Jung Y S, Vassiliev I R, Qiao F, Yang F, Bryant D A, Golbeck J H
Department of Biochemistry, and Center for Biological Chemistry, George W. Beadle Center, University of Nebraska, Lincoln, Nebraska 68588-0664, USA.
J Biol Chem. 1996 Dec 6;271(49):31135-44. doi: 10.1074/jbc.271.49.31135.
The FB and FA electron acceptors in Photosystem I (PS I) are [4Fe-4S] clusters ligated by cysteines provided by PsaC. In a previous study (Mehari, T., Qiao, F., Scott, M. P., Nellis, D., Zhao, J., Bryant, D., and Golbeck, J. H. (1995) J. Biol. Chem. 270, 28108-28117), we showed that when cysteines 14 and 51 were replaced with serine or alanine, the free proteins contained a S = 1/2, [4Fe-4S] cluster at the unmodified site and a mixed population of S = 1/2, [3Fe-4S] and S = 3/2, [4Fe-4S] clusters at the modified site. We show here that these mutant PsaC proteins can be rebound to P700-FX cores, resulting in fully functional PS I complexes. The low temperature EPR spectra of the C14XPsaC.PS I complexes (where X = S, A, or G) show the photoreduction of a wild-type FA cluster and a modified FB' cluster, the latter with g values of 2.115, 1.899, and 1.852 and linewidths of 110, 70, and 85 MHz. Since neither alanine nor glycine contains a suitable side group, an external thiolate provided by beta-mercaptoethanol has likely been recruited to supply the requisite ligand to the [4Fe-4S] cluster. The EPR spectrum of the C51SPsaC.PS I complex differs from that of the C51APsaC.PS I or C51GPsaC.PS I complexes by the presence of an additional set of resonances, which may be derived from the serine oxygen-ligated cluster. In all other mutant PS I complexes, a wild-type spin-coupled interaction spectrum appears when FA and FB are simultaneously reduced. Single turnover flash studies indicate approximately 50% efficient electron transfer to FA/FB in the C14SPsaC.PS I, C51SPsaC.PS I, C14GPsaC.PS I, and C51GPsaC.PS I mutants and less than 40% in the C14APsaC.PS I and C51APsaC.PS I mutants, compared with approximately 76% in the PS I core reconstructed with wild-type PsaC. These data are consistent with the measurements of the rates of cytochrome c6-NADP+ reductase activity, indicating lower rates in the alanine mutants. It is proposed that the chemical rescue of a [4Fe-4S] cluster with a recruited external thiolate at the modified site allows the mutant PsaC proteins to rebind to PS I and to function in forward electron transfer.
光系统I（PS I）中的FB和FA电子受体是由PsaC提供的半胱氨酸连接的[4Fe-4S]簇。在之前的一项研究中（梅哈里，T.，乔，F.，斯科特，M.P.，内利斯，D.，赵，J.，布莱恩特，D.，和戈尔贝克，J.H.（1995年）《生物化学杂志》270，28108 - 28117），我们表明，当半胱氨酸14和51被丝氨酸或丙氨酸取代时，游离蛋白质在未修饰位点含有一个S = 1/2的[4Fe-4S]簇，在修饰位点含有S = 1/2的[3Fe-4S]簇和S = 3/2的[4Fe-4S]簇的混合群体。我们在此表明，这些突变型PsaC蛋白可以重新结合到P700 - FX核心上，从而形成功能完全正常的PS I复合物。C14XPsaC.PS I复合物（其中X = S、A或G）的低温EPR光谱显示野生型FA簇和修饰后的FB'簇发生了光还原，后者的g值为2.115、1.899和1.852，线宽为110、70和85 MHz。由于丙氨酸和甘氨酸都不含有合适的侧链基团，可能已募集了由β-巯基乙醇提供的外部硫醇盐来为[4Fe-4S]簇提供必需的配体。C51SPsaC.PS I复合物的EPR光谱与C51APsaC.PS I或C51GPsaC.PS I复合物的EPR光谱不同，前者存在一组额外的共振峰，这可能源自丝氨酸氧连接的簇。在所有其他突变型PS I复合物中，当FA和FB同时被还原时会出现野生型自旋耦合相互作用光谱。单周转闪光研究表明，在C14SPsaC.PS I、C51SPsaC.PS I、C14GPsaC.PS I和C51GPsaC.PS I突变体中，电子转移到FA/FB的效率约为50%，在C14APsaC.PS I和C51APsaC.PS I突变体中低于40%，而用野生型PsaC重建的PS I核心中的效率约为76%。这些数据与细胞色素c6 - NADP +还原酶活性速率的测量结果一致，表明丙氨酸突变体中的速率较低。有人提出，在修饰位点用募集的外部硫醇盐对[4Fe-4S]簇进行化学挽救，使得突变型PsaC蛋白能够重新结合到PS I上并在正向电子转移中发挥作用。
