Benlloch Reyes, Shevela Dmitriy, Hainzl Tobias, Grundström Christin, Shutova Tatyana, Messinger Johannes, Samuelsson Göran, Sauer-Eriksson A Elisabeth
Department of Forest Genetics and Plant Physiology (R.B) and Department of Plant Physiology (T.S., G.S.), Umeå Plant Science Centre, and Department of Chemistry, Chemistry Biology Centre (D.S., T.H., C.G., J.M., A.E.S.-E.), Umeå University, SE-90187 Umea, Sweden.
Department of Forest Genetics and Plant Physiology (R.B) and Department of Plant Physiology (T.S., G.S.), Umeå Plant Science Centre, and Department of Chemistry, Chemistry Biology Centre (D.S., T.H., C.G., J.M., A.E.S.-E.), Umeå University, SE-90187 Umea, Sweden
Plant Physiol. 2015 Mar;167(3):950-62. doi: 10.1104/pp.114.253591. Epub 2015 Jan 23.
In oxygenic photosynthesis, light energy is stored in the form of chemical energy by converting CO2 and water into carbohydrates. The light-driven oxidation of water that provides the electrons and protons for the subsequent CO2 fixation takes place in photosystem II (PSII). Recent studies show that in higher plants, HCO3 (-) increases PSII activity by acting as a mobile acceptor of the protons produced by PSII. In the green alga Chlamydomonas reinhardtii, a luminal carbonic anhydrase, CrCAH3, was suggested to improve proton removal from PSII, possibly by rapid reformation of HCO3 (-) from CO2. In this study, we investigated the interplay between PSII and CrCAH3 by membrane inlet mass spectrometry and x-ray crystallography. Membrane inlet mass spectrometry measurements showed that CrCAH3 was most active at the slightly acidic pH values prevalent in the thylakoid lumen under illumination. Two crystal structures of CrCAH3 in complex with either acetazolamide or phosphate ions were determined at 2.6- and 2.7-Å resolution, respectively. CrCAH3 is a dimer at pH 4.1 that is stabilized by swapping of the N-terminal arms, a feature not previously observed in α-type carbonic anhydrases. The structure contains a disulfide bond, and redox titration of CrCAH3 function with dithiothreitol suggested a possible redox regulation of the enzyme. The stimulating effect of CrCAH3 and CO2/HCO3 (-) on PSII activity was demonstrated by comparing the flash-induced oxygen evolution pattern of wild-type and CrCAH3-less PSII preparations. We showed that CrCAH3 has unique structural features that allow this enzyme to maximize PSII activity at low pH and CO2 concentration.
在有氧光合作用中，光能通过将二氧化碳和水转化为碳水化合物而以化学能的形式储存起来。为后续二氧化碳固定提供电子和质子的光驱动水氧化发生在光系统II（PSII）中。最近的研究表明，在高等植物中，HCO3 (-) 通过作为PSII产生的质子的移动受体来增加PSII活性。在绿藻莱茵衣藻中，一种腔碳酸酐酶CrCAH3被认为可以改善从PSII中去除质子的过程，可能是通过将二氧化碳快速重新形成HCO3 (-) 来实现的。在本研究中，我们通过膜进样质谱法和X射线晶体学研究了PSII与CrCAH3之间的相互作用。膜进样质谱测量表明，CrCAH3在光照下类囊体腔中普遍存在的微酸性pH值下活性最高。分别以2.6 Å和2.7 Å的分辨率测定了与乙酰唑胺或磷酸根离子复合的CrCAH3的两种晶体结构。CrCAH3在pH 4.1时是二聚体，通过N末端臂的交换而稳定，这是α型碳酸酐酶中以前未观察到的特征。该结构包含一个二硫键，用二硫苏糖醇对CrCAH3功能进行的氧化还原滴定表明该酶可能存在氧化还原调节。通过比较野生型和缺乏CrCAH3的PSII制剂的闪光诱导氧气释放模式，证明了CrCAH3和CO2/HCO3 (-) 对PSII活性的刺激作用。我们表明，CrCAH3具有独特的结构特征，使该酶能够在低pH和CO2浓度下最大限度地提高PSII活性。
