Bramlett, Matthew Richard (2004-12). Mechanistic investigations of the A-cluster of acetyl-CoA synthase. Doctoral Dissertation. Thesis uri icon

abstract

  • The A-cluster of acetyl-CoA synthase (ACS) catalyzes the formation of acetyl- CoA from CO, coenzyme-A, and a methyl group donated by a corrinoid iron-sulfur protein. Recent crystal structures have exhibited three different metals, Zn, Cu, and Ni, in the proximal site, which bridges a square-planar nickel site and a [Fe4S4] cubane. Contradicting reports supported both the nickel and copper containing forms as representing active enzyme. The results presented here indicate that copper is not necessary or sufficient for catalysis and that copper addition to ACS is deleterious. Several proposed mechanisms exist for the synthesis of acetyl-CoA, the two most prominent are the ??paramagnetic?? and ??diamagnetic?? mechanisms. The ??diamagnetic?? mechanism proposes a two electron activation that precedes methylation to produce an EPR silent Ni2+-CH3 species. This then reacts with CO and coenzyme-A to form acetyl- CoA and regenerate the starting species. The ??paramagnetic?? mechanism assumes a one electron activation prior to the methylation of the paramagnetic Ni1+-CO state to form an unstable Ni3+-acetyl species. This is immediately reduced by an electron shuttle. Results are presented here that no shuttle or external redox mediator is necessary for catalysis. This supports the ??diamagnetic?? mechanism, specifically that a two-electron reductive activation is necessary and that the Ni1+-CO species is not an intermediate. The two-electron reductive activation required by the ??diamagnetic?? mechanism results in an unknown electronic state. Two proposals have been made to describe this form of the A-cluster. The first hypothesis from Brunold et al involves a one-electron reduction of the [Fe4S4]2+ cube and a one-electron reduction of the Nip 2+. This should result in a spin-coupled state that is S = integer. The Ni0 hypothesis requires both electrons to localize on the Nip 2+ forming a zero-valent proximal nickel. M??ssbauer spectroscopy has been used to probe the oxidation state and spin state of the [Fe4S4] cube in the reduced active form. No integer spin system is found and this is interpreted as supporting the Ni0 hypothesis. Additionally, spectra are presented that indicate the heterogeneous nature of the A-cluster is not caused by the occupancy of the proximal site.
  • The A-cluster of acetyl-CoA synthase (ACS) catalyzes the formation of acetyl-
    CoA from CO, coenzyme-A, and a methyl group donated by a corrinoid iron-sulfur
    protein. Recent crystal structures have exhibited three different metals, Zn, Cu, and Ni,
    in the proximal site, which bridges a square-planar nickel site and a [Fe4S4] cubane.
    Contradicting reports supported both the nickel and copper containing forms as
    representing active enzyme. The results presented here indicate that copper is not
    necessary or sufficient for catalysis and that copper addition to ACS is deleterious.
    Several proposed mechanisms exist for the synthesis of acetyl-CoA, the two most
    prominent are the ??paramagnetic?? and ??diamagnetic?? mechanisms. The ??diamagnetic??
    mechanism proposes a two electron activation that precedes methylation to produce an
    EPR silent Ni2+-CH3 species. This then reacts with CO and coenzyme-A to form acetyl-
    CoA and regenerate the starting species. The ??paramagnetic?? mechanism assumes a one
    electron activation prior to the methylation of the paramagnetic Ni1+-CO state to form an
    unstable Ni3+-acetyl species. This is immediately reduced by an electron shuttle.
    Results are presented here that no shuttle or external redox mediator is necessary for
    catalysis. This supports the ??diamagnetic?? mechanism, specifically that a two-electron
    reductive activation is necessary and that the Ni1+-CO species is not an intermediate.
    The two-electron reductive activation required by the ??diamagnetic?? mechanism
    results in an unknown electronic state. Two proposals have been made to describe this
    form of the A-cluster. The first hypothesis from Brunold et al involves a one-electron
    reduction of the [Fe4S4]2+ cube and a one-electron reduction of the Nip
    2+. This should
    result in a spin-coupled state that is S = integer. The Ni0 hypothesis requires both
    electrons to localize on the Nip
    2+ forming a zero-valent proximal nickel. M??ssbauer
    spectroscopy has been used to probe the oxidation state and spin state of the [Fe4S4] cube
    in the reduced active form. No integer spin system is found and this is interpreted as
    supporting the Ni0 hypothesis. Additionally, spectra are presented that indicate the
    heterogeneous nature of the A-cluster is not caused by the occupancy of the proximal
    site.

publication date

  • December 2004