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

The carboxylase activity of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBPC/O) decreased when an anaerobic culture of Rhodospirillum 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.     

Partition kinetics of ribulose-1,5-bisphosphate carboxylase from Rhodospirillum rubrum

When the enzymatically generated intermediate 2-carboxy-3-keto-D-arabinitol-1,5-bisphosphate (II) was used as a substrate with fresh enzyme, 70% reacted to produce 3-phosphoglycerate (3PGA). When a reaction mixture of enzyme plus [1-32P]ribulose 1,5-bisphosphate (RuBP) was quenched in the steady state with the tightly bound inhibitor 2-carboxyarabinitol-1,5-bisphosphate, 30% of the enzyme-bound species was released as 3PGA and 70% as RuBP. The major source for this partition was the ternary substrates Michaelis complex. The level of carboxylated intermediate in the steady state was determined to be 8% of active sites under the conditions of substrate saturation. No burst was seen in the appearance of product when 6.5 eq of [1-32P]RuBP was mixed with enzyme plus saturating CO2 and the reaction followed in the steady state. From these data plus the steady-state Vmax and Km of RuBP it is possible to derive the five bulk rate constants represented in the scheme ECO2 + RuBP in equilibrium ERuBPCO2 in equilibrium E X II----E + 2(3PGA).     

Multiple catalytic roles of His 287 of Rhodospirillum rubrum ribulose 1,5-bisphosphate carboxylase/oxygenase.

Active-site His 287 of Rhodospirillum rubrum ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase interacts with the C3-hydroxyl of bound substrate or reaction-intermediate analogue (CABP), water molecules, and ligands for the activator metal-ion (Andersson I, 1996, J Mol Biol 259:160-174; Taylor TC, Andersson I, 1997, J Mol Biol 265:432-444). To test structure-based postulates of catalytic functionality, His 287 was replaced with Asn or Gln. The mutants are not affected adversely in subunit assembly, activation (binding of Mg2+ and carbamylation of Lys 191), or recognition of phosphorylated ligands; they bind CABP with even greater tenacity than does wild-type enzyme. H287N and H287Q are severely impaired in catalyzing overall carboxylation (approximately 10(3)-fold and > 10(5)-fold, respectively) and enolization (each mutant below threshold for detection) of RuBP. H287N preferentially catalyzes decarboxylation of carboxylated reaction intermediate instead of forward processing to phosphoglycerate. Analysis of RuBP turnover that occurs at high concentrations of mutants over extended time periods reveal > 10-fold reduced CO2/O2 specificities, elevated misprotonation of the enediol intermediate, and misprocessing of the oxygenated intermediate of the oxygenase pathway. These results are consistent with multifaceted roles for His 287 in promoting enediol formation, enediol tautomerization, and forward-processing of carboxylated intermediate.     

The oxygenase activity of ribulose-1,5-bisphosphate carboxylase from Rhodospirillum rubrum

Catalysis by pure ribulose bisphosphate carboxylase from $\text{Rhodospirillum rubrum}$, which is a dimer (MW: 114,000) lacking small subunits, is inhibited by oxygen. Oxygen is a competitive inhibitor with respect to carbon dioxide. In the absence of carbon dioxide, the enzyme catalyzes the oxygenolytic cleavage of ribulose-1,5-bisphosphate with consumption of one mole of oxygen per mole of 3-phosphoglycerate produced.     

Ribulose bisphosphate carboxylase/oxygenase in toluene-permeabilized Rhodospirillum rubrum.

