Rapid kinetic studies of acetyl-CoA synthesis: evidence supporting the catalytic intermediacy of a paramagnetic NiFeC species in the autotrophic Wood-Ljungdahl pathway.
CO dehydrogenase/acetyl-CoA synthase (CODH/ACS), a key enzyme in the Wood-Ljungdahl pathway of anaerobic CO(2) fixation, is a bifunctional enzyme containing CODH, which catalyzes the reversible two-electron oxidation of CO to CO(2), and ACS, which catalyzes acetyl-CoA synthesis from CoA, CO, and a methylated corrinoid iron-sulfur protein (CFeSP). ACS contains an active site nickel iron-sulfur cluster that forms a paramagnetic adduct with CO, called the nickel iron carbon (NiFeC) species, which we have hypothesized to be a key intermediate in acetyl-CoA synthesis. This hypothesis has been controversial. Here we report the results of steady-state kinetic experiments; stopped-flow and rapid freeze-quench transient kinetic studies; and kinetic simulations that directly test this hypothesis. Our results show that formation of the NiFeC intermediate occurs at approximately the same rate as, and its decay occurs 6-fold faster than, the rate of acetyl-CoA synthesis. Kinetic simulations of the steady-state and transient kinetic results accommodate the NiFeC species in the mechanism and define the rate constants for the elementary steps in acetyl-CoA synthesis. The combined results strongly support the kinetic competence of the NiFeC species in the Wood-Ljungdahl pathway. The results also imply that the methylation of ACS occurs by attack of the Ni(1+) site in the NiFeC intermediate on the methyl group of the methylated CFeSP. Our results indicate that CO inhibits acetyl-CoA synthesis by inhibiting this methyl transfer reaction. Under noninhibitory CO concentrations (below 100 microM), formation of the NiFeC species is rate-limiting, while at higher inhibitory CO concentrations, methyl transfer to ACS becomes rate-limiting.
Seravalli
Javier
J
Department of Biochemistry, Beadle Center, University of Nebraska, Lincoln, Nebraska 68588-0664, USA.
Kumar
Manoj
M
Ragsdale
Stephen W
SW
D000106:Acetate-CoA Ligase / Q000378:metabolism; D000105:Acetyl Coenzyme A / Q000096:biosynthesis* / Q000737:chemistry; D000445:Aldehyde Oxidoreductases / Q000378:metabolism; D001665:Binding Sites; D002244:Carbon / Q000737:chemistry; D003013:Clostridium / Q000201:enzymology; D004578:Electron Spin Resonance Spectroscopy; D007501:Iron / Q000737:chemistry; D007700:Kinetics; D008745:Methylation; D008956:Models, Chemical; D009097:Multienzyme Complexes / Q000378:metabolism; D009532:Nickel / Q000737:chemistry; D013379:Substrate Specificity
D009097:Multienzyme Complexes; D000105:Acetyl Coenzyme A; D002244:Carbon; D009532:Nickel; D007501:Iron; D000445:Aldehyde Oxidoreductases; C019823:carbon monoxide dehydrogenase; D000106:Acetate-CoA Ligase