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

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 M 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 M 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.Abbreviations APX ascorbate peroxidase - chl chlorophyll - cyt cytochrome - HP high potential - LP low potential - MDA monodehydroascorbate - PQ plastoquinone - PS I and PS II Photosystems I and II