In vivo regulation of form I ribulose 1,5-bisphosphate carboxylase/ oxygenase from Rhodopseudomonas sphaeroides

Abstract

When autotrophically grown cells of Rhodopseudomonas (Rhodobacter) sphaeroides were supplied with an organic carbon source, the activity of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBPC/O) decreased 30 to 60%. The extent of inactivation varied depending on the level of derepression of form I and form II RuBPC/O, and on the nature of the organic carbon source, pyruvate being the most effective. Raising the concentration of CO 2 in the gas phase of autotrophic cultures brought about a similar loss of RuBPC/O activity. Immunological assays of form I and form II RuBPC/O proteins indicated that the synthesis of both enzymes had been repressed. Moreover, it is demonstrated that the observed loss of RuBP carboxylase activity was due to inactivation of the form I enzyme; the form II RuBPC/O was not affected. The isolated inactivated form I RuBPC/O exhibited a fivefold lower specific activity compared to the active form I enzyme. The inactivation was accompanied by changes in the properties as well as the structure of the form I enzyme. In autotrophic cells, form I RuBPC/O appeared to be associated with a phosphate-containing compound that decreased the enzyme's relative mobility in nondenaturing gels and increased its density in sucrose gradients. Form I RuBPC/O was released from an apparent complex or aggregate upon in vivo inactivation and/or after in vitro heat treatment. The inactive form I enzyme was found to reactivate in vitro by a slow reaction that was accelerated by heat treatment. However, experiments showed no evidence for in vivo reactivation after cells were reexposed to autotrophic conditions (1.5% CO 2 in H 2). All these data indicate that R. sphaeroides RuBPC/O activity is controlled at the transcriptional and post-transcriptional levels, through regulatory systems that repress the synthesis of form I and form II RuBPC/O and inactivate the predominant form (form I) when the carbon source no longer becomes limiting for growth.