Crystal Structure of the Rb. sphaeroides Zn~(2+)-binding Reaction Centre Mutant HC(M266)

摘要

In bacterial RCs light-induced electron transfer proceeds from the primary donor, a bacteriochlorophyll dimer, to the first (Q_A) and secondary (Q_B) quinone. As both Q_A and Q_B in the Rb. sphaeroides reaction centre (RC) are ubiquinone-10 molecules their different properties in the electron transfer chain must be the result of interactions with the protein environment. This can be probed by EPR techniques performed on the semiquinone radical anions (for review see [Lubitz & Feher 1999]). However,application of EPR is hindered due to the presence of the high-spin Fe~(2+) that interacts magnetically with these radicals. Here, replacement of the Fe~(2+) by diamagnetic Zn~(2+) [Debus 1986] is possible but the resulting ZnRC_c are very hard to crystallize. It has been shown, however, that a mutant, HC(M266), in which one of the His ligands of the iron is replaced by Cys, incorporates Zn~(2+) from the growth medium [Williams 1991] yielding RCs with >90 percent Zn incorporation (ZnRC_m). In this communication we report on the crystallization of ZnRC_m from the mutant HC(M266). The structure was solved to 2.6 A resolution and compared to that of the wild type FeRC. The data are supplemented by FTIR difference (Q_A~(->)/Q_A) spectroscopy and EPR/ENDORstudies of Q_A~(->).