Stopped-Flow Kinetics of Methyl Group Transfer between the Corrinoid-Iron-Sulfur Protein and Acetyl-Coenzyme A Synthase from Clostridium thermoaceticum

Abstract

Kinetics of methyl group transfer between the Ni-Fe-S-containing acetyl-CoA synthase (ACS) and the corrinoid protein (CoFeSP) from Clostridium thermoaceticum were investigated using the stopped-flow method at 390 nm. Rates of the reaction CH(3)-Co(3+)FeSP + ACS(red) <==> Co(1+)FeSP + CH(3)-ACS(ox) in both forward and reverse directions were determined using various protein and reductant concentrations. Ti(3+)citrate, dithionite, and CO were used to reductively activate ACS (forming ACS(red)). The simplest mechanism that adequately fit the data involved formation of a [CH(3)-Co(3+)FeSP]:[ACS(red)] complex, methyl group transfer (forming [Co(1+)FeSP]:[CH(3)-ACS(ox)]), product dissociation (forming Co(1+)FeSP + CH(3)-ACS(ox)), and CO binding yielding a nonproductive enzyme state (ACS(red) + CO <==> ACS(red)-CO). Best-fit rate constants were obtained. CO inhibited methyl group transfer by binding ACS(red) in accordance with K(D) = 180 +/- 90 microM. Fits were unimproved when >1 CO was assumed to bind. Ti(3+)citrate and dithionite inhibited the reverse methyl group transfer reaction, probably by reducing the D-site of CH(3)-ACS(ox). This redox site is oxidized by 2e(-) when the methyl cation is transferred from CH(3)-Co(3+)FeSP to ACS(red), and is reduced during the reverse reaction. Best-fit K(D) values for pre- and post-methyl-transfer complexes were 0.12 +/- 0.06 and 0.3 +/- 0.2 microM, respectively. Intracomplex methyl group transfer was reversible with K(eq) = 2.3 +/- 0.9 (k(f)/k(r) = 6.9 s(-1)/3.0 s(-1)). The nucleophilicity of the [Ni(2+)D(red)] unit appears comparable to that of Co(1+) cobalamins. Reduction of the D-site may cause the Ni(2+) of the A-cluster to behave like the Ni of an organometallic Ni(0) complex.