血红素蛋白结构影响所导致的硫血红素蛋白血症形成的机理。研究结果将为含硫配体的血红素蛋白的结构与功能的关联、以及血红素蛋白调控的硫化物新陈代谢的机理提供重要的新信息和结构依据。

硫是人和哺乳类动物体的必须元素之一，在半胱氨酸、蛋氨酸、同型半胱氨酸等氨基酸以及各种铁硫电子传递蛋白质中都有硫元素。亚硫酸盐还原反应的终端产物硫化氢（H2S）在人体内也可生物合成，近期研究表明这一具有臭鸡蛋刺鼻气味的气体被作为人体血管舒张的生理信号分子，含硫物种对神经传递、炎症和糖尿病等也具有重要的影响。虽然研究已表明不同的硫化物可以与哺乳动物体内的血红素蛋白发生反应，但至今还没有任何有关人体内血红素蛋白通过铁活性中心介导的含硫物种新陈代谢过程机理的研究报道，这一知识空白极大阻碍了人们对若干与硫的生理或病理相关疾病起因的深入理解。本项目的目标是确定肌球蛋白和血红蛋白铁活性中心对硫化物之间转化反应的控制因素，并研究过量硫化物对该类血红素蛋白结构与功能的影响。 通过改变活性中心氢键数目与位置以及改变血红素大环结构，分析蛋白活性中心对硫化物结构的影响；并研究体内过量硫化物对血红素蛋白结构影响所导致的硫血红素蛋白血症形成的机理。.在本项目执行期间，我们已经取得了以下研究成果：.1. 以野生型马心肌红蛋白质粒pGYM为模版，制备了从单点突变到三点突变共15种重组蛋白的质粒。.2. 已成功表达分离纯化了15种突变体重组蛋白，成功获得了包括hhMb H64V在内的共8中突变型蛋白晶体。.3. 通过观测硫化物与肌红蛋白（野生型和突变型）紫外可见吸收光谱变化发现：1）突变型hhMb硫代血红素得率及稳定度不同；2）突变型hhMb H64V/H93Y基本不与硫化物反应；3)突变型hhMb H93M的硫化物衍生物不稳定，部分形成脱血红素辅基蛋白。.4. 已成功获得L32I MbS在内的3种突变型Mb与硫化物或氮氧化物绑定的衍生物晶体结构。.5. 已成功获得并解析了人体血红蛋白一氧化氮衍生物晶体结构。.6. 已成功获得并报道了第一个有序高分辨率的亚硝基半胱氨酸甲酯衍生物三维晶体结构，并采用密度泛函理论计算了包括亚硝基甲烷在内的4种亚硝基硫醇在气态中的几何构型、成键／反键分子轨道及相对能量等参数。.7. 已成功获得经苯基羟胺诱导产生的首个人体血红蛋白高铁血色原结构，此外beta-Cys93发生翻译后修饰形成亚硝基硫醇结构。

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