title: Identification of an ascorbate-dependent cytochrome b of the tonoplast membrane sharing biochemical features with members of the cytochrome b561 family.
abstract: 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.
authors.affiliation: Laboratory of Molecular Plant Physiology, Department of Biology, University of Bologna, Via Irnerio 42, 40126 Bologna, Italy. fisveg@alma.unibo.it
authors.forename: Valeria
authors.initials: V
authors.lastname: Preger
authors.forename: Sandra
authors.initials: S
authors.lastname: Scagliarini
authors.forename: Paolo
authors.initials: P
authors.lastname: Pupillo
authors.forename: Paolo
authors.initials: P
authors.lastname: Trost
mesh_terms: D000595:Amino Acid Sequence; D001205:Ascorbic Acid / Q000378:metabolism*; D003573:Cytochrome b Group / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D004047:Diethyl Pyrocarbonate; D004579:Electron Transport; D007425:Intracellular Membranes / Q000201:enzymology*; D008861:Microsomes / Q000201:enzymology; D027805:Phaseolus / Q000201:enzymology*
chemical_list: D003573:Cytochrome b Group; C023646:cytochrome b561; D004047:Diethyl Pyrocarbonate; D001205:Ascorbic Acid