Purification and catalytic mechanism of chloroplast dehydroascorbate reductase from spinach leaves

Abstract

Chloroplasts require a high level activity to regenerate ascorbate during photosynthesis to scavenge the active oxygen species produced in photosystem I. One of such enzyme is dehydroascorbate reductase (DHAR). Spinach chloroplast DHAR has been proposed to be trypsin inhibitor. However, its low specific activity cannot explain the high DHAR activity in spinach chloroplasts. In this study, we purified a novel spinach chloroplast DHAR. The Km value for dehydroascorbate and GSH were 70 µM and 1.1 mM, respectively. The specific activity was 360 µmol/min/mg protein. These results suggest that this enzyme can function as DHAR in chloroplasts. The steady-state kinetic studies with recombinant spinach chloroplast DHAR revealed that the catalytic mechanism of spinach chloroplast DHAR was a bi uni uni uni ping pong mechanism. We identified the intermediate complex, in which the enzyme and GSH bound with disulfide bond. We identified the cysteine residue involved in the formation of the intermediate complex with the site-directed mutagenesis study. Spinach chloroplast DHAR has three cysteine residues (Cys-9, Cys-23 and Cys-26). The DHAR activity of C23S DHAR, in which Cys-23 of wild DHAR was changed to serine, was not detected. C9S, C26S and C9S/C26S DHARs, in which Cys-9, Cys-26 and both of Cys-9 and Cys-26 of wild DHAR were replaced to serine residues, respectively, had considerable activities of DHAR. These results indicate that Cys-23 is essential for DHAR to reduce DHA to ascorbate. Cys-23 would be involved in the formation of intermediate complex.