Excitation trap approach to analyze size and pigment-pigment coupling: Reconstitution of LH1 antenna of Rhodobacter sphaeroides with Ni-substituted bacteriochlorophyll

Replacement of the central Mg in chlorophylls by Ni opens an ultrafast (ten s of femtoseconds time range) radiationless de-excitation path, while the p rincipal ground-state absorption and coordination properties of the pigment are retained. A method has been developed for substituting the native bact eriochlorophyll a by Ni-bacteriochlorophyll a ([Ni]-BChl) in the light harv esting antenna of the core complex (LH1) from the purple bacterium, Rhodoba cter (Rb.) sphaeroides, to investigate its unit size and excited state prop erties. The components of the complex have been extracted with an organic s olvent from freeze-dried membranes of an LH1-only strain of Rb. sphaeroides and transferred into the micelles of n-octyl-beta -glucopyranoside (OG). R econstitution was achieved by solubilization in 3.4% OG, followed by diluti on, yielding a complex nearly identical to the native one, in terms of abso rption, fluorescence, and circular dichroism spectra as well as energy tran sfer efficiency from carotenoid to bacteriochlorophyll. By adding increasin g amounts of [Ni]-BChl to the reconstitution mixture, a series of LH1 compl exes was obtained that contain increasing levels of this efficient excitati on trap. In contrast to the nearly unchanged absorption, the presence of [N i]-BChl in LH1 markedly affects the emission properties. Incorporation of o nly 3.2 and 20% [Ni]-BChl reduces the emission by 50% and nearly 100%, resp ectively. The subnanosecond fluorescence kinetics of the complexes were mon oexponential, with the lifetime identical to that of the native complex, an d its amplitude decreasing in parallel with the steady-state fluorescence y ield. Quantitative analysis of the data, based on a Poisson distribution of the modified pigment in the reconstituted complex, suggests that the prese nce of a single excitation trap per LH1 unit suffices for efficient emissio n quenching and that this unit contains 20 +/- 1 BChl molecules.