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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.

SUBMITTER: Lu P

PROVIDER: S-EPMC3918761 | biostudies-literature | 2014 Feb

REPOSITORIES: biostudies-literature

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Structure and mechanism of a eukaryotic transmembrane ascorbate-dependent oxidoreductase.

Lu Peilong P Ma Dan D Yan Chuangye C Gong Xinqi X Du Mingjian M Shi Yigong Y

Proceedings of the National Academy of Sciences of the United States of America 20140121 5

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 fr ...[more]

PMID: 24449903

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Project description:The addition of two electrons and two protons to the C17=C18 bond in protochlorophyllide is catalyzed by a light-dependent enzyme relying on NADPH as electron donor, and by a light-independent enzyme bearing a (Cys)3Asp-ligated [4Fe-4S] cluster which is reduced by cytoplasmic electron donors in an ATP-dependent manner and then functions as electron donor to protochlorophyllide. The precise sequence of events occurring at the C17=C18 bond has not, however, been determined experimentally in the dark-operating enzyme. In this paper, we present the computational investigation of the reaction mechanism of this enzyme at the B3LYP/6-311+G(d,p)//B3LYP/6-31G(d) level of theory. The reaction mechanism begins with single-electron reduction of the substrate by the (Cys)3Asp-ligated [4Fe-4S], yielding a negatively-charged intermediate. Depending on the rate of Fe-S cluster re-reduction, the reaction either proceeds through double protonation of the single-electron-reduced substrate, or by alternating proton/electron transfer. The computed reaction barriers suggest that Fe-S cluster re-reduction should be the rate-limiting stage of the process. Poisson-Boltzmann computations on the full enzyme-substrate complex, followed by Monte Carlo simulations of redox and protonation titrations revealed a hitherto unsuspected pH-dependence of the reaction potential of the Fe-S cluster. Furthermore, the computed distributions of protonation states of the His, Asp and Glu residues were used in conjuntion with single-point ONIOM computations to obtain, for the first time, the influence of all protonation states of an enzyme on the reaction it catalyzes. Despite exaggerating the ease of reduction of the substrate, these computations confirmed the broad features of the reaction mechanism obtained with the medium-sized models, and afforded valuable insights on the influence of the titratable amino acids on each reaction step. Additional comparisons of the energetic features of the reaction intermediates with those of common biochemical redox intermediates suggest a surprisingly simple explanation for the mechanistic differences between the dark-catalyzed and light-dependent enzyme reaction mechanisms.

Dataset Information

Structure and mechanism of a eukaryotic transmembrane ascorbate-dependent oxidoreductase.

Publications

Structure and mechanism of a eukaryotic transmembrane ascorbate-dependent oxidoreductase.

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