STATIC AND TIME-RESOLVED STRUCTURAL ANALYSES OF HEME METABOLIZING ENZYMES

血红素代谢酶的静态和时间分辨结构分析

• 批准号: 8363688
• 负责人: MASAKAZU SUGISHIMA
• 金额: $ 3.65万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-01 至 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8363688
• 关键词: Algorithms; Bilin; Biliverdine; Binding; Chemicals; Complex; Complex Mixtures; Computer software; Cyanobacterium; Data Collection; Data Set; Diffuse; Dissociation; Enzymes; Family; Ferredoxin; Funding; Globin; Grant; Harvest; Heme; Heme Iron; Hemeproteins; High temperature of physical object; Iron; Iron Chelating Agents; Lasers; Light; Lighting; Link; Maps; Methods; Motion; National Center for Research Resources; Organism; Oxidoreductase; Oxygenases; Physiological; Plants; Principal Investigator; Process; Proteins; Reaction; Reagent; Red Algae; Research; Research Infrastructure; Resources; Roentgen Rays; Site; Source; Structure; Techniques; Temperature; Time; United States National Institutes of Health; X ray diffraction analysis; X-Ray Diffraction; cost; cryogenics; in vivo; inhibitor/antagonist; phycobilin; phytochromobilin; research study; structural biology

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Heme oxygenase (HO) catalyzes physiological degradation of heme using O2 and reducing equivalents to produce biliverdin IXalpha (BV) ferrous iron and CO (Tenhunen R. et al. (1968) Proc. Natl. Acad. Sci. USA 61 748-755). The HO reaction proceeds without product inhibition by the CO generated in the second step of the HO reaction although CO is known to be a potent inhibitor of HO and other heme proteins. In fact exogenous CO inhibits the overall HO reaction especially its third step. Therefore HO must have a mechanism by which it escapes inhibition by endogenously produced CO. In order to track how endogenous CO is released from the reaction site we previously collected two X-ray diffraction data sets from the CO-bound form of one crystal of the hemeHO complex. One data set was collected in the dark and the other under illumination by He-Ne CW laser at 35 K. The light minus dark difference Fourier map showed that CO was partially photodissociated from the heme iron and dissociated CO was trapped in a hydrophobic cavity adjacent to the heme pocket (Sugishima M. et al. (2004) J. Mol. Biol. 341 7-13). In contrast to results on globins we could not detect any protein motion upon photodissociation because the extent of photodissociation was low and the cryogenic conditions severely restrict protein motion. To overcome these issues I propose time-resolved Laue experiments at room temperature and cryo-trapping experiments at higher temperatures where protein motion is less restricted. Using these approaches the motions of protein and heme upon CO photodissociation which mimic the endogenous CO dissociation process could be readily detectable. In addition I also propose to identify reaction intermediates during the HO reaction in the crystal without the necessity for chemical or cryotrapping. Previously we initiated the HO reaction in crystals by diffusing a reducing reagent into the initially oxidized form of heme-HO complex crystals. We successfully determined the crystal structure of one of the reaction intermediates the bound form of BV-iron chelate (Sugishima M. et al. (2003) J. Biol. Chem. 278 32352-8). Because a long time (~3hr) is required for the completion of the overall reaction several other intermediate states can be easily generated as the reaction proceeds. We did not analyze the intermediate state structures at that time because we expected that several reaction intermediate states were present in a complex mixture at all time points. However the algorithms and software for analysis of such mixtures have been developed (Schmidt M. et al. (2003) Biophys. J. 84 2112-29). Details of the intermediate structures the overall mechanism and the rate coefficients for interconversion of these intermediates during the HO reaction would be revealed by applying this method to the diffraction datasets obtained during the reaction. Depending on the time required to acquire a complete data set we could use either the Laue or monochromatic oscillation techniques. In plants red algae and cyanobacteria BV produced by HO is reduced to phytochromobilin and phycobilins by ferredoxin-dependent bilin reductases. PcyA one of the enzymes in this family catalyzes the reduction of BV to phycocyanobilin which is incorporated into light-harvesting and light-sensing complexes in red algae and cyanobacteria. Previously we determined the structure of PcyA in complex with BV (Hagiwara Y. et al. (2006) Proc. Natl. Acad. Sci. USA 103 27-32). We found an unexpected covalent link between PcyA and BV under room light illumination; this covalent link was not found under dark conditions. This photochemical reaction may occur in vivo because red algae and cyanobacteria are photosynthetic organisms. To characterize this covalent link I propose time-resolved and cryo-trapping X-ray data collection from PcyA crystals in the BV-bound form in the dark and under laser illumination.