Toluene-permeabilized Rhodospirillum rubrum cells were used to study activation of and catalysis by the dual-function enzyme ribulose bisphosphate carboxylase/oxygenase. Incubation with CO2 provided as HCO3-, followed by rapid removal of CO2 at 2 degrees C and subsequent incubation at 30 degrees C before assay, enabled a determination of decay rates of the carboxylase and the oxygenase. Half-times at 30 degrees C with 20 mM-Mg2+ were 10.8 and 3.7 min respectively. Additionally, the concentrations of CO2 required for half-maximal activation were 56 and 72 microM for the oxygenase and the carboxylase respectively. After activation and CO2 removal, inactivation of ribulose bisphosphate oxygenase in the presence of 1 mM- or 20mM-Mn2+ was slower than that with the same concentrations of Co2+ or Mg2+. Only the addition of Mg2+ supported ribulose bisphosphate carboxylase activity, as Mn2+, Co2+ and Ni2+ had no effect. A pH increase after activation in the range 6.8-8.0 decreased the stability of the carboxylase but in the range 7.2-8.0 increased the stability of the oxygenase. With regard to catalysis. Km values for ribulose 1,5-bisphosphate4- were 1.5 and 67 microM for the oxygenase and the carboxylase respectively, and 125 microM for O2. Over a broad range of CO2 concentrations in the activation mixture, the pH optima were 7.8 and 8-9.2 for the carboxylase and the oxygenase respectively. The ratio of specific activities was constant (9:1 for the carboxylase/oxygenase) of ribulose bisphosphate carboxylase/oxygenase in toluene-treated Rsp. rubrum. Below concentrations of 10 microM-CO2 in the activation mixture, this ratio increased.     

Preliminary X-ray diffraction study of ribulose-1,5-bisphosphate carboxylase from Rhodospirillum rubrum

Crystals from the dimeric enzyme ribulose-1,5-bisphosphate carboxylase of the photosynthetic bacterium Rhodospirillum rubrum have been obtained from the gene product expressed in Escherichia coli. The crystals are of the quarternary complex comprising enzyme: activator CO2 (as a carbamate): Mg2+: 2- carboxyarabinitol -1,5-bisphosphate (as a transition state analog). X-ray diffraction photographs show symmetry consistent with space group P4(1)2(1)2 or the corresponding enantiomorphic space group. Cell parameters are a = b = 82 A, c = 324 A with two subunits per asymmetric unit. The crystals diffract to at least 3 A resolution.     

Derepression of the synthesis of D-ribulose 1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum.

The synthesis of ribulose 1,5-bisphosphate carboxylase/oxygenase in Rhodospirillum rubrum was greatly influenced by the conditions of culture. When grown photolithotrophically in an atmosphere containing low levels of CO2 (1.5 to 2%), enzyme synthesis was derepressed, with the result that the enzyme comprised up to 50% of the soluble protein of the cells as determined by immunological quantitation. This response was not observed when R. rubrum was grown photolithotrophically in an atmosphere of 5% CO2 in hydrogen. Similarly, the derepression of ribulose 1,5-bisphosphate carboxylase/oxygenase was observed in photoheterotrophically (butyrate)-grown cultures only after the HCO3- supply was nearly exhausted. The increase in enzyme activity observed in derepressed cultures was not paralleled by an increase in the in vivo CO2 fixation rate. Apparently, R. rubrum derepresses the synthesis of ribulose 1,5-bisphosphate carboxylase/oxygenase when exposed to low CO2 concentrations to scavenge the limited CO2 available to such cultures.     

New crystal forms of ribulose-1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum

Three crystal forms of the dimeric form of the enzyme ribulose-1,5-bisphosphate carboxylase from the photosynthetic bacterium Rhodospirillum rubrum have been obtained from the gene product expressed in Escherichia coli. Form A crystals formed from the quaternary complex comprising enzyme-activator carbamate-Mg2+-2'-carboxyarabinitol-1,5-bisphosphate are shown here to be devoid of ligands. In contrast, crystals of the quaternary complex formed with the hexadecameric L8S8 enzyme from spinach contain both the activator carbamate and 2'-carboxyarabinitol-1,5-bisphosphate. Form B crystals of the R. rubrum enzyme are monoclinic, space group P2(1) with cell dimensions a = 65.5 A, b = 70.6 A, c = 104.1 A and beta = 92.1 degrees, with two subunits per asymmetric unit. Rotation function calculations show a non-crystallographic 2-fold axis perpendicular to the monoclinic b-axis. Form C crystals are orthorhombic (space group P2(1)2(1)2(1)) with cell dimensions a = 79.4 A, b = 100.1 A and c = 131.0 A. The monoclinic crystal form diffracts to at least 2.0 A resolution on a conventional X-ray source.     

