Partial purification and some properties of a latent CO 2 reductase from green potato tuber chloroplasts

We have partially purified the CO 2 reductase, present in green potato tuber chloroplasts, as a latent form. Illumination of the chloroplasts in the absence of substrate, bicarbonate, activated the enzyme, which could then be obtained in soluble forms. Purification of the enzyme was achieved by (NH 4 ) 2 SO 4 fractionation (0–30%) and adsorption and elution from a DEAE‐Sephadex A‐50 column. The final preparation showed 15‐fold purification and 50% recovery of the activity. The pH optimum for CO 2 reductase was 8.0. Hepes and Tricine buffers showed maximum activity whereas Tris/phosphate or borate failed to show any activity. The enzyme reaction was sensitive to the presence of metal ions like Fe 3+ , Hg 2+ , Cu 2+ , Mo 6+ and Zn 2+, however, a threefold activation was observed with Fe2+ . The metal requirement for CO 2 reductase was evident from the observed inhibition by metal chelators like o ‐phenanthroline α,α′‐dipyridyl, bathocuproine, 8‐hydroxyquinoline etc. Out of these o ‐phenanthroline was the strongest inhibitor and its concentration for 50% inhibition was 40 μM. The presence of Fe 2+ ions in the reaction mixture protected the enzyme from heat denaturation upto 50°C. Maximum enzyme activity was observed at 15°C. The enzyme activity showed a 30‐s lag period and the maximum was reached in 90s. Supplementation of sodium dithionite in the reaction activated enzyme threefold, suggesting involvement of dithiol groups in the catalytic activity. There was strong inhibition by – SH inhibitors like 5,5′‐dithiobis(2‐nitrobenzoic acid) and N ‐ethylmaleimide and – SH reagents like dithiothreitol, 2‐mercaptoehanol and cysteine. Various nucleotide coenzyme tried inhibited the enzyme strongly.