Kinetic dissection of a cysteine desulfurase from Synechocystis sp. PCC 6803

The fully-sequenced genome of the unicellular, photosynthetic, freshwater cyanobacterium, Synechocystis sp. strain PCC 6803 has three genes (slr0387, sll0704, and slr0077) with similarity to those encoding the NifS and IscS cysteine desulfurases. Cysteine desulfurases use pyridoxal-5′-phosphate (PLP) to advance a previously unprecedented nucleophilic attack of an active site cysteinyl thiol on the sulfur of the substrate. The resulting reductive C-S bond cleavage yields alanine and an enzyme cysteinyl persulfide intermediate, which must be cleaved to complete the enzyme's catalytic cycle. It has been proposed that sulfur from the persulfide intermediate is then mobilized for incorporation (via additional accessory proteins) into iron-sulfur clusters, biotin, thiamin, molybdopterin, and thiouridine in transfer RNAs. Our genetic data indicate that, under standard laboratory conditions of photoautotrophic growth, only the product of the slr0077 gene is essential to Synechocystis 6803; Our mechanistic characterization of the cysteine desulfurases initially focused on Slr0387/IscS (CD0387). The mechanism of formation of the cysteineyl persulfide (first-half-reaction) and its cleavage by chemical reductants (second-half-reaction) were dissected by transient-state kinetic methods coupled with site-directed mutagenesis. According to the chemical mechanism previously proposed for the persulfide formation half-reaction (Zheng, 1994), there are at least 13 intermediates. Evidence for six on-pathway intermediate states and one inhibited complex (which, under the proper circumstances, can also be part of the primary reaction pathway) was obtained. The first observed intermediate with λ _max = 350 nm is proposed to be a thiol adduct formed by nucleophilic attack of the sulfhydryl group of the substrate on the imine of the PLP cofactor. The second intermediate has absorption features centered at 340 nm and 420 nm, and forms with k_for = 90–130 s−1 and k_rev = 15–30 s−1. This state is proposed to comprise a mixture of rapidly equilibrating Cys aldimine and Cys ketimine forms of the cofactor. The third intermediate is defined by the development of a 506 nm absorption feature (Ala quinonoid) along with the features of the previous state. It is proposed that the formation of this quinonoid form coincides with cleavage of the C-S bond. The quinonoid then decays with a rate constant of 40 s−1 which is tentatively associated with a conformational change by the enzyme. (Abstract shortened by UMI.)...