Ascorbate-independent electron transfer between cytochrome b561 and a 27 kDa ascorbate peroxidase of bean hypocotyls

Cytochrome b561 (cyt b561) is a trans-membrane cytochrome probably ubiquitous in plant cells. In vitro, it is readily reduced by ascorbate or by juglonol, which in plasma membrane (PM) preparations from plant tissues is efficiently produced by a PM-associated NAD(P)H:quinone reductase activity. In bean hypocotyl PM, juglonol-reduced cyt b561 was not oxidized by hydrogen peroxide alone, but hydrogen peroxide led to complete oxidation of the cytochrome in the presence of a peroxidase found in apoplastic extracts of bean hypocotyls. This peroxidase active on cyt b561 was purified from the apoplastic extract and identified as an ascorbate peroxidase of the cytosolic type. The identification was based on several grounds, including the ascorbate peroxidase activity (albeit labile), the apparent molecular mass of the subunit of 27 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the dimeric native structure, the typical spectral properties of a heme-containing peroxidase, and an N-terminal sequence strongly conserved with cytosolic ascorbate peroxidases of plants. Cyt b561 used in the experiments was purified from bean hypocotyl PM and juglonol was enzymatically produced by recombinant NAD(P)H:quinone reductase. It is shown that NADPH, NAD(P)H:quinone reductase, juglone, cyt b561, the peroxidase interacting with cyt b561, and H2O2, in this order, constitute an artificial electron transfer chain in which cyt b561 is indirectly reduced by NADPH and indirectly oxidized by H2O2.