Purification and Biochemical Properties of Phytochromobilin Synthase from Etiolated Oat Seedlings

Plant phytochromes are dependent on the covalent attachment of the linear tetrapyrrole chromophore phytochromobilin (PΦB) for photoactivity. In planta, biliverdin IXα (BV) is reduced by the plastid-localized, ferredoxin (Fd)-dependent enzyme PΦB synthase to yield 3Z-PΦB. Here, we describe the >50,000-fold purification of PΦB synthase from etioplasts from dark-grown oat (Avena sativa L. cv Garry) seedlings using traditional column chromatography and preparative electrophoresis. Thus, PΦB synthase is a very low abundance enzyme with a robust turnover rate. We estimate the turnover rate to be >100 s(−1), which is similar to that of mammalian NAD(P)H-dependent BV reductase. Oat PΦB synthase is a monomer with a subunit mass of 29 kD. However, two distinct charged forms of the enzymes were identified by native isoelectric focusing. The ability of PΦB synthase to reduce BV is dependent on reduced 2Fe-2S Fds. A K(m) for spinach (Spinacea oleracea) Fd was determined to be 3 to 4 μm. PΦB synthase has a high affinity for its bilin substrate, with a sub-micromolar K(m) for BV.