Mechanism of Cph1 phytochrome assembly from stopped-flow kinetics and circular dichroism
Mechanism of Cph1 phytochrome assembly from stopped-flow kinetics and circular dichroism
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DOI：
10.1021/bi035511n
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发表时间：
2003-11-25
期刊：
影响因子：
2.9
通讯作者：
Lamparter, T
中科院分区：
文献类型：
--
作者：
Borucki, B;Otto, H;Lamparter, T
采用停流法和圆二色谱法研究了集胞藻（Synechocystis）光敏色素holo-Cph 1从载脂蛋白和发色团藻蓝胆素（PCB）和藻红胆素（PEB）中自催化组装的动力学和机理。在1：1化学计量比、pH 7.9和10 ℃下，PCB动力学数据的SVD分析揭示了涉及三个跃迁的三个光谱分量，时间常数τ（1）类似于150 ms，τ（2）类似于2.5 s，τ（3）类似于50 s。τ（1）与从Soret区到680 nm区的主要红移和振子强度转移有关。当半胱氨酸259的巯基被碘乙酰胺封闭，阻止共价加合物的形成时，形成非共价红移复合物（680 nm），时间常数为200 ms。因此，tau（1）可以归因于非共价复合物的形成。tau（1）过程中的吸收变化是由于线性四吡咯的延伸构象的形成及其在结合口袋中的质子化。从浓度和pH依赖性的动力学，我们得到了一个值为1.5 μ M的K-D的这种非共价复合物和一个值为8.4的质子供体的pK（a）。tau（2）组分与约25 nm的蓝移相关，并归因于共价键（P-r）的形成，伴随着环A的3-3'双键的丢失。对于非光致变色的PEB，非共价复合物的形成更快（tau（1）= 70 ms），但共价键的形成比天然发色团PCB慢约80倍（tau（2）= 200 s）。PCB加合物在250-800 nm范围内的CD光谱表明，P-r和P-fr中的发色团几何形状与植物光敏色素中的发色团几何形状相似。P-r和P-fr在最长波段的相反转动强度表明光致异构化引起手性反转。当半胱氨酸259被IAA或体积较大的IAF阻断时，与非共价结合的PCB的Cph 1复合物仍然是光致变色的。因此，与半胱氨酸259的共价连接对于光转化是不需要的。非共价结合的PCB在P-R-和P-FR-样状态的CD光谱定性相似的共价加合物，这表明类似的结构中的结合口袋。与结合口袋的非共价相互作用显然足以将发色团保持在适当的几何形状中以进行光异构化。
The kinetics and mechanism of the autocatalytic assembly of holo-Cph1 phytochrome (from Synechocystis) from the apoprotein and the bilin chromophores phycocyanobilin (PCB) and phycoerythrobilin (PEB) were investigated by stopped flow and circular dichroism. At 1:1 stoichiometry, pH 7.9, and 10 degreesC, SVD analysis of the kinetic data for PCB revealed three spectral components involving three transitions with time constants tau(1) similar to 150 ms, tau(2) similar to 2.5 s, and tau(3) similar to 50 s. tau(1) was associated with a major red shift and transfer of oscillator strength from the Soret region to the 680 nm region. When the sulfhydryl group of cysteine 259 was blocked with iodoacetamide, preventing the formation of a covalent adduct, a noncovalent red-shifted complex (680 nm) was formed with a time constant of 200 ms. tau(1) could thus be assigned to the formation of a noncovalent complex. The absorption changes during tau(1) are due to the formation of the extended conformation of the linear tetrapyrrole and to its protonation in the binding pocket. From the concentration and pH dependence of the kinetics we obtained a value of 1.5 muM for the K-D of this noncovalent complex and a value of 8.4 for the pK(a) of the proton donor. The tau(2) component was associated with a blue shift of about 25 nm and was attributed to the formation of the covalent bond (P-r), accompanied with the loss of the 3-3' double bond to ring A. tau(3) was due to photoconversion to P-fr. For PEB, which is not photochromic, the formation of the noncovalent complex is faster (tau(1) = 70 ms), but the covalent bond formation is about 80 times slower (tau(2) = 200 s) than with the natural chromophore PCB. The CD spectra of the PCB adduct in the 250-800 nm range show that the chromophore geometries in P-r and P-fr are similar to those in plant phytochrome. The opposite rotational strengths of P-r and P-fr in the longest wavelength band suggest that the photoisomerization induces a reversal of the chirality. The Cph1 complex with noncovalently bound PCB was still photochromic when cysteine 259 was blocked with IAA or with the bulkier IAF. The covalent linkage to cysteine 259 is thus not required for photoconversion. The CD spectra of the noncovalently bound PCB in P-r- and P-fr-like states are qualitatively similar to those of the covalent adducts, suggesting analogous structures in the binding pocket. The noncovalent interactions with the binding pocket are apparently sufficient to hold the chromophore in the appropriate geometry for photoisomerization.