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 子项目的主要研究者可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 血红素加氧酶（HO）使用O2和还原当量催化血红素的生理降解以产生胆绿素IX α（BV）亚铁和CO（Tenhunen R.等人（1968）Proc.Natl. Acad. Sci. USA 61 748-755）。虽然已知CO是HO和其它血红素蛋白的有效抑制剂，但是HO反应在没有由HO反应的第二步骤中产生的CO的产物抑制的情况下进行。事实上，外源性CO抑制整个HO反应，尤其是其第三步。因此，HO必须有一种机制，通过这种机制，它可以逃避内源性产生的CO的抑制。 为了跟踪内源性CO是如何从反应位点释放的，我们先前收集了两个X-射线衍射数据集从CO结合形式的一个晶体的血红素HO复合物。一组数据是在黑暗中收集的，另一组是在35 K的He-Ne CW激光照射下收集的。亮暗差傅立叶图显示，CO从血红素铁部分光解离，解离的CO被捕获在邻近血红素口袋的疏水空腔中（Sugishima M.等人（2004）J. Mol. 3417 -13）。与球蛋白的结果相反，我们不能检测到任何蛋白质运动时，光解，因为光解的程度是低的，低温条件下严重限制蛋白质运动。 为了克服这些问题，我建议在室温下的时间分辨劳厄实验和低温捕获实验在更高的温度下，蛋白质运动是不太受限制的。利用这些方法，可以很容易地检测到蛋白质和血红素在模拟内源性CO解离过程的CO光解时的运动。 此外，我还建议，以确定反应中间体在HO反应中的晶体，而不需要化学或cryotracking。以前，我们通过将还原剂扩散到最初氧化形式的血红素-HO复合物晶体中来引发晶体中的HO反应。我们成功地确定了反应中间体之一BV-铁螯合物的结合形式（Sugishima M。等人（2003）J.Biol.Chem.27832352 -8）。由于完成整个反应需要很长的时间（约3小时），随着反应的进行，可以容易地产生几种其他中间状态。我们当时没有分析中间态结构，因为我们预计在所有时间点，在复杂的混合物中存在几个反应中间态。然而，已经开发了用于分析这种混合物的算法和软件（施密特M.等人（2003）Biophys. J. 84 2112-29）。详细的中间体的结构，整体机制和这些中间体在HO反应过程中的相互转化的速率系数将被揭示通过应用这种方法在反应过程中获得的衍射数据集。根据获取完整数据集所需的时间，我们可以使用劳厄或单色振荡技术。 在植物中，由HO产生的红藻和蓝细菌BV被铁氧还蛋白依赖性胆色素还原酶还原为植物色素移动素和藻胆素。PcyA是该家族中的一种酶，催化BV还原为藻蓝胆素，藻蓝胆素被掺入红藻和蓝藻中的光捕获和光传感复合物中。先前我们确定了与BV复合的PcyA的结构（Hagiwara Y.等人（2006）Proc.Natl. Acad. Sci. USA 103 27-32）。我们在室内光照下发现PcyA和BV之间存在意想不到的共价连接;在黑暗条件下未发现这种共价连接。这种光化学反应可能发生在体内，因为红藻和蓝藻是光合生物。为了表征这种共价连接，我建议时间分辨和低温捕获的X射线数据收集从PcyA晶体中的BV结合的形式在黑暗中，在激光照射下。