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 CH₃-Co³⁺FeSP + ACS_red ⇄ Co¹⁺FeSP + CH₃-ACS_ox in both forward and reverse directions were determined using various protein and reductant concentrations. Ti³⁺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 [CH₃-Co³⁺FeSP]:[ACS_red] complex, methyl group transfer (forming [Co¹⁺FeSP]:[CH₃-ACS_ox]), product dissociation (forming Co¹⁺FeSP + CH₃-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 μM. Fits were unimproved when >1 CO was assumed to bind. Ti³⁺citrate and dithionite inhibited the reverse methyl group transfer reaction, probably by reducing the D-site of CH₃-ACS_ox. This redox site is oxidized by 2e^- when the methyl cation is transferred from CH₃-Co³⁺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 μM, 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 {Ni²⁺D_red} unit appears comparable to that of Co¹⁺ cobalamins. Reduction of the D-site may cause the Ni²⁺ of the A-cluster to behave like the Ni of an organometallic Ni⁰ complex.