Oxygen-dependent inactivation of ribulose 1,5-bisphosphate carboxylase/oxygenase in crude extracts of Rhodospirillum rubrum and establishment of a model inactivation system with purified enzyme

Abstract

Ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase (RuBPC/0) was inactivated in crude extracts of Rhodospirilum rubrum under atmospheric levels of oxygen; no inactivation occurred under an atmosphere of argon. RuBP carboxylase activity did not decrease in dialyzed extracts, indicating that a dialyzable factor was required for inactivation. The inactivation was inhibited by catalase. Purified RuBPC/0 is relatively oxygen stable, as no loss of activity was observed after 4 h under an oxygen atmosphere. The aerobic inactivation catalyzed by endogenous factors in crude extracts was mimicked by using a model system containing purified enzyme, ascorbate, and FeSO4 or FeCl3. Dithiothreitol was found to substitute for ascorbate in the model system. Preincubation of the purified enzyme with RuBP led to enhanced inactivation, whereas Mg2+ and HC03-significantly protected against inactivation. Unlike the inactivation catalyzed by endogenous factors from extracts of R. rubrum, inactivation in the model system was not inhibited by catalase. It is proposed that ascorbate and iron, in the presence of oxygen, generate a reactive oxygen species which reacts with a residue at the activation site, rendering the enzyme inactive.Evidence has been presented that aerobic inactivation of ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase (RuBPC/O) in Rhodospirillum rubrum proceeds by a twostep process: the enzyme is first inactivated by an alteration or modification, and then the inactive protein is proteolytically degraded (2). This is very similar to well-described systems, such as glutamine synthetase (GS) and glutamine phosphoribosylpyrophosphate aminotransferase, in which oxidative inactivation precedes the actual degradation of the modified protein (11,21). With regard to GS, inactivation may be mediated by a variety of enzymatic [cytochrome P-450, glucose oxidase, horseradish peroxidase, NAD(P)H oxidase, and xanthine oxidase] and nonenzymatic [ascorbic acid, dihydroxyfumaric acid, and NAD(P)H plus menadione] systems (14). Inactivation was found to result in the loss of 1 of 16 histidine residues, with the subsequent formation of 1 carbonyl group per GS subunit (8). Levine has suggested that a mixed-function oxidation system generates a reactive oxygen species which reacts with a histidine residue to introduce a carbonyl moiety at the active site of the enzyme (9, 10). The oxidatively modified form of GS then becomes increasingly vulnerable to intracellular and exogenous proteases (16)(17)(18).A number of other procaryotic and eucaryotic enzymes are also susceptible to oxidative modification. Pyruvate kinase, creatine kinase, lactate dehydrogenase (14), 3-phosphoglycerate kinase (3), superoxide dismutase (5), and tyrosinase (7) are all susceptible to oxidative inactivation. Thus, loss of catalytic activity due to oxidative modification appears to be a common method of enzymatic regulation. In this study, evidence is presented that RuBPC/O is also susceptible to oxidative modification in vitro.

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.…”

“…We have shown that the RLP disruption strain of R. rubrum is incapable of MTA-dependent growth under aerobic growth conditions. 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).…”

“…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.…”

Degradation of active-oxygen-modified ribulose-1,5-bisphosphate carboxylase/oxygenase by chloroplastic proteases requires ATP-hydrolysis

“…Treatments well known to produce AO in plants cause loss of enzyme activity, oxidative protein modi®cation and/or protein degradation in vivo (Dann and Pell 1989;Eckardt and Pell 1995) or in isolated chloroplasts (Metha et al 1992). Furthermore, oxidative modi®cation of puri®ed Rubisco inhibits enzymatic activity (Cook et al 1988;Eckardt and Pell 1995) and increases the susceptibility of Rubisco to proteolysis by exogenous proteases (PenÄ arrubia and Moreno 1990) in vitro. We recently characterized Rubisco LSU degradation in isolated barley chloroplasts exposed to dierent AO levels (Desimone et al 1996).…”

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

“…The oxygen-mediated control of RuBPC/O in anaerobic photosynthetic bacteria is certainly reminiscent of the oxidative modification of glutamine synthetase in enteric bacteria (6)(7)(8). Other studies of the RuBPC/O inactivation system in R. rubrum have been presented (2).…”