Purification of cytochrome b-561 from bean hypocotyls plasma membrane. Evidence for the presence of two heme centers

Trost, Paolo; Bérczi, Alajos; Sparla, Francesca; Sponza, G.; Marzadori, Beatrice; Asard, Han; Pupillo, Paolo

doi:10.1016/s0005-2736(00)00283-2

Cited by 30 publications

(13 citation statements)

References 25 publications

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“…RecAIR12 sensitivity to the redox potential, with two apparent midpoint redox potentials at +137 and +236 mV, is in line with that of partially purified PM cytochrome b from bean hypocotyls (+135 and +204 mV; Trost et al, 2000) and Arabidopsis leaves (+135 and +180 mV; Bérczi et al, 2003), and consistent with redox titrations of whole PM preparations (Asard et al, 1989;Askerlund and Larsson, 1991;Bérczi et al, 2003). This also indicates that AIR12 represents a major fraction of high potential and ascorbate-reducible cytochromes b of plant PM.…”

Section: Discussionsupporting

confidence: 74%

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Auxin-Responsive Genes AIR12 Code for a New Family of Plasma Membrane b-Type Cytochromes Specific to Flowering Plants

et al. 2009

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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.

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Section: Discussionsupporting

confidence: 74%

“…An ascorbate-reducible cytochrome b from enriched PM preparations was purified as a glycosylated protein of 55 to 63 kD (bean hypocotyls; Trost et al, 2000) or 120 kD (Arabidopsis; Bérczi et al, 2003) in SDS-PAGE. The association to the PM of the bean hypocotyl cytochrome was confirmed by analytical Suc gradient centrifugation (Preger et al, 2005).…”

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Auxin-Responsive Genes AIR12 Code for a New Family of Plasma Membrane b-Type Cytochromes Specific to Flowering Plants

et al. 2009

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“…mETC, mitochondrial electron transfer chain; pETC, photosynthetic electron transfer chain. monodehydroascorbate binding (Trost et al, 2000;Takeuchi et al, 2004;Su and Asard, 2006). The C. reinhardtii proteins retain the conserved residues, making them likely to be true ferrireductases (see Supplemental Figures 4 and 5 online).…”

Section: Iron Assimilation: Transporters and Ferrireductasesmentioning

confidence: 99%

Systems and Trans-System Level Analysis Identifies Conserved Iron Deficiency Responses in the Plant Lineage

et al. 2012

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“…Golgi apparatus, is not ruled out by current data. The PM-associated b-type cytochrome corresponds to the ascorbate-reducible, highpotential cyt b561 of PM vesicles purified by Trost et al (2000), labeled fraction P-I in the present protocol. Upon SDS-PAGE, an apparent molecular weight of 55 kDa has been reported for this cytochrome by Trost et al (2000).…”

Section: Discussionmentioning

confidence: 99%

Identification of an ascorbate-dependent cytochrome�b of the tonoplast membrane sharing biochemical features with members of the cytochrome�b561 family

Preger

Scagliarini

Pupillo

et al. 2004

Planta

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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.

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Purification of cytochrome b-561 from bean hypocotyls plasma membrane. Evidence for the presence of two heme centers

Cited by 30 publications

References 25 publications

Auxin-Responsive Genes AIR12 Code for a New Family of Plasma Membrane b-Type Cytochromes Specific to Flowering Plants

Auxin-Responsive Genes AIR12 Code for a New Family of Plasma Membrane b-Type Cytochromes Specific to Flowering Plants

Systems and Trans-System Level Analysis Identifies Conserved Iron Deficiency Responses in the Plant Lineage

Identification of an ascorbate-dependent cytochrome�b of the tonoplast membrane sharing biochemical features with members of the cytochrome�b561 family

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