Probing the binding site of 800-nm bacteriochlorophyll in the membrane-linked LH2 protein of Rhodobacter capsulatus by local unfolding and chemical modification - Evidence for the involvement of a beta His20 residue

The aim of this study was to investigate the function of beta His20 in the spectral behavior of the 800-nm bacteriochlorophyll (Bchl) of the Rhodobact er capsulatus LH2 protein. In this context, the 800-nm Bchl of the membrane -linked LH2 was used as an intrinsic probe to follow the reversible, denatu rant-elicited unfolding of the neighboring protein region. This band was re versibly shifted to approximate to 770 nm by acidic pH, suggesting that the environment of the pigment, responsible for its native red shift, was sign ificantly disturbed by the protonation of a chemical group. The reversible acid-induced blue shift was only observed in the presence of unfolding agen ts (urea and guanidinium chloride). Thus, dismantling of the protein struct ure facilitated exposure of the basic group to the medium. The acid-base ti trations of the spectral shift indicated an apparent pK approximate to 6.1, a value consistent with His imidazole being the protonatable group respons ible for the acid-induced band shift. The pK values of free N-terminal amin o groups are higher and not expected to be lowered by their local environme nt in the unfolded state of the protein. A similar blue shift of the 800-nm Bchl band was caused by the modifier die thyl pyrocarbonate, which is known to carboxylate the imidazole group of Hi s and free amino groups. It is also shown that the Fourier transform Raman spectrum of diethyl pyrocarbonate-treated LH2 preparations lacks the weak m ode at 1695 cm(-1), suggesting that it should be assigned to the B800 Bchl.