Light energy for photosynthesis is collected by the antenna system, creating an excited state which migrates energetically 'downhill'. To achieve efficient migration of energy the antenna is populated with a series of pigments absorbing at progressively redshifted wavelengths. This variety in absorbing species in vivo has been created in a biosynthetically economical fashion by modulating the absorbance behaviour of one kind of (bacterio)chlorophyll molecule. This modulation is poorly understood but has been ascribed to pigment-pigment and pigment-protein interactions. We have examined the relationship between aromatic residues in antenna polypeptides and pigment absorption, by studying the effects of site-directed mutagenesis on a bacterial antenna complex. A clear correlation was observed between the absorbance of bacteriochlorophyll a and the presence of two tyrosine residues, alpha Tyr44 and alpha Tyr45, in the alpha subunit of the peripheral light-harvesting complex of Rhodobacter sphaeroides, a purple photosynthetic bacterium that provides a well characterized system for site-specific mutagenesis. By constructing single (alpha Tyr44, alpha Tyr45----PheTyr) and then double (alpha Tyr44, alpha Tyr45----PheLeu) site-specific mutants, the absorbance of bacteriochlorophyll was blueshifted by 11 and 24 nm at 77 K, respectively. The results suggest that there is a close approach of tyrosine residues to bacteriochlorophyll, and that this proximity may promote redshifts in vivo.

The photosynthetic apparatus of Rhodobacter sphaeroides is comprised of three types of pigment-protein complex: the photochemical reaction centre and its attendant LH1 and LH2 light-harvesting complexes. To augment existing deletion/insertion mutants in the genes coding for these complexes we have constructed two further mutants, one of which is a novel double mutant which is devoid of all three types of complex. We have also constructed vectors for the expression of either LH1, LH2 or reaction-centre genes. The resulting system allows each pigment-protein complex to be studied either as part of an intact photosystem or as the sole complex in the cell. In this way we have demonstrated that reaction centres can assemble independently of either light-harvesting complex in R. sphaeroides. In addition, the isolation of derivatives of the deletion/insertion mutants exhibiting spontaneous mutations in carotenoid biosynthesis provides an avenue for examining the role of carotenoids in the assembly of the photosynthetic apparatus. We show that the LH1 complex is assembled regardless of the carotenoid background, and that the type of carotenoid present modifies the absorbance of the LH1 bacteriochlorophylls.

The photosynthetic apparatus of the bacterium Rhodobacter sphueroides contains three types of pigment protein: the photochemical reaction centre, and the LH1 and LH2 light-harvesting (LH) complexes. LH1 surrounds and interconnects the reaction centres, forming an LHl-reaction centre (RC) 'core' which receives excitation energy from the peripheral LH2 antenna [l]. This arrangement of complexes presents an experimentally attractive model system for the study of the transfer and trapping of light energy in photosynthesis. There are several reasons for this. First, there is a crystallographically determined structure for the RC [2-41. Second, a wealth of spectroscopic data are available on the RC and I,H complexes. Finally, there is a full range of genetic tools that can be used to produce site-directed mutations in the genes encoding these complexes [5, 61. Light energy is harvested by IJH1 and LH2 complexes, which are both composed of highly aggregated states of a and p polypeptides in close association with bacteriochlorophyll and carotenoid pigments. Secondary-structure predictions indicate that these polypeptides span the membrane once, and form an interconnecting network of several hundred pigments which increase the surface area and range of wavelengths over which light energy can be absorbed. Although this network contains only one type of tetrapyrrole pigment, bacteriochlorophyll a, the antenna system is populated with a series of these molecules which absorb at progressively red-shifted wavelengths so that an excited state at the periphery of a photosynthetic 'unit' migrates in an energetically 'downhill' fashion towards the RC where the energy is trapped and photochemistry takes place.Spectroscopic studies on the antenna of Rb. sphaeroides have been extremely useful in defining the rates of energy transfer between these pigments, and the orientations of these pigments with respect to the plane of the membrane. The availability of mutant strains has also simplified the interpretations of complicated and overlapping spectroscopic signals by contributing a series of strains in which LH2, LH1 or indeed the RC, can be examined in Abbreviations used: LH, light-harvesting complex; RC, reaction centre. isolation from the other complexes, in the membrane environment [ 1,7lo].In this communication, the effects of altering a residue within the LH2 complex will be described.These alterations have made use of a series of deletion/insertion strains, in which the puc genes encoding a and p polypeptides of the LH2 complexes, and the puf genes encoding polypeptide components of the IJH1-RC 'core', have been deleted from the chromosome. In each case, these genes have been replaced by a cassette encoding resistance to an antibiotic. There are two single deletion strains, one of which synthesizes the LH1-RC core only (DRC1; [Ill), and the other which synthesizes LH2 only (DPF2; [ 101). A double deletion strain, DD13, possesses no pigment protein complexes [ 101 and can act as a recipient for either altered LHI, LH2 or RC ge...