Characterisation of a H2O2-oxidisable cytochrome b-559 in intact chloroplasts with a new type of LED Array Spectrophotometer

Abstract

Cytochrome (cyt) b-559 absorbance changes in intact chloroplasts were deconvoluted using a previously described LED-Array-Spectrophotometer (Klughammer et al. (1990), Photosynth Res 25: 317-327). When intact chloroplasts were isolated in the presence of ascorbate, approx. 15% of the total cyt b-559 could be transiently oxidised by 200 μM H2O2 in the dark. This fraction displays low-potential properties, as it can be also oxidised by menadione in the presence of 5 mM ascorbate. Heat pretreatment increased the size of this fraction by a factor of 3-4. Low concentrations of cyanide (in the μM range) prolonged the oxidation time while high concentrations suppressed the oxidation (I50=1.5 mM KCN). The former KCN-effect relates to inhibition of ascorbate dependent H2O2-reduction which is catalysed by ascorbate peroxidase, whereas the latter effect reflects competition between H2O2 and CN(-) for the same binding site at the cytochrome heme. In the light, much lower concentrations of H2O2 were required to obtain oxidation, the amplitude depending on light intensity and on the concentration of the added H2O2, but never exceeding approx. 15% of the total cyt b-559. In the light, but not in the dark, H2O2 also induced the transient oxidation of a cyt f fraction similar in size to the H2O2-oxidisable cyt b-559 fraction. In this case, H2O2 serves as an acceptor of Photosystem I in conjunction with the ascorbate peroxidase detoxification system. Light can also induce oxidation of a 15% cyt b-559 fraction without H2O2-addition, if nitrite is present as electron acceptor and the chloroplasts are depleted of ascorbate. It is concluded that light-induced cyt b-559 oxidation in vivo is likely to be restricted to the H2O2-oxidisable cyt b-559 LP fraction and is normally counteracted by ascorbate.