Open Access

Lucia Cenacchi

• Cytochrome b561 (cyt b561) proteins are members of the recently identified eukaryotic ascorbate reducible protein family named CYBASC (CYtochrome B, ASCorbate reducible). CYBASC proteins are di-heme-b-containing membrane proteins that catalyze the transmembrane electron transfer from ascorbate. The function of the CYBASC proteins has been correlated with ascorbate recycling and/or iron facilitation uptake. Therefore, investigations on this family are of great interest as ascorbate is one of the most powerful antioxidants and iron is essential for cell survival both in animals and plants. As the amino acid sequence conservation of animal and plant CYBASC proteins is relatively high, all CYBASC members are proposed to share the same structural motifs. However, no three-dimensional structure of any representative member of the CYBASC family has been determined to date. In the Arabidopsis thaliana (A. thaliana) genome, two complete putative CYBASC open reading frames (ORFs), artb561-a and artb561-b were identified. In this thesis, these two A. thaliana CYBASC ORFs, encoding for Acytb561-A and Acytb561-B proteins respectively, were investigated and obtained main results are listed. 1. A. thaliana CYBASC proteins were heterologously produced in Pichia pastoris and Escherichia coli and purified by a single-step immobilized metal affinity chromatography (IMAC). To facilitate detection and purification, the recombinant A. thaliana CYBASC proteins were produced in both expression systems with the histidine affinity tag. Pure and stable preparations of the cytochromes were obtained via a single-step IMAC in sufficient amounts to perform biochemical characterizations. 2. Detergent solubilized recombinant Acytb561-A and Acytb561-B are dimers. As previously suggested for other CYBASC proteins, analytical gel filtration experiment suggested that both detergent solubilized cytochromes are dimers. 3. Spectroscopic features of Acytb561-B differed from those of previously described bovine chromaffin granule cyt b561. A distinctive feature of the first identified CYBASC protein, the cyt b561 from bovine chromaffin vesicles of adrenal medulla (Bcytb561-CG), is that its differential visible absorbance spectra (visible-spectra) revealed an asymmetric α-band with a maximum at 562 nm and a clear shoulder at 557 nm. This feature was recently used to discriminate CYBASC proteins from not-CYBASC proteins. However, in this thesis, it is shown for the first time that not all CYBASC proteins display in their reduced-minus-oxidized visible-spectra an asymmetric α- band and therefore, this feature can not be used as a discriminating CYBASC characteristic. 4. Ascorbate dependent reduction of the A. thaliana CYBASC proteins is inhibited by diethylpyrocarbonate (DEPC). As previously reported for the Bcytb561-CG, the ascorbatedependent reduction of the A. thaliana CYBASC proteins was inhibited by DEPC treatment. In addition, the ‘ascorbate protectant’ effect against DEPC that was observed on the Bcytb561-CG was also observed on the Acytb561-A and Acytb561-B proteins. Furthermore, as the physiological electron donor of all CYBASC proteins is supposed to be ascorbate, ascorbate-affinity of Acytb561- A and Acytb561-B was monitored and was found to be in the same range of the one of the Bcytb561- CG. 5. A. thaliana CYBASC proteins are Fe3+-chelate reductases. Recently, the Fe3+-chelate reductase activity of various CYBASC proteins was presented. In this thesis, it is shown that also both A. thaliana CYBASC proteins reduced Fe3+-chelates such as Fe3+-EDTA and Fe3+-citrate. Consistently, heme potentiometric reductive-oxidative titration of purified Acytb561-A and Acytb561-B indicated that the midpoint potential of the two heme centres of both cytochromes was lower than the one of those Fe3+-chelates. The values of both heme centre potentials of Acytb561-A and Acytb561-B are also consistent with the observation that both cytochromes were only partially reducible by ascorbate and were fully reduced with the non-physiological reductant Na-dithionite. In summary, this work describes the heterologous production, purification and initial characterizations of two distinct CYBASC proteins from A. thaliana: Acytb561-A and Acytb561-B. Biochemical characterization of these cytochromes showed that the shape of the α-band in the differential spectra is not a discriminating factor for CYBASC proteins but it is likely the DEPC sensitivity and the Fe3+-chelate reductase activity. Establishment of a purification strategy to obtain sufficient amounts of monodispersed and stable A. thaliana CYBASC proteins has also enabled initial screening of three dimensional crystallization conditions which are a prerequisite for a deeper understanding of this new eukaryotic redox enzyme family.
• In den letzten Jahren hat sich herausgestellt, dass die Cyt b561 Proteine eine neue Ascorbinsäurereduzierende eukaryotische Proteinfamilie, CYBASC (CYtochrome B, ASCorbate reducible), darstellen. CYBASC Proteine sind Di-häm-b-enthaltende Membranproteine, die die Elektronenübertragung von Ascorbinsäure (Elektronendonor) zu einem Elektronenakzeptor auf der gegenüberliegenden Seite der Membran, wie z.B. MonodehydroAscorbinsäure (MDHA) oder Fe3+-Chelate, katalysieren. In Pflanzen und Tieren wurde die Funktion der CYBASC Proteine mit der Regenerierung von Ascorbinsäure in Verbindung gebracht. Das ist von besonderem Interesse, da die Ascorbinsäure eines der stärksten Antioxidantien in Tieren und Pflanzen ist und ihre Beteiligung in verschiedenen wichtigen Stoffwechselwegen, wie z.B. die Hormonbiosynthese und die Immunsystem-Aktivierung, nachgewiesen werden konnte. Zusätzlich reduzieren CYBASC Proteine Fe3+-Chelate und man glaubt deshalb, dass diese die Eisenaufnahme erleichtern. Eisen ist unverzichtbar für eine große Anzahl von biologischen Prozessen wie dem O2-Transport, der DNA-Synthese und dem Elektronentransport. Deshalb ist die Eisenaufnahme für fast alle Lebewesen essenziell. Die CYBASC Proteine von Tieren und Pflanzen haben eine ziemlich hohe Sequenzhomologie. Die Konservierung der Aminosäuresequenz innerhalb der verschiedenen Cytochrome zeigt, dass diese alle die gleichen Strukturmotive teilen, z.B. sechs transmembranäre α-Helizes, vier konservierte Histidine, vermutlich beteiligt an der Koordination der zwei Häm-b Zentren und die Bindestellen für Ascorbinsäure und MDHA. Bis heute wurde noch keine dreidimensionale (3-D) Struktur von einem CYBASC Mitglied bestimmt. Im Genom von Arabidopsis thaliana (A. thaliana) konnten zwei komplette offene CYBASC Leseraster (ORFs) identifiziert werden, artb561-a und artb561-b (Zugangsnummer NM_118689 respektive AB049627). Das Ziel dieses Projektes war die heterologe Expression dieser zwei möglichen A. thaliana CYBASC ORFs und die Reinigung der zwei rekombinanten Acytb561-A und Acytb561-B Proteine zur biochemischen Charakterisierung und auch für 3-D Kristallisationsstudien als Vertretern dieser Familie von eukaryotischen Proteinen. Folgende wesentliche Ergebnisse konnten erhalten werden: ...