Isolation of 34 kDa protein from spinach chloroplasts having ferrooxidase and H2O2-dependent dark O2 evolution activities.
A latent form of 'Ferrooxidase' exhibiting ferrocyanide-dependent O2 uptake was detected in the isolated spinach chloroplasts. Presence of a cationic detergent hexadecyl trimethyl ammonium bromide (CTAB) in the medium was essential to induce this activity. The association of this enzyme activity with photosystem II (PSII) particles as well as the ability of PSII particles to show oxidation of H2O2 (catalase like activity) indicated its possible relationship with water oxidation system. The protein catalysing this activity was purified to homogeneity and its molecular mass was found to be 34 kDa. The purified protein showed a complete dependence on an electron acceptor, namely ferricyanide, for the oxidation of H2O2. While with ferrocyanide in the presence of CTAB, the protein exhibits the ferrooxidase activity. For both activities, a sharp pH optima at 6.1 was observed. The km for H2O2 was 12.2 mM. The purified enzyme protein contained 4 atoms of calcium and 2 atoms of iron per mole of the enzyme. Unlike catalase, the enzyme reaction was insensitive to sodium azide even at 500 microM concentration. The enzyme was found to be sensitive to metal chelators like ethylene-glycol-bis-(beta-aminoethylether) N, N+ tetra acetic acid (EGTA) (2mM), alpha,alpha-dipyridyl (500 microM) and 1,10-orthophenanthroline (200 microM). The sensitivity of the reaction to alpha,alpha-dipyridyl and 1,10-orthophenanthroline suggested the involvement of Fe2+ in the reaction. Inhibition of enzyme activity by EGTA and restoration of activity by supplementation of CaCl2 to the EGTA-dialysed sample confirmed the absolute requirement for calcium for this activity. Calcium was absent in the EGTA-dialysed enzyme. Apart from these inhibitors, NaF and NH2OH were potent inhibitors of the enzyme reaction.(ABSTRACT TRUNCATED AT 250 WORDS)
Nair
P M
PM
Food Technology and Enzyme Engineering Division, Bhabha Atomic Research Centre, Bomaby.
Ramaswamy
N K
NK
D001386:Azides / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002570:Ceruloplasmin / Q000302:isolation & purification / Q000378:metabolism*; D002736:Chloroplasts / Q000201:enzymology*; D003624:Darkness; D004591:Electrophoresis, Polyacrylamide Gel; D005292:Ferricyanides / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000378:metabolism*; D007501:Iron / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D045322:Photosynthetic Reaction Center Complex Proteins / Q000187:drug effects / Q000378:metabolism*; D045332:Photosystem II Protein Complex; D010944:Plants; D019810:Sodium Azide; D012969:Sodium Fluoride / Q000494:pharmacology; D013056:Spectrophotometry, Ultraviolet
D001386:Azides; D005292:Ferricyanides; D045322:Photosynthetic Reaction Center Complex Proteins; D045332:Photosystem II Protein Complex; C007931:hexacyanoferrate III; D012969:Sodium Fluoride; D019810:Sodium Azide; D006861:Hydrogen Peroxide; D007501:Iron; D002570:Ceruloplasmin; D010100:Oxygen; D002118:Calcium