Small-angle neutron scattering studies of chlorophyll micelles: Models for bacterial antenna chlorophyll

Abstract

Micelles of hydrated chlorophyll a (P740), bacteriochlorophyll a (P865), bacteriochlorophyll c (P750), and pheophytin a prepared in organic media have been studied by small-angle neutron scattering to determine their shape, size, and mass per unit length. All of the micelles are hollow cylinders of well-defined size. The P740 and P750 cylinders are essentially monolayers of macrocycles crosslinked by water, probably in an arrangement similar to that of crystals of chlorophyll derivatives. The P865 micelle is more nearly a bilayer of macrocycles. We show that the curvature necessary to form cylinders probably results from intrinsic curvature of the five-coordinated chlorophyll macrocycle. Studies of P740 micelle formation and the disaggregating effects of another nucleophile (pyridine) are described. As the P750 micelles are nearly identical in size and optical spectra to the rod-shaped structures observed in chlorosomes, and the P865 micelles have optical properties very similar to the in vivo properties of the long-wavelength antenna of purple photosynthetic bacteria, we propose that features of the hydrated cylindrical micelles of these chlorophylls provide good models for antenna chlorophyll in photosynthetic bacteria.Many artificial chlorophyll systems (colloidal dispersions, films, monolayers, adsorbates) are of interest because their visible absorption maxima are red-shifted relative to the corresponding monomeric chlorophyll, a feature that is also characteristic of chlorophylls in vivo (1). Of particular importance are the chlorophyll/water micelles formed in nonpolar organic solvents. Chlorophyll a/water micelles so formed are red-shifted from 660 nm (monomer) to 740 nm and are photoactive in red light (2). Bacteriochlorophyll a/water micelles (P865) have red-shifted optical spectra that strongly resemble those of intact purple photosynthetic bacteria (3). As no direct structural information on these systems exists, we have undertaken a small-angle neutron-scattering study of chlorophyll/water micelles in organic media, using the In- MATERIALS AND METHODS Materials. Chlorophyll a (Chla), bacteriochlorophyll a (Bchla), Chlorobium chlorophyll (Bchlc), and pheophytin a (Pheoa) were prepared by literature methods (4). Purity was established by HPLC and by californium-252 plasma desorption mass spectroscopy (5). Deuterated solvents (Aldrich) contained <1% 1H.Chlorophyll/water micelles were prepared by addition of microliter amounts of 'H20 or 2H20 to previously dried chlorophyll dissolved (=10 mg/ml) in a nonpolar solvent.Mild sonication (30 sec) was used to disperse the water. Rigorous drying of the chlorophylls and the solvents (6) before hydration was necessary as the structures formed from undried or partially dried starting materials can vary. Samples for small-angle neutron scattering were sealed in 2-to 5-mm-thick quartz cells. Neutron Scattering. Chlorophylls of natural isotopic composition were studied in deuterated solvents to enhance the scattering intensity in much the same way neutrons h...

A 3-D Structural Model of Solid Self-Assembled Chlorophyll a/H2O from Multispin Labeling and MAS NMR 2-D Dipolar Correlation Spectroscopy in High Magnetic Field

“…The possibility of an antiparallel dimer building block for the aggregate structure is thus rejected. It was inferred from small-angle neutron scattering that hydrated Chl a forms monolayers in a nonpolar solvent that organize into tubular micelles with a fixed radius of ∼57Å, with the phytyl chains on the outside, and with solvent molecules in the interior (6,20 ). Such a tube filled with solvent is very difficult to reconcile with the NMR data, since a response of the n-heptane was not yet observed in any of the datasets.…”

2D13C–13C MAS NMR Correlation Spectroscopy with Mixing by True 1H Spin Diffusion Reveals Long-Range Intermolecular Distance Restraints in Ultra High Magnetic Field

“…When exposed to H 2 O it forms an aggregate. Such aggregates represent a paradigm for chlorophyll stacking in the chlorosome light-harvesting antennae found in some green photosynthetic bacteria (12,13). Thus, chlorophyll aggregates can form proteinfree light-harvesting antennae, which is of potential interest for artificial photosynthesis.…”

Magic Angle Spinning Nuclear Magnetic Resonance of the Chlorosomes

“…Upfi eld aggregation shifts up to ~5 ppm are observed, mostly induced by ring currents. The shift pattern is in agreement with models for the Chl a/water aggregates that assume strong overlap between the rings A and the rings C and E of adjacent molecules forming stacks (Worcester et al, 1986). In addition, in a 2D 13 C-13 C RFDR spectrum recorded with a long mixing time of τ m = 10 ms, several cross-peaks are observed that can be attributed to intermolecular transfer (Fig.…”

“…The crucial evidence is that the in vitro aggregation of BChl c leads to similar structures and spectroscopic properties as occur in the natural system (for review, see Tamiaki et al, 2002). Small-angle neutron scattering studies have indicated cylindrical shaped micelles for aggregates prepared in organic media (Worcester et al, 1986). In addition, various spectroscopic methods suggest a highly ordered structure of both aggregated BChl c and the chlorosomes (Griebenow et al, 1991;Matsuura et al, 1993;Hildebrandt et al, 1994).…”