Ivancich A, Mattioli T A
Departement de Biologie Cellulaire et Moleculaire, CEA and CNRS URA 2096, CEA/Saclay, Gif-sur-Yvette, France.
Biochemistry. 1997 Mar 11;36(10):3027-36. doi: 10.1021/bi962516u.
The primary electron donor (P) of the photosynthetic reaction center (RC) from the purple bacterium Rhodobacter (Rb.) sphaeroides is constituted of two bacteriochlorophyll molecules in excitonic interaction. The C2 acetyl carbonyl group of one of the two bacteriochlorophyll molecules (PL), the one more closely associated with the L polypeptide subunit, is engaged in a hydrogen bond with histidine L168, while the other pi-conjugated carbonyl groups of P are free from such hydrogen-bonding interactions. The three-dimensional X-ray crystal structures of the RC from several strains of Rb. sphaeroides reveal that asparagine L166 probably interacts indirectly with P through His L168. Such an interaction is expected to modulate the hydrogen bond between P and His L168, a residue which is highly conserved in purple bacteria. RC mutants of Rb. sphaeroides where asparagine L166 was genetically replaced by leucine [NL(L166)], histidine [NH(L166)], and aspartate [ND(L166)] were studied using Fourier transform (FT) Raman spectroscopy. All of these mutations resulted in an increase in the strength of the hydrogen bond between His L168 and the acetyl carbonyl group of P(L), as observed in the FT Raman spectrum, by the 2-4 cm(-1) decrease in vibrational frequency of the 1620 cm(-1) band which has been assigned to this specific acetyl carbonyl group [Mattioli, T. A., Lin, X., Allen, J. P., & Williams, J. C. (1995) Biochemistry 34, 6142-6152]. At pH 8, the NH(L166) mutation showed the greatest change in the P0/P.+ redox midpoint potential (515 mV), increasing it by ca. 30 mV compared to that of wild type (485 mV). A similar increase in P0/P.+ redox midpoint potential for NH(L166) compared to that of wild type is also observed at pH 5, 6, and 9.5. The p0/P.+ midpoint potential of the NL(L166) mutant was comparable to that of wild type at all pH values. In contrast, for the ND(L166) mutant, the midpoint potential shows a markedly different pH dependency, being 25 mV higher than wild type at pH 5 but 20 mV lower than wild type at pH 9.5. The hydrogen bond interactions of the primary electron donor from Rhodospirillum (Rsp.) centenum were determined from the FT Raman vibrational spectrum which exhibits a 1616 cm(-1) band similar to what is seen in the NH(L166) and ND(L166) Rb. sphaeroides mutants. Comparison of the sequence of the L subunit determined for the Rsp. centenum RC with that of other species indicates that positions L166 and L168 are occupied by His residues. The stronger hydrogen bond between the conserved His L168 and the acetyl carbonyl group of P(L), observed in the primary donor of Rsp. centenum and of several bacterial species which are known to possess a histidine residue at the analogous L166 position, is proposed to be due to interactions between these two histidine residues.
来自紫色细菌球形红杆菌（Rb.）的光合反应中心（RC）的初级电子供体（P）由处于激子相互作用的两个细菌叶绿素分子组成。两个细菌叶绿素分子之一（PL）的C2乙酰羰基，即与L多肽亚基联系更紧密的那个，与组氨酸L168形成氢键，而P的其他π共轭羰基则不存在这种氢键相互作用。几种球形红杆菌菌株的RC的三维X射线晶体结构表明，天冬酰胺L166可能通过组氨酸L168与P间接相互作用。预计这种相互作用会调节P与组氨酸L168之间的氢键，组氨酸L168是紫色细菌中高度保守的一个残基。对球形红杆菌的RC突变体进行了研究，这些突变体中天冬酰胺L166被亮氨酸[NL(L166)]、组氨酸[NH(L166)]和天冬氨酸[ND(L166)]进行了基因替换，采用傅里叶变换（FT）拉曼光谱法。所有这些突变都导致组氨酸L168与P(L)的乙酰羰基之间的氢键强度增加，如FT拉曼光谱中所示，已指定为该特定乙酰羰基的1620 cm(-1)谱带的振动频率降低了2 - 4 cm(-1) [马蒂奥利，T. A.，林，X.，艾伦，J. P.，& 威廉姆斯，J. C.（1995年）《生物化学》34，6142 - 6152]。在pH 8时，NH(L166)突变在P0/P.+氧化还原中点电位（515 mV）上显示出最大变化，与野生型（485 mV）相比增加了约30 mV。在pH 5、6和9.5时，与野生型相比，NH(L166)的P0/P.+氧化还原中点电位也有类似增加。NL(L166)突变体的p0/P.+中点电位在所有pH值下都与野生型相当。相比之下，对于ND(L166)突变体，中点电位表现出明显不同的pH依赖性，在pH 5时比野生型高25 mV，但在pH 9.5时比野生型低20 mV。来自中心红螺菌（Rsp.）的初级电子供体的氢键相互作用是根据FT拉曼振动光谱确定的，该光谱显示出一个1616 cm(-1)谱带，类似于在NH(L166)和ND(L166)球形红杆菌突变体中看到的谱带。对中心红螺菌RC确定的L亚基序列与其他物种的序列比较表明，L166和L168位置被组氨酸残基占据。在中心红螺菌以及已知在类似L166位置具有组氨酸残基的几种细菌物种的初级供体中观察到的保守组氨酸L168与P(L)的乙酰羰基之间更强的氢键，被认为是由于这两个组氨酸残基之间的相互作用。
