Nabedryk E, Breton J, Joshi H M, Hanson D K
Section de Bioénergétique, Département de Biologie Cellulaire et Moléculaire, CEA/Saclay, 91191 Gif-sur-Yvette Cedex, France.
Biochemistry. 2000 Nov 28;39(47):14654-63. doi: 10.1021/bi0013795.
The photoreduction of the secondary quinone Q(B) in native reaction centers (RCs) of Rhodobacter capsulatus and in RCs from the GluL212 --> Gln and GluL212 --> Ala mutants has been investigated at pH 7 in (1)H(2)O and (2)H(2)O by light-induced Fourier transform infrared (FTIR) difference spectroscopy. The Q(B)(-)/Q(B) FTIR difference spectra reflect changes of quinone-protein interactions and of protonation state of carboxylic acid groups as well as reorganization of the protein upon electron transfer. Comparison of Q(B)(-)/Q(B) spectra of native and mutant RCs indicates that the interactions between Q(B) or Q(B)(-) and the protein are similar in all RCs. A differential signal at approximately 1650/1640 cm(-1), which is common to all the spectra, is associated with a movement of a peptide carbonyl or a side chain following Q(B) reduction. On the other hand, Q(B)(-)/Q(B) spectra of native and mutant RCs display several differences, notably between 1700 and 1650 cm(-1) (amide I and side chains), between 1570 and 1530 cm(-1) (amide II), and at 1728-1730 cm(-1) (protonated carboxylic acid groups). In particular, the latter region in native RCs is characterized by a main positive band at 1728 cm(-1) and a negative signal at 1739 cm(-1). In the L212 mutants, the amplitude of the positive band is strongly decreased leading to a differential signal at 1739/1730 cm(-1) that is insensitive to (1)H/(2)H isotopic exchange. In native RCs, only the 1728 cm(-1) band is affected in (2)H(2)O while the 1739 cm(-1) signal is not. The effects of the mutations and of (1)H/(2)H exchange on the Q(B)(-)/Q(B) spectra concur in the attribution of the 1728 cm(-1) band in native RCs to (partial) proton uptake by GluL212 upon the first electron transfer to Q(B), as previously observed in Rhodobacter sphaeroides RCs [Nabedryk, E., Breton, J., Hienerwadel, R., Fogel, C., Mäntele, W., Paddock, M. L., and Okamura, M. Y. (1995) Biochemistry 34, 14722-14732]. More generally, strong homologies of the Q(B) to Q(B)(-) transition in the RCs from Rb. sphaeroides and Rb. capsulatus are detected by differential FTIR spectroscopy. The FTIR data are discussed in relation with the results from global proton uptake measurements and electrogenic events concomitant with the reduction of Q(B) and with a model of the Q(B) turnover in Rb. sphaeroides RCs [Mulkidjanian, A. Y. (1999) FEBS Lett. 463, 199-204].
利用光诱导傅里叶变换红外（FTIR）差示光谱，在pH 7的条件下，于（1）H₂O和（2）H₂O中研究了荚膜红细菌天然反应中心（RCs）以及GluL212→Gln和GluL212→Ala突变体的RCs中次级醌Q(B)的光还原。Q(B)(-)/Q(B) FTIR差示光谱反映了醌-蛋白质相互作用、羧酸基团质子化状态的变化以及电子转移时蛋白质的重排。天然和突变RCs的Q(B)(-)/Q(B)光谱比较表明，所有RCs中Q(B)或Q(B)(-)与蛋白质之间的相互作用相似。所有光谱共有的约1650/1640 cm⁻¹处的微分信号与Q(B)还原后肽羰基或侧链的移动有关。另一方面，天然和突变RCs的Q(B)(-)/Q(B)光谱显示出一些差异，特别是在1700至1650 cm⁻¹（酰胺I和侧链）、1570至1530 cm⁻¹（酰胺II）以及1728 - 1730 cm⁻¹（质子化羧酸基团）之间。特别是，天然RCs中的后一个区域的特征是在1728 cm⁻¹处有一个主要的正带，在1739 cm⁻¹处有一个负信号。在L212突变体中，正带的幅度大幅降低，导致在1739/1730 cm⁻¹处出现一个对（1）H/（2）H同位素交换不敏感的微分信号。在天然RCs中，只有1728 cm⁻¹的带在（2）H₂O中受到影响，而1739 cm⁻¹的信号不受影响。突变和（1）H/（2）H交换对Q(B)(-)/Q(B)光谱的影响共同表明，天然RCs中1728 cm⁻¹的带归因于GluL212在首次向Q(B)进行电子转移时（部分）摄取质子，这与之前在球形红细菌RCs中观察到的情况一致 [纳贝德里克，E.，布雷顿，J.，希纳瓦德尔，R.，福格尔，C.，曼特尔，W.，帕多克，M. L.，和冈村，M. Y.（1995年）《生物化学》34，14722 - 14732]。更普遍地，通过差示FTIR光谱检测到球形红细菌和荚膜红细菌的RCs中Q(B)到Q(B)(-)转变的强同源性。结合全局质子摄取测量结果、与Q(B)还原伴随的电致事件以及球形红细菌RCs中Q(B)周转模型 [穆尔基贾尼安，A. Y.（1999年）《欧洲生物化学学会联合会快报》463，199 - 204]，对FTIR数据进行了讨论。
