Cecytb-2, a Cytochrome b561 Homolog, Functions as an Ascorbate-Specific Transmembrane Ferric Reductase at Intestinal Lumens of Caenorhabditis elegans

One of the cytochrome b561 family members in C. elegans, named Cecytb-2, was investigated. Purified recombinant Cecytb-2 showed typical visible absorption spectra, EPR signals, and redox midpoint potentials, very similar to those of human Dcytb, which is responsible for intestinal iron acquisition by its ferric reductase activity. Fast kinetic experiments using pulse radiolysis and stopped-flow techniques showed that Cecytb-2 donates electrons to monodehydroascorbate radicals with a much lower reactivity than other cytochrome b561 members, but it can accept electrons from ascorbate (AsA) as rapidly as other members. DEPC treatment of Cecytb-2 caused significant inhibition of electron acceptance from AsA and lowered the midpoint potential of heme bL. MS/MS MASCOT analyses verified that N-carbethoxylations of conserved Lys98 and heme bL axial His101 residues on the cytosolic side were major causes of the inhibition. Reconstituted Cecytb-2 in sealed vesicle membranes, in which AsA was entrapped, showed significant transmembrane ferric reductase activity. In situ hybridization analysis revealed that Cecytb-2 mRNA was distributed in intestinal cells. Immunohistochemical analysis indicated that Cecytb-2 resided in intestinal lumens. Knockdown of the Cecytb-2 gene expression in N2 worms indicated a significant suppression of growth under ferrous ion-deficient conditions. Thus, the ferric reductase activity conferred by Cecytb-2 seems to participate in iron acquisition and is very important for normal growth in low-ferrous conditions, confirming that Cecytb-2 is a genuine Dcytb homolog in C. elegans.