Preliminary structural studies of ribulose-1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum

Ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) from Rhodospirillum rubrum has been crystallized in a form that is suitable for structural studies by x-ray diffraction. The asymmetric unit of the crystal contains one dimeric enzyme molecule of molecular mass 101,000 Da. The enzyme was activated prior to crystallization and is presumed to be in the CO2-activated state in the crystal. The method of hydrophobicity correlation has been used to compare the amino acid sequence of this molecule (466 residues) to that of the large subunit of a higher plant ribulose-1,5-bisphosphate carboxylase/oxygenase (477 residues in Nicotiana tabacum). The pattern of residue hydrophobicities is similar along the two polypeptides. This suggests that the three-dimensional folding of the large polypeptide chains may be similar in plant and bacterial enzymes. If this is so, knowing the structure of either the plant or bacterial ribulose-1,5-bisphosphate carboxylase/oxygenase should aid in learning the structure of the other.     

A site-specific mutation within the active site of ribulose-1,5-bisphosphate carboxylase of Rhodospirillum rubrum

In vitro mutagenic techniques have generated an asp→glu substitution at residue 198 adjacent to the carbamate-divalent metal ion binding site of Rhodospirillum rubrum ribulose 1,5-bisphosphate carboxylase. A single C→A nucleotide change in the coding strand created the mutant and introduced a new EcoRI restriction site on the expression plasmid pRR2119. Although the carboxylase:oxygenase ratio remained the same, the mutant enzyme had slightly altered kinetic properties. The e.p.r. spectra of the quaternary complexes enzyme.activator carbamate.Mn²⁺.2-carboxyarabinitol 1,5-bisphosphate and enzyme.activator carbamate.Mn²⁺.4-carboxyarabinitol 1,5-bisphosphate for mutant and wild-type enzymes were different, indicating that the metal ion was in a slightly altered environment. These findings are consistent with the hypothesis that, besides the carbamate at lys 201, the carboxyl group of asp 198 contributes to the formation of the divalent metal ion binding site.     

Ribulose 1,5-bisphosphate and activation of the carboxylase in the chloroplast

Ribulose 1,5-bisphosphate in the chloroplast has been suggested to regulate the activity of the ribulose bisphosphate carboxylase/oxygenase. To generate high levels of ribulose bisphosphate, isolated and intact spinach chloroplasts were illuminated in the absence of CO₂. Under these conditions, chloroplasts generate internally up to 300 nanomoles ribulose 1,5-bisphosphate per milligram chlorophyll if O₂ is also absent. This is equivalent to 12 millimolar ribulose bisphosphate, while the enzyme, ribulose bisphosphate carboxylase, offers up to 3.0 millimolar binding sites for the bisphosphate in the chloroplast stroma. During illumination, the ribulose bisphosphate carboxylase is deactivated, due mostly to the absence of CO₂ required for activation. The rate of deactivation of the ribulose bisphosphate carboxylase was not affected by the chloroplast ribulose bisphosphate levels. Upon addition of CO₂, the carboxylase in the chloroplast was completely reactivated. Of interest, addition of 3-phosphoglycerate stopped deactivation of the carboxylase in the chloroplast while ribulose bisphosphate accumulated. With intact chloroplasts in light, no correlation between deactivation of the carboxylase and ribulose bisphosphate levels could be shown.     

Ribulose 1,5-bisphosphate carboxylase-oxygenase. I. Structural, immunochemical and catalytic properties

Some structural, immunochemical and catalytic properties are examined for ribulose 1,5-bisphosphate carboxylase-oxygenase from various cellular organisms including bacteria, cyanobacteria, algae and higher plants. The native enzyme molecular masses and the subunit polypeptide compositions vary according to enzyme sources. The molecular masses of the large and small subunits from different cellular organisms, on the other hand, show a relatively high homology due to their well-conserved primary amino acid sequence, especially that of the large subunit. In higher plants, the native enzyme and the large subunit are recognized by the antibodies raised against either the native or large subunit, whereas the small subunit apparently cross-reacts only with the antibodies directed against itself. A wide diversity exists, however, in the serological response of the native enzyme and its subunits with antibodies directed against the native enzyme or its subunits from different cellular organisms. According to numerous kinetic studies, the carboxylase and oxygenase reactions of the enzyme with ribulose 1,5-bisphosphate and carbon dioxide or oxygen require activation by carbon dioxide and magnesium prior to catalysis with ribulose 1,5-bisphosphate and carbon dioxide or oxygen. The activation and catalysis are also under the regulation of other metal ions and a number of chloroplastic metabolites. Recent double-labeling experiments using radioactive ribulose 1,5-bisphosphate and 14CO2 have elucidated the carboxylase/oxygenase ratios of the enzymes from different organisms. Another approach, i.e., genetic experiments, has also been used to examine the modification of the carboxylase/oxygenase ratio.     

