Oxygen regulation of ribulose 1,5-bisphosphate carboxylase activity in Rhodospirillum rubrum

Abstract

The carboxylase activity of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBPC/O) decreased when an anaerobic culture of RhodospiriUum rubrum was exposed to atmospheric levels of oxygen. From 70 to 80% of the activity was lost within 12 to 24 h. Inactivation was apparent when the enzyme was assayed in situ (in whole cells) and when activity was measured in dialyzed crude extracts. The quantity of enzyme protein, as estimated from sodium dodecyl sulfate-polyacrylamide gels or as quantified immunologically, did not decrease within 24 h of exposure to air. Following extended exposure to aerobic conditions (48 to 72 h), degradation of enzyme occurred. These results indicate that the inactivation of RuBPC/O in R. rubrum may be due to an alteration or modification of the preformed enzyme, followed by eventual degradation of the inactive enzyme. When shifted back to anaerobic conditions (under an argon atmosphere), the RuBPC/O activity increased rapidly. This increase appeared to be due to de novo synthesis of enzyme. The increase in activity was not observed when the culture was maintained in the dark or in the absence of a suitable carbon source. Thus, the oxygen-mediated inactivation of RuBPC/O appeared to be due to some form of irreversible modification. The cloned R. rubrum RuBPC/O gene, expressed in Escherichia coli, yielded functional enzyme that was not affected by oxygen, indicating that inactivation in R. rubrum is mediated by a gene product(s) not found in E. coli.Ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase (RuBPC/O) catalyzes the first reaction of both the reductive and oxidative photosynthetic carbon cycles (10). In the absence of oxygen, RuBPC/O catalyzes the carboxylation and cleavage of RuBP, resulting in the formation of two molecules of 3-phosphoglyceric acid. In the presence of oxygen, RuBPC/O catalyzes the oxygenolysis of RuBP, resulting in the formation of 1 molecule of 3-phosphoglyceric acid and 1 molecule of phosphoglycolate. Usually the phosphoglycolate formed is further metabolized via the glycolate pathway, resulting in the wasteful loss of carbon and nitrogen. The oxygenase function of the enzyme therefore results ultimately in a futile cycle, with loss of CO2 and enormous energy expenditure (5). The ability of RuBPC/O to catalyze these two distinct reactions necessitates careful metabolic regulation of enzyme activity. Rhodospirillum rubrum, like all members of the family Rhodospirillaceae, synthesizes high levels of RuBPC/O when grown under anaerobic photosynthetic conditions with butyrate or hydrogen as the electron donor (1,13,15,16,18). In the present investigation, it is shown that RuBPC/O is metabolically regulated in R. rubrum following exposure to oxygen. The evidence suggests that inactivation occurs by some form of alteration or modification of the enzyme when cells are exposed to atmospheric levels of oxygen. The physiological significance of this response is evident and may be related to the need to inactivate RuBPC/O in order to limit the wasteful loss of carbon a...

Roles of RubisCO and the RubisCO-Like Protein in 5-Methylthioadenosine Metabolism in the Nonsulfur Purple Bacterium Rhodospirillum rubrum

“…Although an intact cbbM gene is present in this strain, it is barely expressed under aerobic growth conditions (6). Further, R. rubrum cells that do contain exogenously expressed RubisCO were found to oxidatively inactivate and then subsequently degrade this protein under aerobic conditions (6,7). It was thus not surprising that the rlpA-disruption strain falied to metabolize MTA in the presence of oxygen.…”

“…However, to this point there is no experimental evidence for the existence of a functional MSP (21) in R. rubrum. Thus, in this study, we sought to determine the role of the RLP and RubisCO protein in sulfur salvage since each protein catalyzes different reactions and RubisCO is known to be synthesized only under anaerobic conditions (6,7). Moreover, it is well appreciated that R. rubrum possesses a versatile metabolic capacity and is able to grow under both anaerobic and aerobic conditions, using a variety of carbon sources.…”

A Conserved Mechanism Controls Translation of Rubisco Large Subunit in Different Photosynthetic Organisms

“…R. rubrum is photosynthetic only when grown under anaerobic conditions (Anderson and Fuller, 1967). Exposure of an anaerobic culture of R. rubrum to atmospheric air reduced the activity of Rubisco to less than 20% within 12 h, while the steady-state level of the enzyme remained constant (Cook and Tabita, 1988). Extensive degradation was observed only after extended exposure to aerobic conditions (48 to 72 h).…”

Expression of endogenous and foreign ribulose 1,5-bisphosphate carboxylase-oxygenase (RubisCO) genes in a RubisCO deletion mutant of Rhodobacter sphaeroides

“…For example, R. rubrum has been shown to inactivate RubisCO in the presence of oxygen by endogenous factors, and the system described here could demonstrate whether this inactivation is strain dependent (6). The R. sphaeroides RubisCO deletion strain and RubisCO promoter system will also be useful in characterizing mutagenized RubisCO proteins in vivo and in the selection for altered RubisCO molecules after random mutagenesis of RubisCO genes or gene regions.…”