title: Tunnel mutagenesis and Ni-dependent reduction and methylation of the alpha subunit of acetyl coenzyme A synthase/carbon monoxide dehydrogenase.
abstract: Two isolated alpha subunit mutants (A110C and A222L) of the alpha(2)beta(2) acetyl coenzyme A synthase (ACS)/carbon monoxide dehydrogenase (CODH) from Moorella thermoacetica were designed to block the CO-migrating tunnel in the alpha subunit, allowing comparison with equivalent mutants in ACS/CODH. After Ni activation, both mutants exhibited electron paramagnetic resonance spectra indicating that the A-cluster was properly assembled. ACS activities were similar to those of the wild-type recombinant Ni-activated alpha subunit, suggesting that CO diffuses directly to the A-cluster from solvent rather than through the tunnel as is observed for the "majority" activity of ACS/CODH. Thus, CO appears to migrate to the A-cluster through two pathways, one involving and one not involving the tunnel. The kinetics and extent of reduction of the Fe(4)S(4) cubane in the apo-alpha subunit and the Ni-activated alpha subunit upon exposure to titanium(III) citrate were examined using the stopped-flow method. The extent of reduction was independent of Ni, whereas the kinetics of reduction was Ni-dependent. Apo-alpha subunit reduction was monophasic while Ni-activated alpha subunit reduction was biphasic, with the more rapid phase coincident with that of apo-alpha subunit reduction. Thus, binding of Ni to the A-cluster slows the reduction kinetics of the [Fe(4)S(4)](2+) cubane. An upper limit of two electrons per alpha subunit are transferred from titanium(III) citrate to the Ni subcomponent of the A-cluster during reductive activation. These electrons are accepted quickly relative to the reduction of the [Fe(4)S(4)](2+) cubane. This reduction is probably a prerequisite for methyl group transfer. CO appears to bind to reduced nonfunctional subunits, thereby inhibiting reduction (or promoting reoxidation) of the cubane subcomponent of the A-cluster.
authors: Departments of Chemistry and of Biochemistry and Biophysics, Texas A&M University, College Station, TX, 77843-3255, USA.
authors: Xiangshi
authors: X
authors: Tan
authors: Paul A
authors: PA
authors: Lindahl
mesh_terms: D000445:Aldehyde Oxidoreductases / Q000235:genetics* / Q000378:metabolism*; D001051:Apoenzymes / Q000378:metabolism; D019343:Citric Acid / Q000378:metabolism; D004583:Electrons; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D007506:Iron-Sulfur Proteins / Q000737:chemistry; D007700:Kinetics; D008745:Methylation; D008958:Models, Molecular; D009097:Multienzyme Complexes / Q000235:genetics* / Q000378:metabolism*; D016296:Mutagenesis / Q000187:drug effects*; D009532:Nickel / Q000494:pharmacology*; D010084:Oxidation-Reduction; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000494:pharmacology
chemical_list: D001051:Apoenzymes; D007506:Iron-Sulfur Proteins; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; C419387:titanium citrate; D019343:Citric Acid; D009532:Nickel; D000445:Aldehyde Oxidoreductases; C019823:carbon monoxide dehydrogenase