Exchanging cofactors in the core antennae from purple bacteria: Structure and properties of Zn-bacteriopheophytin-containing LH1

The core light-harvesting LH1 complex of Rhodospirillum rubrum consists of an assembly of membrane-spanning alpha and beta polypeptides, each of which binds one bacteriochlorophyll (BChl) a molecule. In this work, we describe a technique that allows the replacement of the natural, Mg BChl a cofactor s present in this protein by Zn-bacteriopheophytin (Zn-Bpheo). This techniq ue makes use of the well-characterized, reversible dissociation of LH1 indu ced by the detergent beta-octylglucoside. Incubation of partially dissociat ed LH1 with exogeneous pigments induces an equilibrium between the protein- bound BChl and the exogeneous pigment. This results in the binding of chemi cally modified pigments to LH1, in amounts which depend on the pigment comp osition and concentration of the exchange buffer. This method carl yield in formation on the relative affinities of the LH1 protein-binding sites for t he different pigments EChl and Zn-Bpheo and can also be used to obtain full y reassociated LH1 proteins, with a variable content of modified pigment, w hich may be precisely monitored. Absorption and FT-Raman spectroscopy indic ate that this exchange procedure leads to LH1 proteins containing modified pigments, bur retaining a binding site structure identical to that of nativ e LH1. Furthermore, examination of the binding curves suggests that there a re two distinguishable binding sites, probably corresponding to the two pol ypeptides, with very different properties. One of these two binding sites s hows a marked preference for Zn-Bpheo over BChl, while the other binding si te appears to prefer BChl.