Ribulose diphosphate oxygenase. V. Presence in ribulose diphosphate carboxylase from Rhodospirillum rubrum

Ribulose-1,5-diphosphate carboxylase was purified fifteenfold from Rhodospirillum rubrum grown autotrophically under H₂ and CO₂. There was RuDP oxygenase activity associated with the carboxylase. The oxygenase had maximal activity at pH 9.4. Although these bacterial RuDP oxygenase and carboxylase activities were cold labile, activity could not be restored by treatment at 50° in the presence of Mg⁺⁺ and a sulfhydryl reagent, in contrast to results with the enzyme from eukaryotes.     

Crystallographic refinement and structure of ribulose-1,5-bisphosphate carboxylase from Rhodospirillum rubrum at 1.7 A resolution

The amino acid sequence of ribulose-1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum has been fitted to the electron density maps. The resulting protein model has been refined to a nominal resolution of 1.7 A using the constrained-restrained least-squares refinement program of Sussman and the restrained least-squares refinement program of Hendrickson & Konnert. The crystallographic refinement, based on 76,452 reflections with F greater than sigma (F) in the resolution range 5.5 to 1.7 A resulted in a crystallographic R-factor of 18.0%. The asymmetric unit contains one dimeric ribulose-1,5-biphosphate carboxylase molecule, consisting of 869 amino acid residues and 736 water molecules. The geometry of the refined model is close to ideal, with root-mean-square deviations of 0.018 A in bond lengths and 2.7 degrees in bond angles. Two loop regions, comprising residues 54 to 63 and 324 to 335, and the last ten amino acid residues at the C terminus are disordered in our crystals. The expected trimodal distribution is obtained for the side-chain chi 1-angles with a marked preference for staggered conformation. The hydrogen-bonding pattern in the N-terminal beta-sheet and the parallel sheet in the beta/alpha-barrel is described. A number of hydrogen bonds and salt bridges are involved in domain-domain and subunit-subunit interactions. The subunit-subunit interface in the dimer covers an area of 2800 A2. Considerable deviations from the local 2-fold symmetry are found at both the N terminus (residues 2 to 5) and the C terminus (residues 422 to 457). Furthermore, loop 8 in the beta/alpha-barrel domain has a different conformation in the two subunits. A number of amino acid side-chains have different conformations in the two subunits. Most of these residues are located at the surface of the protein. An analysis of the individual temperature factors indicates a high mobility of the C-terminal region and for some of the loops at the active site. The positions and B-factors for 736 solvent sites have been refined (average B: 45.9 A2). Most of the solvent molecules are bound as clusters to the protein. The active site of the enzyme, especially the environment of the activator Lys191 in the non-activated enzyme is described. Crystallographic refinement at 1.7 A resolution clearly revealed the presence of a cis-proline at the active site. This residue is part of the highly conserved region Lys166-Pro167-Lys168.     

Three-dimensional structure of ribulose-1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum at 2.9 A resolution

The three-dimensional structure of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from Rhodospirillum rubrum has been determined at 2.9 Å resolution by X-ray crystallographic methods. The MIR-electron density map was substantially improved by two-fold non-crystallographic symmetry averaging. The polypeptide chains in the dimer were traced using a graphics display system with the help of the BONES option in FRODO. The dimer has approximate dimensions of 50 x 72 x 105 Å. The enzyme subunit is a typical two-domain protein. The smaller, N-terminal domain consists of 137 amino acid residues and forms a central, mixed five-stranded β-sheet with α-helices on both sides of the sheet. The larger C-terminal domain consists of 329 amino acid residues. This domain has an eight-stranded parallel α/β barrel structure as found in triosephosphate isomerase and a number of other functionally non-related proteins. The active site in Rubisco determined by difference Fourier techniques and fitting of active site residues to the electron density map, is located at the carboxy-end of the β-strands in the α/β barrel of the C-terminal domain. There are few domain–domain interactions within the subunit. The interactions at the interface between the two subunits of the dimer are tight and extensive. There are tight contacts between the two C-terminal domains, which build up the core of the molecule. There are also interactions between the N-terminal domain of one subunit and the C-terminal domain of the second subunit, close to the active site.     

