Structure and mechanism of a eukaryotic transmembrane ascorbate-dependent oxidoreductase

Abstract

Vitamin C, also known as ascorbate, is required in numerous essential metabolic reactions in eukaryotes. The eukaryotic ascorbate-dependent oxidoreductase cytochrome b561 (Cyt b561), a family of highly conserved transmembrane enzymes, plays an important role in ascorbate recycling and iron absorption. Although Cyt b561 was identified four decades ago, its atomic structure and functional mechanism remain largely unknown. Here, we report the high-resolution crystal structures of cytochrome b561 from Arabidopsis thaliana in both substrate-free and substrate-bound states. Cyt b561 forms a homodimer, with each protomer consisting of six transmembrane helices and two heme groups. The negatively charged substrate ascorbate, or monodehydroascorbate, is enclosed in a positively charged pocket on either side of the membrane. Two highly conserved amino acids, Lys(81) and His(106), play an essential role in substrate recognition and catalysis. Our structural and biochemical analyses allow the proposition of a general electron transfer mechanism for members of the Cyt b561 family.

Fig. 1.

Overall structure of Cyt b₅₆₁. (A) Overall structure of WT, full-length Cyt b₅₆₁. The structure of Cyt b₅₆₁ is shown in three successive views. There are two molecules of Cyt b₅₆₁ in each asymmetrical unit, named Mol A (green) and Mol B (blue). (B) Surface features of the Cyt b₅₆₁ homodimer by electrostatic potential. The three views shown correspond to those in A. Two cavities on either side are surrounded by positively charged amino acids. All structural figures were prepared using PyMOL Molecular Graphics System, Version 1.5 (Schrödinger, LLC).

Fig. 2.

Heme-binding sites of Cyt b₅₆₁. (A) Ribbon representation of the Cyt b₅₆₁ homodimer. Cyt b₅₆₁ is rainbow-colored, with its N terminus in blue and its C terminus in red. (B) Heme arrangement in Cyt b₅₆₁. The heme groups are in a ball-and-stick configuration, with the iron atoms as red spheres. The four histidines that coordinate heme iron are shown. (C) Location of a water molecule and Phe¹²⁹ between two heme groups in each protomer. The electron density maps, contoured at 1.0σ, are colored blue for waters and magenta for the heme groups. (D) Close-up view of the intervening water molecule and Phe¹²⁹ between the two heme groups in each protomer of Cyt b₅₆₁. H-bonds are indicated by red dashed lines. (E) Close-up view of the heme group on the noncytoplasmic side. (F) Close-up view of the heme group on the cytoplasmic side.

Fig. 3.

Recognition of ascorbate by conserved amino acids. (A) Recognition of ascorbate on the cytoplasmic side. Ascorbate is bound on the cytoplasmic side of Mol B, coordinated by the heme group and residues from TM3, TM4, and TM5. The same position of Mol A is occupied by a sulfate ion and the side chain of Arg¹⁴⁷ from a neighboring molecule, Mol A′. The F_obs − F_calc electron density for ascorbate (magenta) is contoured at 2.0σ. (B) Recognition of monodehydroascorbate on the noncytoplasmic side. Monodehydroascorbate is bound on the noncytoplasmic side of the Cyt b₅₆₁ dimer, coordinated by residues from TM3, Loop3, and TM5. The F_obs − F_calc electron density for ascorbate (cyan) is contoured at 2.5σ.

Fig. 4.

Proposed model for electron transfer. (A) Proposed electron transfer path. This illustration shows the electron transfer path from ascorbate in the cytoplasm to monodehydroascorbate on the noncytoplasmic side. (B) Proposed mechanism of ascorbate recycling and ascorbate-dependent ferric reduction catalyzed by Cyt b₅₆₁. The reactions occur on both sides of the membrane, with Cyt b₅₆₁ catalyzing the transfer of electrons.