Selective 15N-Isotope Labelling of Histidines in PSII Reaction Centers of Spirodela Oligorrhiza

Abstract

The Dl-D2 polypeptide heterodimer in Photosystem II (PSII) binds an unusual variety of cofactors. These include chlorophylls (Chl), pheophytins, QA, non-heme iron, QB and probably also the inorganic cofactors associated with oxygen evolution: manganese, calcium and chloride (1). However, detailed information about the interaction between these co-factors and highly conserved amino acids of the D1 and D2 polypeptides is still missing. Histidine (His) is one of the important and highly conserved amino acids in the D1 and D2 polypeptides which is believed to take part in interaction with many of these redox components e.g. as axial ligands to the magnesium atoms of the putative primary donor Chls (His 198 for both D1 and D2 proteins) and to the ferrous-iron in the quinone (QA and QB)-iron acceptor complex (His-215 and His-272 on the D1 protein and His-215 and His-269 on D2 protein) (1,2). These interactions could be crucial for primary photochemistry in PSII. His is often found in active sites of enzymes where hydrogen bonding and switching between protonation states of the imidazole side chain can control reactivity of active sites (3,4). Because of the tautomeric nature of the imidazole ring of His, it is not clear which nitrogen of the imidazole ring is actually co-ordinating with redox components. Specific 15N labelling at either of the two nitrogen sites (τ:tele or π:pros) of the imidazole ring of His followed by incorporation of this specifically labelled His in the PSII reaction center can provide a tool for identification of interactions between the His nitrogen and redox components by isotope-sensitive spectroscopic techniques such as solid-state NMR. 15N CP/MAS NMR (cross-polarization/Magic angle spinning, nuclear magnetic resonance) spectroscopy of selectively enriched samples is the method of choice to obtain NMR access with atomic selectivity for large membrane protein (5). In the present study we developed methods to incorporate specific 15N-labelled His into PSII reaction centers in Spirodela oligorrhiza.