Date of Award
Doctor of Philosophy (PhD)
Rhodopseudomonas viridis grows by means of nitrogen fixation under anaerobic, photosynthetic conditions. In batch culture, nitrogenase activity was highest at early-logarithmic phase, lower during mid- to late-logarithmic phase, and nearly zero during stationary phase. Nitrogen-fixing cells were morphologically and ultrastructurally similar to non-nitrogen-fixing cells as determined by electron microscopy. Electron spin resonance (esr) spectroscopy of nitrogen-fixing whole cells yielded g4.26 and g3.66 signals indicating the presence of nitrogenase molybdenum-iron (MoFe) protein. Ammonia switch-off occurred upon addition of 0.2 mM NH(,4)Cl, however, nitrogenase activity did not reappear for nearly four hours. Esr spectroscopy of whole cell multilayers (WCM) of Azotobacter vinelandii and Rhodospirillum rubrum was used to detect structural associations between nitrogenase MoFe protein and cell membrane. Conditions were defined for observing MoFe protein esr signals in whole cell preparations of each organism. The orientation of membranes in A. vinelandii WCM was demonstrated using doxyl stearate spin label and orientation of intracytoplasmic membrane was demonstrated in R. rubrum WCM by the orientation dependence of the bacteriochlorophyll a dimer triplet signal, (BChl a)(,2)('T), from the intracytoplasmic membrane-bound primary donor unit. The low field MoFe protein signals, g4.3 and g3.6, showed orientation dependent characteristics in WCM of both organisms, although the properties of each were not identical. As the normal to the membrane plane was rotated from perpendicular to parallel with the esr magnetic field, the amplitude of the g3.6 signal decreased from maximum to 37% of maximum in A. vinelandii and from maximum to 88% of maximum in R. rubrum. The angular dependence of the g4.3 peak during rotation varied in A. vinelandii, but decreased from maximum to 63% of maximum in R. rubrum. These properties suggest that the MoFe protein of nitrogenase was oriented in response to the physical orientation of cellular membranes and that a structural association exists between this nitrogenase component and membrane in these organisms.
Howard, Karen Smith, "Structural Interaction Between Nitrogenase and Cell Membrane in Free-Living, Nitrogen-Fixing Bacteria (Rhodopseudomonas, Viridis, Azotobacter, Vinelandii, Rhodospirillum, Rubrum)." (1982). LSU Historical Dissertations and Theses. 3807.