Ascorbate-independent electron transfer between cytochrome b(561) and a 27kDa ascorbate peroxidase of bean hypocotyls

Cytochrome b(561) (cyt b(561)) is a trans-membrane cytochrome probably ubiq uitous in plant cells. In vitro, it is readily reduced by ascorbate or by j uglonol, which in plasma membrane (PM) preparations from plant tissues is e fficiently produced by a PM-associated NAD(P)H:quinone reductase activity. In bean hypocotyl PM, juglonol-reduced cyt b(561) was not oxidized by hydro gen 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 b(561) was purified from the apoplastic extract and identified as an ascorbate peroxidase of the cytoso lic type. The identification was based on several grounds, including the as corbate peroxidase activity (albeit labile), the apparent molecular mass of the subunit of 27 kDa by sodium dodecyl sulfate-polyacrylamide gel electro phoresis, 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 b(561) used in the exp eriments 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 b(561), the peroxidase interacting with cvt b(561), and H2O2, in this order, constitute an artificial electro n transfer chain in which cyt b(561) is indirectly reduced by NADPH and ind irectly oxidized by H2O2.