Assuntos
Complexo de Proteínas do Centro de Reação Fotossintética/química , Sequência de Aminoácidos , Bacterioclorofilas/química , Bacterioclorofilas/genética , Bacterioclorofilas/efeitos da radiação , Luz , Complexos de Proteínas Captadores de Luz , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Complexo de Proteínas do Centro de Reação Fotossintética/genética , Complexo de Proteínas do Centro de Reação Fotossintética/efeitos da radiação , Engenharia de Proteínas , Rhodobacter sphaeroides/química , Rhodobacter sphaeroides/genética , EspectrofotometriaRESUMO
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.
Assuntos
Complexo de Proteínas do Centro de Reação Fotossintética/genética , Rhodobacter sphaeroides/genética , Carotenoides/genética , Conjugação Genética , Escherichia coli , Teste de Complementação Genética , Vetores Genéticos , Mutagênese , Plasmídeos , Mapeamento por RestriçãoRESUMO
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.
