Single-crystal resonance Raman spectroscopy of site-directed mutants of cytochrome c peroxidase.
Single-crystal resonance Raman spectroscopy of site-directed mutants of cytochrome c peroxidase.
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细胞色素 c 过氧化物酶定点突变体的单晶共振拉曼光谱。
DOI：
10.1021/bi00483a004
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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;Mauro,JM;Wang,JM;Fishel,LA;Kraut,J;Spiro,TG
摘要：报道了细胞色素c过氧化物酶（CCP）突变体的单晶共振拉曼光谱，采用配备变温载物台的显微镜。光谱与溶液中突变蛋白质的光谱相似，但存在与血红素的配位和自旋状态有关的可检测差异。Asn-235突变体含有六配位的高自旋态和低自旋态的混合物，其中前者的比例比溶液中高得多。在冷却到223 K时，血红素大部分转化为低自旋形式。Phe-191突变体同样显示出高/低自旋六坐标混合物，以及五坐标血红素的优势群体。冷却后，高自旋的六配位粒子数立即转化为低自旋形式，而五配位粒子数转化得更慢。这种行为介于天然CCP和Asn-235突变体之间，与正常Trp-191-Asp-235氢键在血红素Fe近端锚定中的辅助作用一致。Phe-51突变体在单晶中显示出占主导地位的高自旋五坐标血红素群体，而在溶液中，六坐标形式占主导地位。通过向溶液中加入2-甲基-2，4-戊二醇（MPD）来模拟这种差异，并将其归因于结晶过程中存在的MPD的脱水效应。在降低温度时，五坐标血红素部分转化为六坐标高自旋形式。这种突变体以及天然CCP的老化形式是独特的，在低温下具有稳定的高自旋六配位血红素。光谱显示选择性取向和偏振效应的拉曼带强度，这可以定量地理解的基础上的血红素取向相对于晶轴（定向气体模型）。这些效应有助于区分来自不同对称性振动模式的带，并且它们给出了关于蛋白质中血红素电子跃迁矩的定位的信息。定点诱变提供了关于细胞色素c过氧化物酶（CCP）活性位点的分子相互作用的有价值的信息（Mauro et al.，1989; Smulevich等人，1988 a，B）。该酶催化细胞色素c还原过氧化氢（Yonetani，1976）。注意力自然集中在促进过氧化物0-0键断裂的因素上，这是一个具有重大生物化学意义的过程。高分辨率晶体结构可用于来自面包酵母的CCP（Poulos等人，1980; Finzel等人，1984），并揭示了血红素结合口袋中的一些残基，这些残基准备与血红素Fe原子的轴向配体相互作用。定点诱变使得有可能通过系统地改变这些侧链的化学性质来评价这些相互作用（Fishel等人，1987; Mauro等人，1988年）。我们已经利用血红素基团的共振拉曼（RR）光谱通过已知为配位和自旋状态或轴向配体状态的标记的谱带来监测这些相互作用（Smulevich等人，1986 a，B，1988 a，B，1989，1990）。从这些研究中出现的是Fe原子处于动态张力状态的图像，该动态张力状态是由于通过氢键相互作用施加在轴向配体上的力，
Revised Manuscript Received March 29, 1990 abstract: ResonanceRaman spectra are reported for single crystals of cytochrome c peroxidase (CCP) mutants, taken by using a microscope equipped with a variable-temperature stage. The spectra are similar to those observed for the mutant proteins in solution, but there are detectable differences having to do with the coordination and spin state of the heme. The Asn-235 mutant contains a mixture of six-coordinate highand low-spin states with a delectably higher fraction of the former thanin solution. Upon cooling even to 223 K, the heme is converted mostly to the low-spin form. The Phe-191 mutant likewise shows a high/low-spin six-coordinate mixture, together with a preponderant population of five-coordinate heme. Upon cooling, the high-spin six-coordinate population converts immediately to the low-spin form, while the five-coordinate population does so more slowly. This behavior is intermediate between that of native CCP and the Asn-235 mutant, consistent with an ancillary role for the normalTrp-191-Asp-235 H-bond inthe proximal anchoring of the heme Fe. The Phe-51 mutant shows a dominant high-spin five-coordinate heme population in the single crystal, whereas in solution the six-coordinate form is dominant. This difference is mimicked by adding 2-methyl-2, 4-pentanediol (MPD) to the solution and is attributed to the dehydrating effect of MPD, which is present during crystallization. Upon lowering the temperature, the five-coordinate heme converts partially to a six-coordinate high-spin form. This mutant, as well as an aged form of native CCP, is unique in having a stable high-spin six-coordinate heme at low temperature. The spectra show selective orientation and polarization effects on the Raman band intensities, which can be understood quantitatively on the basis of the heme orientation relative to the crystal axes (oriented gas model). These effects help to discriminate among bands arisingfrom vibrational modes of differentsymmetry, and they give information aboutthe localization of the heme electronictransition moments in the protein.Site-directed mutagenesis is providing valuable information about molecular interactions at the active site of cytochrome c peroxidase (CCP)(Mauro et al., 1989; Smulevich et al., 1988a, b). This enzyme catalyzes the reduction of hydrogen peroxide by cytochrome c (Yonetani, 1976). Attention nat-urally focuses on the factors promoting the cleavage of the peroxide 0-0 bond, a process of great biochemical signifi-cance. High-resolution crystal structures are available for CCP from bakers’ yeast (Poulos et al., 1980; Finzel et al., 1984), and reveal a number of residues in the heme-binding pocket, which are poised to interact with the axial ligands of the heme Fe atom. Site-directed mutagenesismakes it possible to evaluate these interactions by systematic alteration of the chemical nature of these side chains (Fishel et al., 1987; Mauro et al., 1988). We have utilized resonance Raman (RR) spectra of the heme group to monitor these interactions via bands that are known to be markers of the coordination and spin state or of the status of the axial ligands (Smulevich et al., 1986a, b, 1988a, b, 1989, 1990). What emerges from these studies is a picture of an Fe atomin a state of dynamic tension due to forces exerted on the axial ligands by H-bond interactions from
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温度和甘油对细胞色素 c 过氧化物酶和选定突变体的共振拉曼光谱的影响。
影响因子：
2.9
作者：
G. Smulevich;A. Mantini;A. English;J. Mauro
通讯作者：
J. Mauro
细胞色素c过氧化物酶氰化物的质子超精细磁共振的温度和pH依赖性。
DOI：
--
发表时间：
1983
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Satterlee,JD;Erman,JE
通讯作者：
Erman,JE
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。
影响因子：
2.9
作者：
Smulevich,G;Wang,Y;Edwards,SL;Poulos,TL;English,AM;Spiro,TG
通讯作者：
Spiro,TG
瞬态共振拉曼光谱显示细胞色素 c 过氧化物酶 CO 光解离后出现未松弛的血红素。
DOI：
10.1016/0167-4838(86)90193-7
发表时间：
1986
期刊：
Biochimica et biophysica acta
影响因子：
--
作者：
Smulevich,G;Dasgupta,S;English,A;Spiro,TG
通讯作者：
Spiro,TG
DOI：
10.1016/s0021-9258(18)67355-7
发表时间：
1986-08
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
S. Hashimoto;J. Teraoka;T. Inubushi;T. Yonetani;T. Kitagawa
通讯作者：
S. Hashimoto;J. Teraoka;T. Inubushi;T. Yonetani;T. Kitagawa