J Phys Chem B. 2018 May 3;122(17):4611-4624. doi: 10.1021/acs.jpcb.8b00032. Epub 2018 Apr 18.
We provide an analysis of the pigment composition of reconstituted wild type CP29 complexes. The obtained stoichiometry of 9 ± 0.6 Chls a and 3 ± 0.6 Chls b per complex, with some possible heterogeneity in the carotenoid binding, is in agreement with 9 Chls a and 3.5 Chls b revealed by the modeling of low-temperature optical spectra. We find that ∼50% of Chl b614 is lost during the reconstitution/purification procedure, whereas Chls a are almost fully retained. The excitonic structure and the nature of the low-energy (low-E) state(s) are addressed via simulations (using Redfield theory) of 5 K absorption and fluorescence/nonresonant hole-burned (NRHB) spectra obtained at different excitation/burning conditions. We show that, depending on laser excitation frequency, reconstituted complexes display two (independent) low-E states (i.e., the A and B traps) with different NRHB and emission spectra. The red-shifted state A near 682.4 nm is assigned to a minor (∼10%) subpopulation (sub. II) that most likely originates from an imperfect local folding occurring during protein reconstitution. Its lowest energy state A (localized on Chl a604) is easily burned with λ = 488.0 nm and has a red-shifted fluorescence origin band near 683.7 nm that is not observed in native (isolated) complexes. Prolonged burning by 488.0 nm light reveals a second low-E trap at 680.2 nm (state B) with a fluorescence origin band at ∼681 nm, which is also observed when using a direct low-fluence excitation near 650 nm. The latter state is mostly delocalized over the a611, a612, a615 Chl trimer and corresponds to the lowest energy state of the major (∼90%) subpopulation (sub. I) that exhibits a lower hole-burning quantum yield. Thus, we suggest that major sub. I correspond to the native folding of CP29, whereas the red shift of the Chl a604 site energy observed in the minor sub. II occurs only in reconstituted complexes.
我们分析了重组野生型 CP29 复合物的色素组成。每个复合物含有 9 ± 0.6 个叶绿素 a 和 3 ± 0.6 个叶绿素 b，可能存在类胡萝卜素结合的不均一性，这与低温光谱模型显示的 9 个叶绿素 a 和 3.5 个叶绿素 b 一致。我们发现，在重组/纯化过程中，约 50%的叶绿素 b614 丢失，而叶绿素 a 几乎全部保留。通过在不同激发/燃烧条件下获得的 5 K 吸收和荧光/非共振孔烧蚀（NRHB）光谱的模拟（使用 Redfield 理论），我们研究了激子结构和低能（低 E）态的性质。我们表明，根据激光激发频率，重组复合物显示两个（独立的）低 E 态（即 A 和 B 陷阱），具有不同的 NRHB 和发射光谱。近 682.4nm 的红移态 A 被分配给一个小（约 10%）亚群（亚群 II），该亚群很可能源自蛋白重组过程中发生的局部折叠不完全。其最低能量态 A（位于叶绿素 a604 上）容易被 λ=488.0nm 烧蚀，且荧光起源带在近 683.7nm 处红移，在天然（分离）复合物中未观察到。用 488.0nm 光长时间烧蚀会在 680.2nm 处产生第二个低 E 陷阱（B 态），其荧光起源带约为 681nm，当用近 650nm 的直接低通量激发时也能观察到。后一态主要定域在 a611、a612、a615 叶绿素三聚体上，对应于主要（约 90%）亚群（亚群 I）的最低能量态，其孔烧蚀量子产率较低。因此，我们认为主要的亚群 I 对应于 CP29 的天然折叠，而在较小的亚群 II 中观察到叶绿素 a604 位置能量的红移仅发生在重组复合物中。
