title: Evidence that ferredoxin interfaces with an internal redox shuttle in Acetyl-CoA synthase during reductive activation and catalysis.
abstract: Acetyl-CoA synthase (ACS), a subunit of the bifunctional CO dehydrogenase/acetyl-CoA synthase (CODH/ACS) complex of Moorella thermoacetica requires reductive activation in order to catalyze acetyl-CoA synthesis and related partial reactions, including the CO/[1-(14)C]-acetyl-CoA exchange reaction. We show that the M. thermoacetica ferredoxin(II) (Fd-II), which harbors two [4Fe-4S] clusters and is an electron acceptor for CODH, serves as a redox activator of ACS. The level of activation depends on the oxidation states of both ACS and Fd-II, which strongly suggests that Fd-II acts as a reducing agent. By the use of controlled potential enzymology, the midpoint reduction potential for the catalytic one-electron redox-active species in the CO/acetyl-CoA exchange reaction is -511 mV, which is similar to the midpoint reduction potential that was earlier measured for other reactions involving ACS. Incubation of ACS with Fd-II and CO leads to the formation of the NiFeC species, which also supports the role of Fd-II as a reductant for ACS. In addition to being a reductant, Fd-II can accept electrons from acetylated ACS, as observed by the increased intensity of the EPR spectrum of reduced Fd-II, indicating that there is a stored electron within an "electron shuttle" in the acetyl-Ni(II) form of ACS. This "shuttle" is proposed to serve as a redox mediator during activation and at different steps of the ACS catalytic cycle.
authors.affiliation: Department of Biological Chemistry, University of Michigan, Ann Arbor, 48109, United States.
authors.forename: Güneş
authors.initials: G
authors.lastname: Bender
authors.forename: Stephen W
authors.initials: SW
authors.lastname: Ragsdale
mesh_terms: D000107:Acetylation; D000445:Aldehyde Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D055664:Electrochemical Techniques; D004578:Electron Spin Resonance Spectroscopy; D004789:Enzyme Activation; D005288:Ferredoxins / Q000737:chemistry / Q000378:metabolism*; D008745:Methylation; D058845:Moorella / Q000737:chemistry / Q000201:enzymology*; D009097:Multienzyme Complexes / Q000737:chemistry / Q000378:metabolism*; D010084:Oxidation-Reduction; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet
chemical_list: D005288:Ferredoxins; D009097:Multienzyme Complexes; D021122:Protein Subunits; D000445:Aldehyde Oxidoreductases; C019823:carbon monoxide dehydrogenase