title: Heterologous production and characterisation of two distinct dihaem-containing membrane integral cytochrome b(561) enzymes from Arabidopsis thaliana in Pichia pastoris and Escherichia coli cells.
abstract: Cytochrome (cyt) b(561) proteins are dihaem-containing membrane proteins, belonging to the CYBASC (cytochrome-b(561)-ascorbate-reducible) family, and are proposed to be involved in ascorbate recycling and/or the facilitation of iron absorption. Here, we present the heterologous production of two cyt b(561) paralogs from Arabidopsis thaliana (Acytb(561)-A, Acytb(561)-B) in Escherichia coli and Pichia pastoris, their purification, and initial characterisation. Spectra indicated that Acytb(561)-A resembles the best characterised member of the CYBASC family, the cytochrome b(561) from adrenomedullary chromaffin vesicles, and that Acytb(561)-B is atypical compared to other CYBASC proteins. Haem oxidation-reduction midpoint potential (E(M)) values were found to be fully consistent with ascorbate oxidation activities and Fe(3+)-chelates reductase activities. The ascorbate dependent reduction and protein stability of both paralogs were found to be sensitive to alkaline pH values as reported for the cytochrome b(561) from chromaffin vesicles. For both paralogs, ascorbate-dependent reduction was inhibited and the low-potential haem E(M) values were affected significantly by incubation with diethyl pyrocarbonate (DEPC) in the absence of ascorbate. Modification with DEPC in the presence of ascorbate left the haem E(M) values unaltered compared to the unmodified proteins. However, ascorbate reduction was inhibited. We concluded that the ascorbate-binding site is located near the low-potential haem with the Fe(3+)-chelates reduction-site close to the high-potential haem. Furthermore, inhibition of ascorbate oxidation by DEPC treatment occurs not only by lowering the haem E(M) values but also by an additional modification affecting ascorbate binding and/or electron transfer. Analytical gel filtration experiments suggest that both cyt b(561) paralogs exist as homodimers.
authors.affiliation: Max Planck Institute of Biophysics, Department of Molecular Membrane Biology, Frankfurt, Germany.
authors.forename: Lucia
authors.initials: L
authors.lastname: Cenacchi
authors.forename: Manuela
authors.initials: M
authors.lastname: Busch
authors.forename: Philipp G
authors.initials: PG
authors.lastname: Schleidt
authors.forename: Florian G
authors.initials: FG
authors.lastname: Müller
authors.forename: Tina V M
authors.initials: TV
authors.lastname: Stumpp
authors.forename: Werner
authors.initials: W
authors.lastname: Mäntele
authors.forename: Paolo
authors.initials: P
authors.lastname: Trost
authors.forename: C Roy D
authors.initials: CR
authors.lastname: Lancaster
mesh_terms: D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D029681:Arabidopsis Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D001205:Ascorbic Acid / Q000737:chemistry; D003573:Cytochrome b Group / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D004047:Diethyl Pyrocarbonate / Q000737:chemistry; D004579:Electron Transport; D004926:Escherichia coli; D005656:Fungal Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D015870:Gene Expression; D006418:Heme / Q000737:chemistry; D010084:Oxidation-Reduction; D010843:Pichia / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics
chemical_list: D029681:Arabidopsis Proteins; D003573:Cytochrome b Group; D005656:Fungal Proteins; D011994:Recombinant Proteins; C023646:cytochrome b561; D006418:Heme; D004047:Diethyl Pyrocarbonate; D001205:Ascorbic Acid