Yakovlev Andrei G, Shkuropatova Tatiana A, Vasilieva Luidmila G, Shkuropatov Anatoli Ya, Gast Peter, Shuvalov Vladimir A
Department of Photobiophysics, Belozersky Institute of Chemical and Physical Biology, Moscow State University, Moscow 119899, Russian Federation.
Biochim Biophys Acta. 2006 May-Jun;1757(5-6):369-79. doi: 10.1016/j.bbabio.2006.05.025. Epub 2006 May 23.
Femtosecond absorption difference spectroscopy was applied to study the time and spectral evolution of low-temperature (90 K) absorbance changes in isolated reaction centers (RCs) of the HM182L mutant of Rhodobacter (Rb.) sphaeroides. In this mutant, the composition of the B-branch RC cofactors is modified with respect to that of wild-type RCs by replacing the photochemically inactive BB accessory bacteriochlorophyll (BChl) by a photoreducible bacteriopheophytin molecule (referred to as PhiB). We have examined vibrational coherence within the first 400 fs after excitation of the primary electron donor P with 20-fs pulses at 870 nm by studying the kinetics of absorbance changes at 785 nm (PhiB absorption band), 940 nm (P*-stimulated emission), and 1020 nm (BA- absorption band). The results of the femtosecond measurements are compared with those recently reported for native Rb. sphaeroides R-26 RCs containing an intact BB BChl. At delay times longer than approximately 50 fs (maximum at 120 fs), the mutant RCs exhibit a pronounced BChl radical anion (BA-) absorption band at 1020 nm, which is similar to that observed for Rb. sphaeroides R-26 RCs and represents the formation of the intermediate charge-separated state P+ BA-. Femtosecond oscillations are revealed in the kinetics of the absorption development at 1020 nm and of decay of the P*-stimulated emission at 940 nm, with the oscillatory components of both kinetics displaying a generally synchronous behavior. These data are interpreted in terms of coupling of wave packet-like nuclear motions on the potential energy surface of the P* excited state to the primary electron-transfer reaction P*-->P+ BA- in the A-branch of the RC cofactors. At very early delay times (up to 80 fs), the mutant RCs exhibit a weak absorption decrease around 785 nm that is not observed for Rb. sphaeroides R-26 RCs and can be assigned to a transient bleaching of the Qy ground-state absorption band of the PhiB molecule. In the range of 740-795 nm, encompassing the Qy optical transitions of bacteriopheophytins HA, HB, and PhiB, the absorption difference spectra collected for mutant RCs at 30-50 fs resemble the difference spectrum of the P+ PhiB- charge-separated state previously detected for this mutant in the picosecond time domain (E. Katilius, Z. Katiliene, S. Lin, A.K.W. Taguchi, N.W. Woodbury, J. Phys. Chem., B 106 (2002) 1471-1475). The dynamics of bleaching at 785 nm has a non-monotonous character, showing a single peak with a maximum at 40 fs. Based on these observations, the 785-nm bleaching is speculated to reflect reduction of 1% of PhiB in the B-branch within about 40 fs, which is earlier by approximately 80 fs than the reduction process in the A-branch, both being possibly linked to nuclear wave packet motion in the P* state.
飞秒吸收差分光谱法被用于研究球形红杆菌(Rb.)HM182L突变体的分离反应中心(RCs)在低温(90K)下吸光度变化的时间和光谱演化。在这个突变体中,B分支RC辅因子的组成相对于野生型RCs有所改变,用一个可光还原的细菌脱镁叶绿素分子(称为PhiB)取代了光化学惰性的BB辅助细菌叶绿素(BChl)。我们通过研究785nm(PhiB吸收带)、940nm(P受激发射)和1020nm(BA-吸收带)处吸光度变化的动力学,考察了在870nm处用20飞秒脉冲激发初级电子供体P后最初400飞秒内的振动相干性。飞秒测量结果与最近报道的含有完整BB BChl的天然球形红杆菌R-26 RCs的结果进行了比较。在延迟时间长于约50飞秒(在120飞秒时达到最大值)时,突变体RCs在1020nm处呈现出明显的BChl自由基阴离子(BA-)吸收带,这与在球形红杆菌R-26 RCs中观察到的相似,代表了中间电荷分离态P+BA-的形成。在1020nm处吸收发展的动力学以及940nm处P受激发射的衰减中揭示了飞秒振荡,两种动力学的振荡成分总体上呈现同步行为。这些数据根据P激发态势能面上类似波包的核运动与RC辅因子A分支中初级电子转移反应P→P+BA-的耦合来解释。在非常早的延迟时间(长达80飞秒),突变体RCs在785nm附近呈现出微弱的吸光度下降,这在球形红杆菌R-26 RCs中未观察到,可归因于PhiB分子Qy基态吸收带的瞬态漂白。在740 - 795nm范围内,涵盖细菌脱镁叶绿素HA、HB和PhiB的Qy光学跃迁,在30 - 50飞秒时为突变体RCs收集的吸收差分光谱类似于先前在皮秒时间域中为该突变体检测到的P+PhiB-电荷分离态的差分光谱(E. Katilius,Z. Katiliene,S. Lin,A.K.W. Taguchi,N.W. Woodbury,J. Phys. Chem., B 106 (2002) 1471 - 1475)。785nm处漂白的动力学具有非单调特性,在40飞秒时出现一个最大值的单峰。基于这些观察结果,推测785nm处的漂白反映了在约40飞秒内B分支中1%的PhiB被还原,这比A分支中的还原过程早约80飞秒,两者可能都与P*态中的核波包运动有关。