Two membrane-bound, ascorbate-dependent b-type cytochromes were identified in etiolated bean (Phaseolus vulgaris L.) hypocotyls. Following solubilization of microsomal membranes and anion-exchange chromatography at pH 8.0, two major cytochrome peaks (P-I and P-II) were separated. Both cytochromes were reduced by ascorbate and re-oxidized by monodehydroascorbate, but P-I reduction by ascorbate was higher and saturated at far lower concentrations of ascorbate with respect to P-II. The alpha-band was symmetrically centered at 561 nm in P-I, but it was asymmetric in P-II with a maximum at 562 nm and shoulder at 557 nm. Ascorbate reduction of P-II, but not P-I, was inhibited by diethyl pyrocarbonate. Reduced P-II but not P-I was readily oxidized by certain ferric chelates, including FeEDTA and Fe-nitrilotriacetic acid. Purified P-I, associated with the plasma membrane, showed up as a 63-kDa glycosylated protein during sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and behaved as a monomer of about 70 kDa during size-exclusion chromatography. P-I identified with a previously purified ascorbate-dependent b-type cytochrome of bean hypocotyl plasma membranes. Partially purified P-II, on the other hand, correlated with a heme-protein of 27 kDa in SDS-PAGE gels, was dimeric (60 kDa) during size-exclusion chromatography, and was associated with the tonoplast marker V-ATPase in sucrose gradients. The sequence of a peptide of 11 residues obtained by tryptic digestion of P-II was found to be identical to a segment of a putative cytochrome b561 of Zea mays and highly conserved in other related plant sequences, including that of Arabidopsis thaliana cytochrome b561-1 (CAA18169). The biochemical features fully support the assignment of P-II cytochrome to the family of cytochrome b561, ascorbate-dependent (CYBASC) cytochromes, which also includes cytochrome b561 of animal chromaffin granules. The presence of a cytochrome reducing ferric chelates on the tonoplast is consistent with the role of plant vacuoles in iron homeostasis.

We report here on the identification of the major plasma membrane (PM) ascorbate-reducible b-type cytochrome of bean (Phaseolus vulgaris) and soybean (Glycine max) hypocotyls as orthologs of Arabidopsis (Arabidopsis thaliana) AIR12 (for auxin induced in root cultures). Soybean AIR12, which is glycosylated and glycosylphosphatidylinositol-anchored to the external side of the PM in vivo, was expressed in Pichia pastoris in a recombinant form, lacking the glycosylphosphatidylinositol modification signal and purified from the culture medium. Recombinant AIR12 is a soluble protein predicted to fold into a b-sandwich domain and belonging to the DOMON (for dopamine b-monooxygenase N terminus) domain superfamily. It is shown to be a b-type cytochrome with a symmetrical a-band at 561 nm, fully reduced by ascorbate, and fully oxidized by monodehydroascorbate radical. AIR12 is a high-potential cytochrome b showing a wide bimodal dependence from the redox potential between +80 mV and +300 mV. Optical absorption and electron paramagnetic resonance analysis indicate that AIR12 binds a single, highly axial low-spin heme, likely coordinated by methionine-91 and histidine-76, which are strongly conserved in AIR12 sequences. Phylogenetic analyses reveal that the auxin-responsive genes AIR12 represent a new family of PM b-type cytochromes specific to flowering plants. Circumstantial evidence suggests that AIR12 may interact with other redox partners within the PM to constitute a redox link between cytoplasm and apoplast.

Quinone oxidoreductase activities dependent on pyridine nucleotides are associated with the plasma membrane (PM) i n ZUC-chini (Cucurbifa pepo L.) hypocotyls. I n the presence of NADPH, lipophilic ubiquinone homologs with up t o three isoprenoid units were reduced by intact PM vesicles with a K,,, of 2 t o 7 p M. Affinities for both NADPH and N A D H were similar (K, of 62 and 51 p ~ , respectively). Two NAD(P)H:quinone oxidoreductase forms were identified. The first, labeled as peak I i n gel-filtration experiments, behaves as an intrinsic membrane complex of about 300 kD, it slightly prefers N A D H over NADPH, it is markedly sensitive t o the inhibitor diphenylene iodonium, and it is active with lipophilic quinones. The second form (peak II) is an NADPH-preferring oxidoreductase of about 90 kD, weakly bound t o the PM. Peak II is diphenylene iodonium-insensitive and resembles, in many properties, the soluble NAD(P)H:quinone oxidoreductase that is also present i n the same tissue. Following purification of peak I, however, the latter gave rise t o a quinone oxidoreductase of the soluble type (peak II), based o n substrate and inhibitor specificities and chromatographic and electrophoretic evidence. It is proposed that a redox protein of the same class as the soluble NAD(P)H:quinone oxidoreductase (F. Sparla, C. Tedeschi, and P. Trost 11 9961 Plant Physiol. 1 1 2:249-258) is a component of the diphenylene iodonium-sensitive PM complex capable of reducing lipophilic quinones. ~ ~ Plant cells appear to contain severa1 types of NAD(P)H-dependent quinone oxidoreductases in addition to those of energy-conserving reactions of plastids and mitochondria. The purification of some of these plant proteins has been accomplished, but their biochemical characterization is still at an initial stage. Recently, NAD(P)H-QR from plant tissues was purified and characterized (Rescigno et al., 1995; Trost et al., 1995; Sparla et al., 1996). This plant oxidoreduc-tase represents a functional equivalent of animal DT-diaphorase, since it reduces short-chain quinones to quin-01s by two-electron donation without semiquinone intermediates, thereby resulting in enhanced quinone conjugation and low probability of formation of active oxygen species (Trost et al., 1995). However, a number of properties are different from animal-type DT-diaphorase, i.e. the NAD(P)H-QR contains FMN, it has a mass of about 90 kD with subunits of 21.4 kD (by MS), and the hydride transfer

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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.

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