Giuffra E, Zucchelli G, Sandonà D, Croce R, Cugini D, Garlaschi F M, Bassi R, Jennings R C
Centro CNR Biologia Cellulare e Molecolare delle Piante, Dipartimento di Biologia, Università di Milano, Via Celoria 26, 20133 Milano, Italy.
Biochemistry. 1997 Oct 21;36(42):12984-93. doi: 10.1021/bi9711339.
The minor photosystem II antenna complex CP29(Lhcb-4) has been reconstituted in vitro with the Lhcb-4 apoprotein, overexpressed in Escherichia coli, and the native pigments. Modulation of the pigment composition during reconstitution yields binding products with markedly different chlorophyll a/b binding ratios even though the total number of bound chlorophylls (a plus b) remains constant at eight. A thermodynamic analysis of steady state absorption and fluorescence spectra demonstrates that all chlorophylls are energetically coupled, while the kinetics of chlorophyll photooxidation indicate that triplet chlorophyll-carotenoid coupling is also conserved during pigment binding in vitro. The influence of the chlorophyll a/b binding ratio on the absorption spectra measured at 72 and 300 K is analyzed for the Qy absorption region. Increased chlorophyll b binding leads to large increases in absorption in the 640-660 nm region, while absorption in the 675-690 nm interval decreases markedly. These changes are analyzed in terms of a Gaussian decomposition description in which the eight subbands display a temperature-dependent broadening in agreement with the weak electron-phonon coupling demonstrated for other antenna chlorophyll spectral forms. In this way, we demonstrate that increased chlorophyll b binding leads to increased absorption intensity associated with the subbands at 640, 648, 655, and 660 nm and decreased intensity for the long wavelength subbands at 678 and 684 nm. The wavelength position of all subbands is unchanged. The above data are interpreted to indicate that CP29 has eight chlorophyll binding sites, many or all of which can be occupied by either chlorophyll a or chlorophyll b according to the conditions in which pigment binding occurs. Chlorophyll b absorption is primarily associated with four subbands located at 640, 648, 655, and 660 nm. The invariance of the wavelength position of the absorption bands in recombinant products with different chlorophyll a/b binding stoichiometries is discussed in terms of the mechanism involved in the formation of spectral bands. We conclude that pigment-protein interactions dominate in the determination of spectral heterogeneity with probably only minor effects on absorption associated with pigment-pigment interactions.
小型光系统II天线复合物CP29（Lhcb - 4）已在体外与在大肠杆菌中过表达的Lhcb - 4脱辅基蛋白和天然色素进行了重组。重组过程中色素组成的调节产生了叶绿素a/b结合比明显不同的结合产物，尽管结合的叶绿素（a加b）总数保持恒定为八个。对稳态吸收和荧光光谱的热力学分析表明，所有叶绿素在能量上都是耦合的，而叶绿素光氧化动力学表明，在体外色素结合过程中，三线态叶绿素 - 类胡萝卜素耦合也得以保留。针对Qy吸收区域，分析了叶绿素a/b结合比对在72K和300K下测量的吸收光谱的影响。叶绿素b结合的增加导致640 - 660nm区域的吸收大幅增加，而675 - 690nm区间的吸收明显减少。这些变化根据高斯分解描述进行分析，其中八个子带显示出与其他天线叶绿素光谱形式所证明的弱电子 - 声子耦合一致的温度依赖性展宽。通过这种方式，我们证明叶绿素b结合的增加导致与640、648、655和660nm处的子带相关的吸收强度增加，而678和684nm处的长波长子带的强度降低。所有子带的波长位置不变。上述数据被解释为表明CP29有八个叶绿素结合位点，其中许多或全部位点可根据色素结合发生的条件被叶绿素a或叶绿素b占据。叶绿素b吸收主要与位于640、648、655和660nm处四个子带相关。根据光谱带形成所涉及的机制，讨论了具有不同叶绿素a/b结合化学计量比的重组产物中吸收带波长位置的不变性。我们得出结论，色素 - 蛋白质相互作用在光谱异质性的决定中占主导地位，可能对与色素 - 色素相互作用相关的吸收只有微小影响。
