Identification and characterization of multiple rubisco activases in chemoautotrophic bacteria

Abstract

Ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) is responsible for almost all biological CO2 assimilation, but forms inhibited complexes with its substrate ribulose-1,5-bisphosphate (RuBP) and other sugar phosphates. The distantly related AAA+ proteins rubisco activase and CbbX remodel inhibited rubisco complexes to effect inhibitor release in plants and α-proteobacteria, respectively. Here we characterize a third class of rubisco activase in the chemolithoautotroph Acidithiobacillus ferrooxidans. Two sets of isoforms of CbbQ and CbbO form hetero-oligomers that function as specific activases for two structurally diverse rubisco forms. Mutational analysis supports a model wherein the AAA+ protein CbbQ functions as motor and CbbO is a substrate adaptor that binds rubisco via a von Willebrand factor A domain. Understanding the mechanisms employed by nature to overcome rubisco's shortcomings will increase our toolbox for engineering photosynthetic carbon dioxide fixation.

Figure 1. The oligomeric state of A. ferrooxidans rubisco proteins.

(a) Arrangement of the CO₂-fixation operons encoding form I and form II rubisco in A. ferrooxidans. (b) Native PAGE analysis of purified AfLS and AfM complexes compared to hexadecameric Rhodobacter sphaeroides form I (RsLS) and dimeric Rhodospirillum rubrum form II (RrM) rubisco. (c) Determination of native molecular weight of rubisco enzymes by analytical gel filtration. RpM, Rhodopseudomonas palustris form II rubisco (hexameric). Ten microgram of protein was loaded per experiment. (d) Gel-filtration analysis of AfM at varying concentrations in the apo form (E) and in the CABP bound form (ECMC).

Figure 2. Analysis of the CbbQO hetero-oligomers.

(a) Gel-filtration analysis of the recombinant, purified CbbQ and CbbQO complexes. Ten microgram of protein was loaded per experiment. (b,c) Characteristic (b) and selected (c) unbiased 2D class averages of AfQ2 (b) and Q2O2ΔC444 (c) incubated with 5 mM Mg-ATP. Scale bar, 100 Å. (d) SDS–PAGE analysis of Q1O1, Q2O2 and Q2O2ΔC444 (4 μg protein loaded per lane).

Figure 3. The CbbQO complexes function as rubisco activases.

(a) The relationship between rubisco complexes used in this study. The apoenzyme E needs to bind CO₂ and Mg²⁺ cofactors to form the active ECM. Both E and ECM can be inactivated by binding to RuBP (forming ER) or CABP (forming ECMC), respectively. Rubisco activases release inhibitors favouring ECM formation. (b–e) The CbbQO complexes activate their respective rubisco enzymes. Rubisco activity assays of activated (ECM) and inactivated (ER and ECMC) AfLS (0.3 μM active sites, 20 mM NaHCO₃) (b,c) or AfM (0.1 μM active sites, 5 mM NaHCO₃) complexes (d,e) in the absence and presence of Q1O1 (b,c) or Q2O2 (d,e) (0. 27 μM oligomer). PEG indicates addition of 5% v/v polyethylene glycol 3350. Error bars indicate the mean and s.d. of at least three independent experiments.

Figure 4. Inhibited rubisco complexes stimulate CbbQO ATPase activity.

(a,b) ATPase activity assays of Q1O1 (a) and Q2O2 (b) (0.27 μM oligomer) in the presence of varying concentrations of inhibited AfLS (a) or AfM (b) complexes. (c) The ATPase stimulation is isoform specific. ATPase activity of 0.27 μM QO complex was measured in the presence (coloured bars) and absence (black bars) of the indicated rubisco complex (3 μM active sites). Error bars indicate the mean and s.d. of at least three independent experiments.

Figure 5. The mechanism of CbbQO mediated rubisco activation is conserved.

(a–f) Identification of residues in Q2O2 and both forms of rubisco that affect activase function and the stimulation of ATPase activity. ATPase activity assays of the indicated QO complexes (0.27 μM oligomer) in the presence and absence of active and inactive rubisco complexes (3 μM active sites) (a,c,e) and normalized rubisco activase activity (b,d,f) carried out using mutated Q2O2 (a,b), AfLS (c,d) and AfM (e,f). Protein concentrations used for activase assays: QO complexes, 0.27 μM oligomer; AfLS, 0.3 μM active sites; AfM, 0.1 μM active sites. Activase assays were performed using ECMC complexes. Error bars indicate the mean and s.d. of at least three independent experiments.

Figure 6. Proposed model of CbbQO function visualized using form I rubisco.

CbbQO binds to inhibited rubisco complexes via the acidic surface residue (Asp 82, coloured yellow) of the rubisco large subunit using the MIDAS binding site located on CbbO. The AAA+ hexamer formed by CbbQ may also interact with rubisco directly, possibly via the C terminus of the large subunit (indicated by yellow dashed double arrow). Productive binding, which involves both Asp 82 and the C terminus, results in a stimulation of ATPase activity. This provides the energy used to remodel the inhibited rubisco active site allowing release of the inhibitor. The CbbQO model is not drawn to scale. The form I rubisco model is from Halothiobacillus neapolitanus (pdb:1SVD).