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

Research output: Contribution to journalArticlepeer-review

37 Scopus citations


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
Issue number50
StatePublished - Dec 22 2009

All Science Journal Classification (ASJC) codes

  • Biochemistry


Dive into the research topics of 'Kinetic analysis of cysteine desulfurase CD0387 from Synechocystis sp. PCC 6803: Formation of the persulfide intermediate'. Together they form a unique fingerprint.

Cite this