Antonkine M L, Bentrop D, Bertini I, Luchinat C, Shen G, Bryant D A, Stehlik D, Golbeck J H
Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park 16802, USA.
J Biol Inorg Chem. 2000 Jun;5(3):381-92. doi: 10.1007/pl00010667.
The PsaC subunit of Photosystem I (PS I) is a 9.3-kDa protein that binds two important cofactors in photosynthetic electron transfer: the [4Fe-4S] clusters FA and FB. The g-tensor orientation of FA- and FB- is believed to be correlated to the preferential localization of the mixed-valence and equal-valence (ferrous) iron pairs in each [4Fe-4S]+ cluster. The preferential position of the mixed-valence and equal-valence pairs, in turn. can be inferred from the study of the temperature dependence of contact-shifted resonances by 1H NMR spectroscopy. For this, a sequence-specific assignment of these signals is required. The 1H NMR spectrum of reduced, unbound PsaC from Synechococcus sp. PCC 7002 at 280.4 K in 99% D2O solution shows 18 hyperfine-shifted resonances. The non-solvent-exchangeable, hyperfine-shifted resonances of reduced PsaC are clearly identified as belonging to the cysteines coordinating the clusters FA- and FB- by their downfield chemical shifts, by their temperature dependencies, and by their short T1 relaxation times. The usual fast method of assigning the 1H NMR spectra of reduced [4Fe-4S] proteins through magnetization transfer from the oxidized to the reduced state was not feasible in the case of reduced PsaC. Therefore, a de novo self-consistent sequence-specific assignment of the hyperfine-shifted resonances was obtained based on dipolar connectivities from 1D NOE difference spectra and on longitudinal relaxation times using the X-ray structure of Clostridium acidi urici 2[4Fe-4S] cluster ferredoxin at 0.94 A resolution as a model. The results clearly show the same sequence-specific distribution of Curie and anti-Curie cysteines for unbound, reduced PsaC as established for other [4Fe-4S]-containing proteins; therefore, the mixed-valence and equal-valence (ferrous) Fe-Fe pairs in FA- and FB- have the same preferential positions relative to the protein. The analysis reveals that the magnetic properties of the two [4Fe-4S] clusters are essentially indistinguishable in unbound PsaC, in contrast to the PsaC that is bound as a component of the PS I complex.
光系统I（PS I）的PsaC亚基是一种9.3 kDa的蛋白质，在光合电子传递中结合两个重要的辅因子：[4Fe-4S]簇FA和FB。据信，FA和FB的g张量取向与每个[4Fe-4S]+簇中混合价和等价位（亚铁）铁对的优先定位相关。反过来，混合价和等价位对的优先位置可以通过1H NMR光谱对接触位移共振的温度依赖性研究来推断。为此，需要对这些信号进行序列特异性归属。来自聚球藻属PCC 7002的还原态、未结合的PsaC在99% D2O溶液中于280.4 K时的1H NMR谱显示出18个超精细位移共振。还原态PsaC的非溶剂可交换超精细位移共振通过其低场化学位移、温度依赖性以及短T1弛豫时间，被明确鉴定为属于与簇FA和FB配位的半胱氨酸。通过从氧化态到还原态的磁化转移来归属还原态[4Fe-4S]蛋白的1H NMR谱的常用快速方法在还原态PsaC的情况下不可行。因此，基于1D NOE差谱的偶极连接性以及使用分辨率为0.94 Å的尿酸梭菌2[4Fe-4S]簇铁氧还蛋白的X射线结构作为模型的纵向弛豫时间，获得了超精细位移共振的从头自洽序列特异性归属。结果清楚地表明，未结合的还原态PsaC中居里和反居里半胱氨酸的序列特异性分布与其他含[4Fe-4S]的蛋白质相同；因此，FA和FB中的混合价和等价位（亚铁）Fe-Fe对相对于蛋白质具有相同的优先位置。分析表明，与作为PS I复合物组分结合的PsaC相比，未结合的PsaC中两个[4Fe-4S]簇的磁性基本无法区分。
