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Free keywords:
Arabidopsis thaliana; Biochemical characterization; Cytochrome b561 paralog; Heterologous production; Membrane protein
Abstract:
Cytochrome (cyt) b561 proteins are dihaem-containing membrane proteins, belonging to the CYBASC (cytochrome-b561-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 b561 paralogs from Arabidopsis thaliana (Acytb561-A, Acytb561-B) in Escherichia coli and Pichia pastoris, their purification, and initial characterisation. Spectra indicated that Acytb561-A resembles the best characterised member of the CYBASC family, the cytochrome b561 from adrenomedullary chromaffin vesicles, and that Acytb561-B is atypical compared to other CYBASC proteins. Haem oxidation–reduction midpoint potential (EM) values were found to be fully consistent with ascorbate oxidation activities and Fe3+-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 b561 from chromaffin vesicles. For both paralogs, ascorbate-dependent reduction was inhibited and the low-potential haem EM 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 EM 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 Fe3+-chelates reduction-site close to the high-potential haem. Furthermore, inhibition of ascorbate oxidation by DEPC treatment occurs not only by lowering the haem EM values but also by an additional modification affecting ascorbate binding and/or electron transfer. Analytical gel filtration experiments suggest that both cyt b561 paralogs exist as homodimers.
