Pheophytin−Protein Interactions in Photosystem II Studied by Resonance Raman Spectroscopy of Modified Reaction Centers†

Soret-excited resonance Raman spectra of two types of pheophytin-exchanged photosystem II RCs are reported. The cofactor composition of the reaction centers was modified by exchanging pheophytin a for 131-deoxo-131-hydroxypheophytin a, yielding one preparation with selective replacement of the photochemically inactive pheophytin (HB) and a second one exhibiting total replacement of HB and 40% replacement of HA, the primary electron acceptor. Resonance Raman spectra indicate that the other bound cofactors present are not significantly perturbed by Pheo substitution. The resonance Raman contributions from HA and HB in the carbonyl stretching region are identified at 1679 and 1675 cm-1, respectively, indicating that both pheophytin molecules in the photosystem II reaction center have hydrogen-bonded keto-carbonyl groups. This conclusion differs from what is observed in the functionally related RCs of purple non-sulfur bacteria, where the keto-carbonyl group of HB is not hydrogen bonded, but confirms predicti...