Resonance Raman spectroscopy of cytochrome c peroxidase single crystals on a variable-temperature microscope stage.
Resonance Raman spectroscopy of cytochrome c peroxidase single crystals on a variable-temperature microscope stage.
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变温显微镜台上细胞色素 c 过氧化物酶单晶的共振拉曼光谱。
DOI：
10.1021/bi00462a022
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发表时间：
1990
期刊：
影响因子：
2.9
通讯作者：
Spiro,TG
中科院分区：
文献类型：
--
作者：
Smulevich,G;Wang,Y;Edwards,SL;Poulos,TL;English,AM;Spiro,TG
Firenze大学Dipartimento di Chimica，Via G.Capponi 9,50121 Firenze，意大利，普林斯顿大学化学系，新泽西州普林斯顿，08544-1009，马里兰大学生物技术研究所高级生物技术研究中心，马里兰大学，Shady Grove，9600 Gudelsky Drive，Rockville，马里兰20850，以及加拿大康科迪亚大学化学系，蒙特利尔，魁北克，加拿大H3G 1M8收到2月15日，；11月1日收到修订稿件，摘要：在显微镜下获得了高质量的细胞色素C过氧化物酶单晶(0.2X0.5X1 mm)的共振拉曼(RR)光谱。观察到晶体取向和极化效应，根据卟啉振动模的对称性来区分RR带。利用定向气体中的DiH生色团模型，利用晶体结构原子坐标，对完全对称和非完全对称模式的孤立带的退偏率进行了精确的标定。计算表明，电子跃迁力矩大致沿连接亚甲基桥的线方向，这表明乙烯基团的电子转向效应。观察到与卟啉V10和乙烯基C=C伸缩模式相关的带的偏离，这可能是由于它们的近共振相互作用所致。谱带频率与先前在溶液中观察到的五配位高自旋Fe111血红素的谱带频率相对应，与X射线结构一致，表明Fe原子在近端的血红素平面外，而远端的水分子位于非键距离2.4？用焦耳-汤普森低温恒温器对封在玻璃毛细管中的晶体测定了RR谱的温度依赖性。随着温度的降低，光谱转换为低自旋的Fe111血红素的一种特征。这种转变是相当渐进的，这是很容易逆转的。在-50℃可以检测到，但即使在-190℃也是不完全的。提出了一种对蛋白质结构的温度效应，允许Fe原子接近血红素平面，并结合远端水分子或远端组氨酸。由于蛋白质分子结构的信息主要取决于X射线晶体结构决定，因此发展能够比较单晶、溶液或其生物基质中的蛋白质的光谱技术是非常必要的。拉曼光谱学是进行这种比较的一个有吸引力的候选者，因为振动光谱只对分子振荡器的局部环境敏感，并且与介质无关。振动频率对分子结构很敏感，人们可以通过共振拉曼效应将激发光的波长调节到适当的电子跃迁来探测特定的发色部位(Carey，1982)。在晶体中，生色团通常在振动上彼此很好地隔离。因此，分子散射张量的性质可以通过分子和晶轴之间的几何关系从晶体散射张量的性质中计算出来。激光光源使我们有可能在晶面的背向散射中获得拉曼光谱，其小到激光的横截面。然而，蛋白质晶体对拉曼光谱学家来说是一个巨大的挑战，因为它们很脆弱，很容易被激光破坏，特别是在共振拉曼光谱的情况下，当样品吸收光时，…
Dipartimento di Chimica, Universitá di Firenze, Via G. Capponi 9, 50121 Firenze, Italy, Department of Chemistry, Princeton University, Princeton, New Jersey 08544-1009, Center for Advanced Research in Biotechnology of the Maryland Biotechnology Institute, University of Maryland, Shady Grove, 9600Gudelsky Drive, Rockville, Maryland 20850, and Department of Chemistry, Concordia University, Montreal, Quebec, CanadaH3G 1M8 Received February 15, 1989; Revised Manuscript Received November 1, 1989 abstract: Good quality resonance Raman (RR) spectra have been obtained for cytochrome c peroxidase single crystals (0.2 X 0.5 X 1 mm) lying on their 110 faces on a microscope stage. Crystal orientation and polarization effects are observed which differentiate the RR bands on the basis of the symmetries of the porphyrin vibrational modes. The measured depolarization ratios are accurately calibrated for isolated bands of both totally symmetric and non totally symmetric modes by using a model of Dih chromophores in an oriented gas using the crystal structure atomic coordinates. The calculations indicatethat the electronic transition moments are approximately along thelines connecting the methine bridges, suggesting an electronic steering effect of the vinyl groups. Deviations are observed for bands associated with the porphyrin v10 and the vinyl C= C stretching modes, which may be due to their near-resonant interaction. The band frequencies correspond to those of a five-coordinate high-spin Fe111 heme, as previously observed in solution, consistent with the X-ray structure showing the Fe atom to be out of the heme plane on the proximal side with a distal water molecule located at a nonbonded distance, 2.4 Á. The temperature dependence of the RR spectrum was determined with a Joule-Thompson cryostat on crystals sealed in glass capillaries. As the temperature is lowered, the spectrum converts to one characteristic of a low-spin Fe111 heme. The conversion, which is readily reversible, is quite gradual. It is detectable at-50 Cbut is incomplete even at-190 C. A temperature effect on the protein structure is proposed which permits the Fe atomto approach the heme plane and bind the distal water molecule, or the distal histidine.Since information on the molecular architecture of proteins depends overwhelmingly on X-ray crystal structure determi-nations, it is highly desirable to develop spectroscopic techniques capable of comparing proteins in single crystals and in solution or in their biological matrices. Ramanspectroscopy is an attractive candidate for such comparisons because the vibrational spectrum is sensitive only to the local environment of the molecular oscillators and is independent of the medium. The vibrational frequencies are sensitive to molecular structure, and one can probe for specific chromophoric sites via the resonance Raman effect by tuning the wavelength of the ex-citing light to an appropriate electronic transition (Carey, 1982). In the crystal the chromophores are in general vibrationally wellisolated from one another. The properties of the molecular scattering tensor can be therefore calculated from the properties of the crystal scattering tensor by means of the geometric relationships between the molecular and the crystal axes. Laser light sources make it possible to obtain a Raman spectrum in backscattering from a crystal face which is as small as the laser beam cross section. Nevertheless, protein crystals are a formidable challenge to Raman spec-troscopists because they are fragile and easily destroyed by the laser beam, especially in the case of resonance Raman spectroscopy when the sample absorbs the light …