Evidence that NiNi acetyl-CoA synthase is active and that the CuNi enzyme is not.
The bifunctional CO dehydrogenase/acetyl-CoA synthase (CODH/ACS) plays a central role in the Wood-Ljungdahl pathway of autotrophic CO(2) fixation. One structure of the Moorella thermoacetica enzyme revealed that the active site of ACS (the A-cluster) consists of a [4Fe-4S] cluster bridged to a binuclear CuNi center with Cu at the proximal metal site (M(p)) and Ni at the distal metal site (M(d)). In another structure of the same enzyme, Ni or Zn was present at M(p). On the basis of a positive correlation between ACS activity and Cu content, we had proposed that the Cu-containing enzyme is active [Seravalli, J., et al. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 3689-3694]. Here we have reexamined this proposal. Enzyme preparations with a wider range of Ni (1.6-2.8) and Cu (0.2-1.1) stoichiometries per dimer were studied to reexamine the correlation, if any, between the Ni and Cu content and ACS activity. In addition, the effects of o-phenanthroline (which removes Ni but not Cu) and neocuproine (which removes Cu but not Ni) on ACS activity were determined. EXAFS results indicate that these chelators selectively remove M(p). Multifrequency EPR spectra (3-130 GHz) of the paramagnetic NiFeC state of the A-cluster were examined to investigate the electronic state of this proposed intermediate in the ACS reaction mechanism. The combined results strongly indicate that the CuNi enzyme is inactive, that the NiNi enzyme is active, and that the NiNi enzyme is responsible for the NiFeC EPR signal. The results also support an electronic structure of the NiFeC-eliciting species as a [4Fe-4S](2+) (net S = 0) cluster bridged to a Ni(1+) (S = (1)/(2)) at M(p) that is bridged to planar four-coordinate Ni(2+) (S = 0) at M(d), with the spin predominantly on the Ni(1+). Furthermore, these studies suggest that M(p) is inserted during cell growth. The apparent vulnerability of the proximal metal site in the A-cluster to substitution with different metals appears to underlie the heterogeneity observed in samples that has confounded studies of CODH/ACS for many years. On the basis of this principle, a protocol to generate nearly homogeneous preparations of the active NiNi form of ACS was achieved with NiFeC signals of approximately 0.8 spin/mol.
Seravalli
Javier
J
Department of Biochemistry, University of Nebraska-Lincoln, Lincoln, Nebraska 68588-0664, USA.
Xiao
Yuming
Y
Gu
Weiwei
W
Cramer
Stephen P
SP
Antholine
William E
WE
Krymov
Vladimir
V
Gerfen
Gary J
GJ
Ragsdale
Stephen W
SW
D000106:Acetate-CoA Ligase / Q000737:chemistry*; D000445:Aldehyde Oxidoreductases / Q000737:chemistry*; D002614:Chelating Agents / Q000737:chemistry; D003013:Clostridium / Q000201:enzymology / Q000254:growth & development; D003300:Copper / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy; D004789:Enzyme Activation; D009097:Multienzyme Complexes / Q000737:chemistry*; D009532:Nickel / Q000737:chemistry*; D010618:Phenanthrolines / Q000737:chemistry; D013057:Spectrum Analysis; D014965:X-Rays
D002614:Chelating Agents; D009097:Multienzyme Complexes; D010618:Phenanthrolines; D003300:Copper; D009532:Nickel; C002701:neocuproine; D000445:Aldehyde Oxidoreductases; C019823:carbon monoxide dehydrogenase; D000106:Acetate-CoA Ligase; C025205:1,10-phenanthroline