Functional Assembly of <i>Caenorhabditis elegans</i> Cytochrome b-2 (Cecytb-2) into Phospholipid Bilayer Nanodisc with Enhanced Iron Reductase Activity
Hamed A. Abosharaf,
Yuki Sakamoto,
Aliaa M. Radwan,
Keisuke Yuzu,
Mika Fujimura,
Thoria Diab,
Tarek M. Mohamed,
Eri Chatani,
Tetsunari Kimura,
Motonari Tsubaki
Affiliations
Hamed A. Abosharaf
Department of Chemistry, Graduate School of Science, Kobe University, Nada-ku, Kobe, Hyogo 657-8501, Japan
Yuki Sakamoto
Department of Chemistry, Graduate School of Science, Kobe University, Nada-ku, Kobe, Hyogo 657-8501, Japan
Aliaa M. Radwan
Department of Chemistry, Graduate School of Science, Kobe University, Nada-ku, Kobe, Hyogo 657-8501, Japan
Keisuke Yuzu
Department of Chemistry, Graduate School of Science, Kobe University, Nada-ku, Kobe, Hyogo 657-8501, Japan
Mika Fujimura
Department of Chemistry, Graduate School of Science, Kobe University, Nada-ku, Kobe, Hyogo 657-8501, Japan
Among seven homologs of cytochrome b561 in a model organism C. elegans, Cecytb-2 was confirmed to be expressed in digestive organs and was considered as a homolog of human Dcytb functioning as a ferric reductase. Cecytb-2 protein was expressed in Pichia pastoris cells, purified, and reconstituted into a phospholipid bilayer nanodisc. The reconstituted Cecytb-2 in nanodisc environments was extremely stable and more reducible with ascorbate than in a detergent-micelle state. We confirmed the ferric reductase activity of Cecytb-2 by analyzing the oxidation of ferrous heme upon addition of ferric substrate under anaerobic conditions, where clear and saturable dependencies on the substrate concentrations following the Michaelis–Menten equation were observed. Further, we confirmed that the ferric substrate was converted to a ferrous state by using a nitroso-PSAP assay. Importantly, we observed that the ferric reductase activity of Cecytb-2 became enhanced in the phospholipid bilayer nanodisc.
