人体血红蛋白-亚硝基化合物的结构生物学研究

An estimated 1 in 5 adult Chinese suffer from one or more types of cardiovascular disease (CVD) and, every year about 3 million Chinese die of CVD which is now the leading cause of death. It is now widely recognized that the simplest nitrogen oxide, NO, biosynthesized by a mammalian heme-containing enzyme (NO synthase, NOS), plays a major role in regulating normal blood pressure and maintaining proper cardiac function. .Nitrosothiols (RSNO) are implicated as physiological NO donors. On the other hand, the related alkylnitrites (RONO) are known to oxidize deoxyHb and oxyHb to metHb which is not able to bind oxygen. Nitrosoalkane/arene compounds (RNO; R = alkyl or aryl) are toxic due to their binding to the heme sites of ferrous Hb to form stable HbRNO complexes which prevent oxygen access to Hb. Such RNO compounds result from the Hb-mediated (or cyt P450 mediated) conversions of nitroaromatic or amine drugs. Organonitro compounds represent a diverse class of compounds that have been utilized as explosives in the mining industry and in combat. TNT (2,4,6-trinitrotoluene) is commercially available and is a high-impact explosive. It has been known for quite some time that TNT can attack the Fe cnters in proteins such as Hb, and can also cause liver cancer, cataract formation, toxic hepatitis, etc. While the physiology of NO continues to be studied in earnest, there is an urgent need for chemical information regarding the factors that direct various nitroso compounds (as well as organonitro) as behavior in various heme protein environments towards productive (e.g., signaling) or destructive (e.g., cell death) consequences. .The existence of this gap in knowledge is an impediment towards a better mechanistic understanding of nitroso compounds biochemistry and also inhibits a complete understanding of the adverse effects that organonitro munitions have on life in general, and how to reverse these adverse effects. The overall objective of this proposal is to use a structural biology approach to examine the effects of the interactions of human hemoglobin with nitroso compounds as well as organonitro compounds (TNT). Specifically, we will employ spectroscopy, macromolecular X-ray crystallography, and correlated single-crystal spectroscopy/crystallography to determine the crystal structures of Hb derivatives that have been modified by physiologically important nitroso compounds, and wil examine associated protein conformation changes.

近年来我国心血管疾病的发病率和死亡率呈上升趋势，据统计，每5个成年人中就有1人患有心血管疾病。因此作为血管舒张因子的一氧化氮（NO）的生理过程及其反应机理显然是该领域的研究重点之一。其中化学因素导致或控制包括NO在内的亚硝基化合物在各种含血红素的蛋白环境中的行为研究则显得尤为重要，因为这些行为将直接影响到这些化合物在体内是发挥良性作用(如信号传导或其本身合成的反馈抑制过程)或破坏性作用(如导致细胞死亡)。本项目拟制备和解析若干具有生物重要性的亚硝基化合物（包括有机-氮氧化物）与人体内血红蛋白结合的晶体结构，研究结果将为血红蛋白-亚硝基化合物的结构与功能的关系、血红蛋白调控这些重要亚硝基化合物新陈代谢机理的阐明、以及这些亚硝基化合物对心血管疾病相关过程的影响提供重要的新信息和结构依据。

心血管疾病是威胁人类健康的头号杀手，据统计，我国有近2.7亿心血管疾病患者，该病死亡人数300万／年，占总死亡病因的51%。因此作为血管舒张因子的一氧化氮（NO）的生理过程及其反应机理显然是该领域的研究重点之一。其中化学因素导致或控制包括NO在内的亚硝基化合物在各种含血红素的蛋白环境中的行为研究则显得尤为重要，因为这些行为将直接影响到这些化合物在体内是发挥良性作用(如信号传导或其本身合成的反馈抑制过程)或破坏性作用(如导致细胞死亡)。该项目的目标为制备和解析若干具有生物重要性的一氧化氮及亚硝基化合物（包括有机-氮氧化物）与人体内血红蛋白结合的晶体结构，研究结果将为血红蛋白-亚硝基化合物的结构与功能的关系、血红蛋白调控这些重要亚硝基化合物新陈代谢机理的阐明、以及这些亚硝基化合物对心血管疾病相关过程的影响提供重要的新信息和结构依据。.在本项目执行期间，我们已经取得了以下研究成果：.1. 人体血红蛋白一氧化氮衍生物晶体结构解析（已发表）：通过晶体浸泡法获得HbNO结构，晶体衍射数据经过数据处理，相位获取，模型构建及结构精修等步骤，最终获得1.9Å分辨率晶体数据。.2. 亚硝基硫醇结构链接异构化研究（已发表）: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，显示为顺式构型。.3. 血红蛋白与苯基羟胺结构功能学研究（未发表，请勿公开）：通过晶体浸泡法制备metHb-苯基羟胺反应衍生物的晶体，适当大小的metHb单晶转入含5～8%甘油的冻存液，加入少量苯基羟胺，室温下浸泡一个月。形状规整的单晶冻存，送至上海光源收集X-射线晶体衍射数据，最终获得分辨率为1.85Å晶体结构。结构显示高铁血红蛋白经苯基羟胺诱导在beta亚基活性中心形成高铁血色原结构，即近远端组氨酸残基的咪唑环同时与血红素铁原子绑定。此外，bCys93被发生翻译后修饰，形成亚硝基硫醇结构。.4. Hb与TNT反应衍生物的结构分析（未发表，请勿公开）：为进一步探索TNT与血红蛋白在还原剂条件下的相互作用机理，通过晶体浸泡、共结晶、反应后结晶三种不同的方法，尝试制备TNT与Hb反应衍生物的单晶。将metHb晶体与TNT、Na2S2O4混合浸泡11个月后，获得一套最高分辨率为2.15 Å的衍生物晶体结构数据，但由于部分分子结构无法构建，仍在结构精修步骤。