Kinetic analysis of cysteine desulfurase CD0387 from Synechocystis sp. PCC 6803: Formation of the persulfide intermediate

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Abstract

Stopped-flow absorption and isotope effect experiments have been used to dissect the mechanism of formation of the enzyme cysteinyl persulfide intermediate in the reaction of a cysteine desulfurase (CD), CD0387 from Synechocystis sp. strain PCC 6803. Seven accumulating intermediates have been identified and tentatively mapped onto the CD chemical mechanism originally proposed by Dean, White, and co-workers [Zheng, L., White, R. H., Cash, V. L., and Dean, D. R. (1994) Biochemistry 33, 4714-4720]. The first intermediate with λmax ∼ 350 nm is assigned as either a gem-diamine complex or a thiol adduct formed by nucleophilic attack of either the amine group or the sulfhydryl group of the substrate on the internal aldimine form of the pyridoxal 5′-phosphate (PLP) cofactor. The second intermediate, with absorption features at ∼417 and ∼340 nm, is assigned as Cys aldimine and Cys ketimine forms in rapid equilibrium. In agreement with this assignment, a significant substrate α-deuterium equilibrium isotope effect ( 2H-EIE) favoring the aldimine form (417 nm) is observed in the second state produced in either wild-type CD0387 or the inactive C326A variant protein, which lacks the nucleophilic cysteine residue and is thus unable to proceed beyond this state unless "rescued" by a high concentration of an exogenous thiol. The third intermediate has an additional ∼506 nm feature, characteristic of a quinonoid form, along with the features of the previous state. Its assignment as Ala aldimine, quinonoid, and ketimine forms in rapid equilibrium, which associates its formation with C-S bond cleavage and persulfide formation, is supported by its failure to develop in the C326A variant and the normal kinetic isotope effect (2H-KIE) on its formation, which is similar in magnitude to the 2H-EIE disfavoring Cys-ketimine (from which the third state forms) in the second state. Decay of the Ala quinonoid absorption is tentatively attributed to a conformational change by the enzyme that disfavors this form in its equilibrium with Ala aldimine and Ala ketimine. Subsequent decay of the ketimine absorption (∼340 nm) is attributed to release of Ala from the cofactor with an observed rate constant of 10 s-1, the slowest step in the persulfide-forming half-reaction. The enzyme-persulfide·Ala complex dissociates rapidly with a Kd of 98 mM. The final state with λmax ∼350 nm is assigned as a dead-end complex between the enzyme-persulfide and a second L-cysteine, which adds to the cofactor via its sulfhydryl group, possibly forming a cyclic thiazolidine species.

Original languageEnglish (US)
Pages (from-to)12014-12023
Number of pages10
JournalBiochemistry
Volume48
Issue number50
DOIs
StatePublished - Dec 22 2009

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Synechocystis
Kinetics
Isotopes
Enzymes
Sulfhydryl Compounds
Cysteine
Thiazolidines
Gems
Biochemistry
Pyridoxal Phosphate
Diamines
Deuterium
Substrates
Amines
Rate constants
cysteine desulfurase
persulfides
ketimine
Proteins
Experiments

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

@article{2e7562ea773b4ed8bedd886fd635a846,
title = "Kinetic analysis of cysteine desulfurase CD0387 from Synechocystis sp. PCC 6803: Formation of the persulfide intermediate",
abstract = "Stopped-flow absorption and isotope effect experiments have been used to dissect the mechanism of formation of the enzyme cysteinyl persulfide intermediate in the reaction of a cysteine desulfurase (CD), CD0387 from Synechocystis sp. strain PCC 6803. Seven accumulating intermediates have been identified and tentatively mapped onto the CD chemical mechanism originally proposed by Dean, White, and co-workers [Zheng, L., White, R. H., Cash, V. L., and Dean, D. R. (1994) Biochemistry 33, 4714-4720]. The first intermediate with λmax ∼ 350 nm is assigned as either a gem-diamine complex or a thiol adduct formed by nucleophilic attack of either the amine group or the sulfhydryl group of the substrate on the internal aldimine form of the pyridoxal 5′-phosphate (PLP) cofactor. The second intermediate, with absorption features at ∼417 and ∼340 nm, is assigned as Cys aldimine and Cys ketimine forms in rapid equilibrium. In agreement with this assignment, a significant substrate α-deuterium equilibrium isotope effect ( 2H-EIE) favoring the aldimine form (417 nm) is observed in the second state produced in either wild-type CD0387 or the inactive C326A variant protein, which lacks the nucleophilic cysteine residue and is thus unable to proceed beyond this state unless {"}rescued{"} by a high concentration of an exogenous thiol. The third intermediate has an additional ∼506 nm feature, characteristic of a quinonoid form, along with the features of the previous state. Its assignment as Ala aldimine, quinonoid, and ketimine forms in rapid equilibrium, which associates its formation with C-S bond cleavage and persulfide formation, is supported by its failure to develop in the C326A variant and the normal kinetic isotope effect (2H-KIE) on its formation, which is similar in magnitude to the 2H-EIE disfavoring Cys-ketimine (from which the third state forms) in the second state. Decay of the Ala quinonoid absorption is tentatively attributed to a conformational change by the enzyme that disfavors this form in its equilibrium with Ala aldimine and Ala ketimine. Subsequent decay of the ketimine absorption (∼340 nm) is attributed to release of Ala from the cofactor with an observed rate constant of 10 s-1, the slowest step in the persulfide-forming half-reaction. The enzyme-persulfide·Ala complex dissociates rapidly with a Kd of 98 mM. The final state with λmax ∼350 nm is assigned as a dead-end complex between the enzyme-persulfide and a second L-cysteine, which adds to the cofactor via its sulfhydryl group, possibly forming a cyclic thiazolidine species.",
author = "Elham Behshad and {Bollinger, Jr.}, {Joseph M.}",
year = "2009",
month = "12",
day = "22",
doi = "10.1021/bi802161u",
language = "English (US)",
volume = "48",
pages = "12014--12023",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
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}

Kinetic analysis of cysteine desulfurase CD0387 from Synechocystis sp. PCC 6803 : Formation of the persulfide intermediate. / Behshad, Elham; Bollinger, Jr., Joseph M.

