Structure and mechanism of a eukaryotic transmembrane ascorbate-dependent oxidoreductase.
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.
Ministry of Education Protein Science Laboratory and State Key Laboratory of Biomembrane and Membrane Biotechnology, Joint Center for Life Sciences, Center for Structural Biology, School of Life Sciences and School of Medicine, Tsinghua University, Beijing 100084, China.
Peilong
P
Lu
Dan
D
Ma
Chuangye
C
Yan
Xinqi
X
Gong
Mingjian
M
Du
Yigong
Y
Shi
D000596:Amino Acids / Q000378:metabolism; D017360:Arabidopsis / Q000201:enzymology*; D001205:Ascorbic Acid / Q000378:metabolism*; D001665:Binding Sites; D002462:Cell Membrane / Q000201:enzymology*; D017124:Conserved Sequence; D003573:Cytochrome b Group / Q000737:chemistry* / Q000378:metabolism*; D004583:Electrons; D005057:Eukaryotic Cells / Q000166:cytology / Q000201:enzymology; D006418:Heme / Q000378:metabolism; D008954:Models, Biological; D008958:Models, Molecular; D010088:Oxidoreductases / Q000737:chemistry* / Q000378:metabolism*
D000596:Amino Acids; D003573:Cytochrome b Group; C023646:cytochrome b561; D006418:Heme; D010088:Oxidoreductases; D001205:Ascorbic Acid