Truncation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from Rhodospirillum rubrum affects the holoenzyme assembly and activity.

Truncations of the subunit of ribulose bisphosphate carboxylase/oxygenase (Rubisco) from Rhodospirillum rubrum were generated by site-directed mutagenesis to examine the role of the C-terminal tail section. Removal of the last and the penultimate alpha-helices in the tail section changes the quaternary structure of the protein. Electrophoretic and electron microscope analysis revealed that the truncated subunits assemble into an octamer, whereas the wild-type enzyme has a dimeric structure. The octomerization of the mutant protein is due to a hydrophobic patch exposed to the solvent by truncation of the subunit. The mutant protein thus consists of four dimers, bound end-to-end by hydrophobic interactions. Insertion of a polar amino acid in the hydrophobic patch by a L424 to N424 substitution restores the familiar dimeric structure. Truncation of the subunit is associated with a considerable decrease in catalytic activity. The mutants undergo carbamylation but bind the reaction intermediate analog, 2-carboxy arabinitol-1,5-bisphosphate, poorly. This indicates that loss of activity in the mutant is due to weakened substrate binding. These findings suggest that the mutations in the tail section of the subunit are transmitted to the active site, although the C-terminal region is far from the active site. On the basis of the crystal structure of Rubisco, we propose a model for how the truncations of the enzyme subunit induce conformational changes in one of the two phosphate binding sites.     

Isolation and sequence of the pyridoxal 5'-phosphate active-site peptide from Rhodospirillum rubrum ribulose-1,5-bisphosphate carboxylase/oxygenase

Ribulose-1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum was modified with pyridoxal 5'-phosphate and then reduced with sodium borohydride. Both carboxylase and oxygenase activities were lost when one molecule of pyridoxal 5'-phosphate was bound per enzyme dimer. Peptide maps of modified enzyme showed one N6-(phosphopyridoxal)lysine-containing peptide. This peptide was isolated by gel filtration and cation-exchange chromatography and its sequence determined as Ala-Leu-Gly-Arg-Pro-Glu-Val-Asp-(PLP-Lys)-Gly-Thr-Leu-Val-Ile-Lys. Since activation of the enzyme with Mg2+/CO2 enhances pyridoxal 5'-phosphate modification and subsequent inactivation and the substrate ribulose bisphosphate protects against modification, the modified lysyl group is most certainly at the catalytic site and not at the activation site of the enzyme.     

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.

Ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase (RuBPC/O) was inactivated in crude extracts of Rhodospirillum 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/O 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 HCO3- 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.     

Small-angle X-ray study on the structure of ribulose-1,5-bisphosphate carboxylase-oxygenase from Rhodospirillum rubrum

The quaternary structure of ribulose-1,5-bisphosphate carboxylase-oxygenase (rubisco) from Rhodospirillum rubrum, an enzyme consisting of two large subunits, L₂, was investigated by small-angle X-ray scattering. In the presence of HCO ₃ ^- and Mg²⁺, rubisco is in the active state and displays a radius of gyration of 2.96 nm, a maximum diameter of 9.5 nm and a volume of 170 nm³. A model is presented where the subunits are arranged back-to-back, rotated relative to each other by 90°, and shifted by 1.3 nm. Upon inactivation by removal of HCO ₃ ^- and Mg²⁺, the model swells slightly without any distinct changes in configuration. This contrasts with our previous observations with rubisco from Alcaligenes eutrophus, an enzyme composed of small (S) and large (L) subunits, L₈S₈, where inactivation gives rise to substantial changes in configuration.Abbreviations RuBP Ribulose-1,5-bisphosphate - 3-PGA 3-phosphoglyceric acid