In: Biochemistry, Vol. 48, No. 50, 22.12.2009, p. 12014-12023.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Kinetic analysis of cysteine desulfurase CD0387 from Synechocystis sp. PCC 6803

T2 - Formation of the persulfide intermediate

AU - Behshad, Elham

AU - Bollinger, Jr., Joseph M.

PY - 2009/12/22

Y1 - 2009/12/22

N2 - Stopped-flow absorption and isotope effect experiments have been used to dissect the mechanism of formation of the enzyme cysteinyl persulfide intermediate in the reaction of a cysteine desulfurase (CD), CD0387 from Synechocystis sp. strain PCC 6803. Seven accumulating intermediates have been identified and tentatively mapped onto the CD chemical mechanism originally proposed by Dean, White, and co-workers [Zheng, L., White, R. H., Cash, V. L., and Dean, D. R. (1994) Biochemistry 33, 4714-4720]. The first intermediate with λmax ∼ 350 nm is assigned as either a gem-diamine complex or a thiol adduct formed by nucleophilic attack of either the amine group or the sulfhydryl group of the substrate on the internal aldimine form of the pyridoxal 5′-phosphate (PLP) cofactor. The second intermediate, with absorption features at ∼417 and ∼340 nm, is assigned as Cys aldimine and Cys ketimine forms in rapid equilibrium. In agreement with this assignment, a significant substrate α-deuterium equilibrium isotope effect ( 2H-EIE) favoring the aldimine form (417 nm) is observed in the second state produced in either wild-type CD0387 or the inactive C326A variant protein, which lacks the nucleophilic cysteine residue and is thus unable to proceed beyond this state unless "rescued" by a high concentration of an exogenous thiol. The third intermediate has an additional ∼506 nm feature, characteristic of a quinonoid form, along with the features of the previous state. Its assignment as Ala aldimine, quinonoid, and ketimine forms in rapid equilibrium, which associates its formation with C-S bond cleavage and persulfide formation, is supported by its failure to develop in the C326A variant and the normal kinetic isotope effect (2H-KIE) on its formation, which is similar in magnitude to the 2H-EIE disfavoring Cys-ketimine (from which the third state forms) in the second state. Decay of the Ala quinonoid absorption is tentatively attributed to a conformational change by the enzyme that disfavors this form in its equilibrium with Ala aldimine and Ala ketimine. Subsequent decay of the ketimine absorption (∼340 nm) is attributed to release of Ala from the cofactor with an observed rate constant of 10 s-1, the slowest step in the persulfide-forming half-reaction. The enzyme-persulfide·Ala complex dissociates rapidly with a Kd of 98 mM. The final state with λmax ∼350 nm is assigned as a dead-end complex between the enzyme-persulfide and a second L-cysteine, which adds to the cofactor via its sulfhydryl group, possibly forming a cyclic thiazolidine species.

AB - Stopped-flow absorption and isotope effect experiments have been used to dissect the mechanism of formation of the enzyme cysteinyl persulfide intermediate in the reaction of a cysteine desulfurase (CD), CD0387 from Synechocystis sp. strain PCC 6803. Seven accumulating intermediates have been identified and tentatively mapped onto the CD chemical mechanism originally proposed by Dean, White, and co-workers [Zheng, L., White, R. H., Cash, V. L., and Dean, D. R. (1994) Biochemistry 33, 4714-4720]. The first intermediate with λmax ∼ 350 nm is assigned as either a gem-diamine complex or a thiol adduct formed by nucleophilic attack of either the amine group or the sulfhydryl group of the substrate on the internal aldimine form of the pyridoxal 5′-phosphate (PLP) cofactor. The second intermediate, with absorption features at ∼417 and ∼340 nm, is assigned as Cys aldimine and Cys ketimine forms in rapid equilibrium. In agreement with this assignment, a significant substrate α-deuterium equilibrium isotope effect ( 2H-EIE) favoring the aldimine form (417 nm) is observed in the second state produced in either wild-type CD0387 or the inactive C326A variant protein, which lacks the nucleophilic cysteine residue and is thus unable to proceed beyond this state unless "rescued" by a high concentration of an exogenous thiol. The third intermediate has an additional ∼506 nm feature, characteristic of a quinonoid form, along with the features of the previous state. Its assignment as Ala aldimine, quinonoid, and ketimine forms in rapid equilibrium, which associates its formation with C-S bond cleavage and persulfide formation, is supported by its failure to develop in the C326A variant and the normal kinetic isotope effect (2H-KIE) on its formation, which is similar in magnitude to the 2H-EIE disfavoring Cys-ketimine (from which the third state forms) in the second state. Decay of the Ala quinonoid absorption is tentatively attributed to a conformational change by the enzyme that disfavors this form in its equilibrium with Ala aldimine and Ala ketimine. Subsequent decay of the ketimine absorption (∼340 nm) is attributed to release of Ala from the cofactor with an observed rate constant of 10 s-1, the slowest step in the persulfide-forming half-reaction. The enzyme-persulfide·Ala complex dissociates rapidly with a Kd of 98 mM. The final state with λmax ∼350 nm is assigned as a dead-end complex between the enzyme-persulfide and a second L-cysteine, which adds to the cofactor via its sulfhydryl group, possibly forming a cyclic thiazolidine species.

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