Ferredoxin-Dependent Photoreduction of the Monodehydroascorbate Radical in Spinach Thylakoids

Abstract

Thylakoid-bound and stromal ascorbate peroxidases scavenge the hydrogen peroxide that is photoproduced in PSI of chloroplast thylakoids. The primary oxidation product of ascorbate in the reaction catalyzed by ascorbate peroxidase, the monodehydroascorbate (MDA) radical, is photoreduced by thylakoids [Miyake and Asada (1992) Plant Cell Physiol. 33: 541]. We have now shown that the photoreduction of MDA radical in spinach thylakoids is largely dependent on ferredoxin (Fd), as determined by the monitoring the MDA radical by electron paramagnetic resonance. Further, the reduced Fd generated by NADPH and Fd-NADP reductase could reduce the MDA radical at a rate of over 10⁶ M⁻¹ s⁻¹, indicating that the photoreduced Fd in PSI directly reduces the MDA radical to ascorbate. Photoreduction of NADP⁺ by spinach thylakoids was suppressed by the MDA radical and conversely that of MDA radical was suppressed by NADP⁺, indicating a competition between the MDA radical and NADP⁺ for the photoreduced Fd in PSI. The ratio of the rate constant for the photoreduction of MDA radical to that for the photoreduction of NADP⁺ was estimated to be more than 30 to 1. Thus, MDA radical is preferentially photoreduced as compared to NADP⁺. From these results, we propose that the thylakoid-bound ascorbate peroxidase and the Fd-dependent photoreduction of MDA radical in PSI are the primary system for the scavenging of the hydrogen peroxide that is photoproduced in the thylakoids.