The function and molecular architecture of the UQH2:cyt c2 oxidoreductase of R. spheroides are being studied by a combined approach using molecular genetics, biochemistry and biophysics. We are modifying the genes coding for the enzyme, and assaying the functional and structural consequences of specific changes in the proteins. By using new protein structural prediction algorithms, and information from the mapping at the residue level of lesions leading to inhibitor resistance, we have proposed an 8-transmembrane felix model, and have located the two quinone reactive catalytic sites. We have started to modify residues at the putative sites which might be important in catalysis. Our goal is to map the contribution of specific residues by detailed biophysical studies of mutants generated by site-specific or random mutagenesis, and to use this information to analyse mechanism and the architecture of the sites. We are using similar approaches to robe the structure, assembly, and topology of the enzyme. Studies of the topology of cytochrome b subunit using the phoA-fusion technique have supported our 8 membrane helic model, and we have done control experiments using R. sphaeroides reaction center L-sub-nit to demonstrate the validity of the phoa fusion technique We have completed the DNA-sequencing of the fbc operon coding for the three polypeptides (bis-cyt b, cyt cl and 2Fe.2S containing subunits) and derived the amino acid sequences. We have developed a preparative protocol to allow large-scale purification of a lightly active enzyme, and have completed an initial biochemical and biophysical charact- erization. Preliminary N-terminal sequencing of the subunits has confirmed the data derived from DNA-sequencing, and shown some post translational processing. By introducing a kanamycin cassette in place of the fbcf gene, we have prepared a strain lacking a functional fbc operon which was unable to grow photosynthetically (pho-). Complementation with the fbc operon on plasmid led to restoration of pho+ phenotype. We have made a number of site-directed mutants, and have started the detailed biophysical characterization of cinetic and thermodynamic parameters. We have also selected inhibitor resistant mutants, and made a preliminary characterization of myxothiazol resistant strains. We have constructed an apparatus for computer-aided fluorescence video imaging to aid in the screening and preliminary characterization of mutant strains. The software developed allows us to select strains with specific phenotypic attributes using false color coding of images, and measurement of induction kinetics of individual colonies.
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