Biophysical investigations of photosynthetic reaction centres from Rhodobacter sphaeroides

Fyfe, Paul Kelman (1997) Biophysical investigations of photosynthetic reaction centres from Rhodobacter sphaeroides. PhD thesis, University of Glasgow.

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Site-directed mutagenesis was used to produce a series of mutant reaction centres from the purple bacterium Rb. sphaeroides. These mutants were investigated using a combination of functional assays and X- ray crystallography in order to correlate structure/function relationships. In particular the effect of the protein environment on the carotenoid pigment in the reaction complex has been investigated. The carotenoid is held within a largely hydrophobic pocket within the reaction centre complex, assuming a twisted 15,15' cis conformation. A small number of polar amino acid residues are positioned towards one end, forming almost a ring around the pigment molecule. Each of these amino acids was replaced with a non-polar residue of an approximately similar size: Trp M115-Phe, Ser M119-Ala, Met M122-Leu, Trp M157-Phe and Tyr M177-Phe. The mutant reaction centres were expressed in a background lacking light-harvesting complexes, permitting observation of the spectral characteristics of the complex in the bacterial cell and chromatophores. A range of techniques were then applied to attempt to identify the effects of the mutations on the reaction centre carotenoid. One additional mutant complex was investigated, in which an amino acid in close proximity to the BChl special pair was mutated (Phe to Arg M197). Limited investigation of this mutant complex had already identified altered spectral characteristics, although the precise mechanism by which this spectral shift was produced by the mutation was unclear. The investigations performed fell into three groups. The initial focus was concentrated on establishing whether the mutant reaction centre complexes were still being properly assembled by the bacteria. To this end, the ability of the bacteria to insert complexes into the membrane, the stability of the complexes upon removal from the membrane, and the ratio of pigments bound within the complex were determined. The second group of investigations utilised specialised spectroscopic techniques. Circular dichroism studies showed that the carotenoid was apparently binding in a similar fashion in the mutant and wild-type complexes, and that the blue-shifted P band in the absorption spectra of RM197 strains was also manifested in the CD spectra. ESR was the final spectroscopic technique utilised. It was used to investigate the ability of the various RC complexes to transfer the triplet energy from BChl to carotenoid. All the carotenoid binding site mutants returned wild-type signals. However, those carrying the RM197 mutation showed marked differences from the wild-type. The mutation at RM197 affected the values of the ESR field splitting parameters, and allowed the triplet energy to be transferred at temperatures much lower than that determined for the wild-type. (Abstract shortened by ProQuest.).

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Microbiology, photosynthetic bacteria, Rhodobacter sphaeroides.
Subjects: Q Science > QR Microbiology
Colleges/Schools: College of Medical Veterinary and Life Sciences
Supervisor's Name: Cogdell, Professor Richard
Date of Award: 1997
Depositing User: Enlighten Team
Unique ID: glathesis:1997-71294
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 10 May 2019 10:49
Last Modified: 05 Sep 2022 08:35
Thesis DOI: 10.5525/gla.thesis.71294

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