Faculty of Engineering and Natural Sciences
CHARACTERISATION OF GENETICALLY MODIFIED CYTOCHROME SYSTEMS AND THEIR APPLICATION TO BIOHYDROGEN PRODUCTION IN RHODOBACTER CAPSULATUS
Facultative phototrophic bacterium Rhodobacter capsulatus has two c-type electron carrier cytochromes (cyt); the soluble cyt c2 and the membrane-attached cyt cy, that act as electron carriers during respiratory and photosynthetic growth of this species. Previously, a soluble form of cyt cy was constructed by fusing genetically the signal sequence of cyt c2 to the cyt c domain of cyt cy. The obtained novel soluble cyt cy (cyt S-cy) was unable to support photosynthetic growth of R. capsulatus but yielded photosynthetically functional (Ps+) revertants frequently. In the first part of this study, photosynthetic electron transfer properties of some of Ps+ revertants of cyt S-cy were analyzed by biochemical and biophysical methods and compared with the cyt cy and cyt c2. Reduction-oxidation titration of membrane supernatants showed that the redox midpoint potential of cyt S-cy was +338 mV which is similar to midpoint potentials of cyt cy or the cyt c2. However, light-activated, time resolved spectroscopy revealed that reaction center mediated oxidation kinetics of cyt S-cy exhibited only a slow phase, unlike cyt c2 which has both fast and slow phases. It therefore appeared that during electron transfer cyt S-cy does not interact with the reaction centre as tightly as cyt c2. These findings imply that attaching electron carrier cyts to the membrane allowed them to weaken their interactions with their partners, while restricting their spatial diffusion, so that they accomplish rapid multiple turnovers.
In the second part of this study, hydrogen production of various R. capsulatus strains harboring the genetically modified electron carrier cytochromes, cyt cbb3 deleted and Qox deleted strains were compared with the wild type. Under photoheterotrophic growth conditions with limiting nitrogen source, the excess reducing equivalents generated by organic acid oxidation are consumed to reduce protons into hydrogen by the activity of nitrogenase in R. capsulatus. The results indicated that the hydrogen production of mutant strains with modified electron carrier cytochromes decreased 3-5 folds, and the hydrogen production rate of the cyt cbb3- mutant increased significantly. Moreover in this study, the hydrogen production efficiency of different R. capsulatus strains was increased by the chromosomal inactivation of uptake hydrogenase genes and enzymatic activity of uptake hydrogenase of R. capsulatus strains were determined.
Bio: BS 1998, METU Biology department. - MS 2001 METU Biotechnology department. Thesis subject; "Construction and analysis of a soluble version of the membrane attached cytochrome cy in Rhodobacter capsulatus " – PhD 2005 METU Biotechnology department. Thesis subject; "Characterisation of the Genetically Modified Cytochrome Systems and Their Application to Biohydrogen Production in Rhodobacter Capsulatus”.
June 18, 2008, 13:40, FENS G032