Literature summary for 7.2.1.3 extracted from

Nakanishi, N.; Rahman, M.M.; Sakamoto, Y.; Takigami, T.; Kobayashi, K.; Hori, H.; Hase, T.; Park, S.-Y.; Tsubaki, M.
Importance of the conserved lysine 83 residue of Zea mays cytochrome b561 for ascorbate-specific transmembrane electron transfer as revealed by site-directed mutagenesis studies (2009), Biochemistry, 48, 10665-10678 .

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Cloned(Commentary)

Cloned (Comment)	Organism
recombinant expression of His6-tagged enzyme in Pichia pastoris under control of a methanol-inducible promoter AOX1	Zea mays

Protein Variants

Protein Variants	Comment	Organism
K83A	site-directed mutagenesis, mutation of a cytosolic loop residue, the mutant shows a significant decrease in the final heme reduction level in an acidic pH region	Zea mays
K83D	site-directed mutagenesis, mutation of a cytosolic loop residue, mutant K83D shows a significant reduction in the electron-accepting activity with ascorbate as reductant	Zea mays
K83E	site-directed mutagenesis, mutation of a cytosolic loop residue, the mutant activity is similar to the wild-type enzyme	Zea mays
S118A	site-directed mutagenesis, mutation of a conserved residue in the putative monodehydroascorbate radical binding site, the mutant electron transfer activity to the monodehydroascorbate radical is very similar to those of the wild-type protein, about 70% heme reduction level compared to wild-type	Zea mays
W122A	site-directed mutagenesis, mutation of a conserved residue in the putative monodehydroascorbate radical binding site, the mutant electron transfer activity to the monodehydroascorbate radical is very similar to those of the wild-type protein, about 70% heme reduction level compared to wild-type	Zea mays

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism	Structure
additional information	-	additional information	stopped-flow kinetic analysis at pH 5.0-7.0	Zea mays

Localization

Localization	Comment	Organism	GeneOntology No.	Textmining
microsome	-	Zea mays	-	-

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
ascorbate[side 1] + Fe(III)[side 2]	Zea mays	-	monodehydroascorbate[side 1] + Fe(II)[side 2]	-	r

Organism

Organism	UniProt	Comment	Textmining
Zea mays	Q5D8X4	-	-

Posttranslational Modification

Posttranslational Modification	Comment	Organism
additional information	the N-terminal Met residue is removed posttranslationally. No partial proteolytic digestion at the C-terminal part of wild-type Zmb561	Zea mays
no glycoprotein	although the deduced amino acid sequence of Zmb561 contains two potential N-glycosylation sites (109NES111 and 203NFT205), periodic acid-Schiff staining of the purified wild-type-Zmb561 in SDS-PAGE gels do not show any indication of glycosyl groups	Zea mays

Purification (Commentary)

Purification (Comment)	Organism
native enzyme from microsomal membranes by anion exchange and concanavalin A affinity chromatography, recombinant His6-tagged enzyme from Pichia pastoris by anion exchange and nickel affinity chromatography and gel filtration, elution with a solution containing 1.0% w/v n-octyl beta-glucoside	Zea mays

Reaction

Reaction	Comment	Organism	Reaction ID
ascorbate[side 1] + Fe(III)[side 2] = monodehydroascorbate[side 1] + Fe(II)[side 2]	reaction mechanism	Zea mays	a

Source Tissue

Source Tissue	Comment	Organism	Textmining
nervous system	-	Zea mays	-

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
ascorbate[side 1] + Fe(III)[side 2]	-	Zea mays	monodehydroascorbate[side 1] + Fe(II)[side 2]	-	r
additional information	a concerted proton/electron transfer mechanism is operative in Zea mays cytochrome b561, electron transfer from ascorbate to the cytosolic heme center	Zea mays	?	-	?

Subunits

Subunits	Comment	Organism
?	x * 25000, about, SDS-PAGE	Zea mays
More	N-terminal amino acid sequencing of the purified cytochrome b561	Zea mays

Synonyms

Synonyms	Comment	Organism
CYB561	-	Zea mays
Zmb561	-	Zea mays

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
5	-	oxidation rate by monodehydroascorbate radical	Zea mays
6	-	reduction rate by ascorbate	Zea mays

Cofactor

Cofactor	Comment	Organism	Structure
ascorbate	additions of ascorbate to oxidized wild-type Zmb561 and His6-tagged recombinant Zmb561 causes a quick reduction of heme b reaching the final reduction level of about 80%, suggesting that Zmb561 might utilize ascorbate as a physiological reductant in maize cells	Zea mays
heme b	cytosolic heme b prosthetic group, the transmembrane electron transport protein cytochromes b561 has two heme ligation sites	Zea mays

General Information

General Information	Comment	Organism
additional information	conserved Lys83 residue in a cytosolic loop plays a very important role for the binding of ascorbate and the succeeding electron transfer via electrostatic interactions. Lys83 might also be responsible for the intramolecular electron transfer to the intravesicular heme. Conserved residues Ser118 and Trp122, located in the putative monodehydroascorbate radical binding site, do not have major roles for the redox events on the intravesicular side	Zea mays
physiological function	cytochrome b561 exists in the neurosecretory vesicle membranes of the nervous system of higher animals. The cytochrome receives an electron equivalent from cytosolic ascorbate (AsA)1 and donates it to the intravesicular monodehydroascorbate radical to regenerate AsA after the transmembrane electron transfer. Transmembrane electron transfer catalyzed by cytochrome b561 is essential for the biosynthesis of neurotransmitters	Zea mays

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Release 2023.1 (January 2023)