Journal of Biomolecular NMR 28: 157–164, 2004. KLUWER/ESCOM © 2004 Kluwer Academic Publishers. Printed in the Netherlands. 157 Heteronuclear 2D ( 1 H- 13 C) MAS NMR resolves the electronic structure of coordinated histidines in light-harvesting complex II: Assessment of charge transfer and electronic delocalization effect Alia a , Jörg Matysik a , Ido de Boer a , Peter Gast b , Hans J. van Gorkom b & Huub J.M. de Groot a,∗ a Leiden Institute of Chemistry, Gorlaeus Laboratoria, P.O. Box 9502, 2300 RA Leiden, The Netherlands; b Department of Biophysics, Huygens Laboratorium, P.O. Box 9504, 2300 RA Leiden, The Netherlands Received 20 June 2003; Accepted 3 September 2003 Key words: electronic structure, histidine, light-harvesting complex II, MAS NMR, membrane protein, ring currents Abstract In a recent MAS NMR study, two types of histidine residues in the light-harvesting complex II (LH2) of Rhodopseudomonas acidophila were resolved: Type 1 (neutral) and Type 2 (positively charged) (Alia et al. J. Am. Chem. Soc.). The isotropic 13 C shifts of histidines coordinating to B850 BChl a are similar to fully positively charged histidine, while the 15 N shift anisotropy shows a predominantly neutral character. In addition the possibility that the ring currents are quenched by overlap in the superstructure of the complete ring of 18 B850 molecules in the LH2 complex could not be excluded. In the present work, by using two-dimensional heteronuclear ( 1 H- 13 C) dipolar correlation spectroscopy with phase-modulated Lee–Goldburg homonuclear 1 H decoupling applied during the t 1 period, a clear and unambiguous assignment of the protons of histidine interacting with the magnesium of a BChl a molecule is obtained and a significant ring current effect from B850 on the coordinating histidine is resolved. Using the ring current shift on 1 H, we refine the 13 C chemical shift assignment of the coordinating histidine and clearly distinguish the electronic structure of coordinating histidines from that of fully positively charged histidine. The DFT calculations corroborate that the coordinating histidines carry ∼0.2 electronic equivalent of positive charge in LH2. In addition, the data indicate that the ground state electronic structures of individual BChl a /His complexes is largely independent of supermolecular π interactions in the assembly of 18 B850 ring in LH2. Introduction Histidine (His) residues play an important role in bio- catalytic molecular processes of many proteins. Their imidazole side chains can occur in different proton- ation and charge states and form hydrogen bonds to the surroundings. Interaction of histidine with Mg 2+ has been suggested in all BChl-protein complexes with known structures (Prince et al., 1997; Matthews et al., 1997; Deisenhofer et al., 1985). Although histidine residues are the main ligands to B(Chl) in all known photosynthetic reaction centres, and many antenna complexes, detailed knowledge ∗ To whom correspondence should be addressed. E-mail: ssnmr@chem.leidenuniv.nl about the protonation and electronic state of these residues is still lacking. High resolution solid-state NMR in combination with isotope labelling provides clear access to the local spatial and electronic struc- ture around amino acid side chains in large membrane protein complexes (Castellani et al., 2002; Egorova- Zachernyuk et al., 2001). Using MAS/NMR studies in conjunction with site-directed isotope labelling, we provided recently a conclusive 15 N chemical shift assignment of histidine nitrogens coordinating with Mg 2+ in the light-harvesting complex II (LH2) of Rhodopseudomonas (Rps.) acidophila (Alia et al., 2001). LH2 is a peripheral antenna complex that serves to absorb light and to transfer the excited state energy to the LH1-reaction centre complex. The high