Evidence that NiNi Acetyl-CoA Synthase Is Active and that the CuNi Enzyme Is Not

Javier Seravalli, Yuming Xiao, Weiwei Gu, Stephen P. Cramer, William E. Antholine, Vladimir Krymov, Gary J. Gerfen, Stephen W. Ragsdale

Research output: Contribution to journalArticle

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Abstract

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 Mp. 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 ∼0.8 spin/mol.

Original languageEnglish (US)
Pages (from-to)3944-3955
Number of pages12
JournalBiochemistry
Volume43
Issue number13
DOIs
StatePublished - Apr 6 2004

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Acetyl Coenzyme A
carbon monoxide dehydrogenase
Enzymes
Metals
Moorella
Paramagnetic resonance
Cell growth
Electronic states
Carbon Monoxide
Chelating Agents
Stoichiometry
Dimers
Electronic structure
Catalytic Domain
Wood
Substitution reactions
Growth

ASJC Scopus subject areas

  • Biochemistry

Cite this

Seravalli, J., Xiao, Y., Gu, W., Cramer, S. P., Antholine, W. E., Krymov, V., ... Ragsdale, S. W. (2004). Evidence that NiNi Acetyl-CoA Synthase Is Active and that the CuNi Enzyme Is Not. Biochemistry, 43(13), 3944-3955. https://doi.org/10.1021/bi036194n

Evidence that NiNi Acetyl-CoA Synthase Is Active and that the CuNi Enzyme Is Not. / Seravalli, Javier; Xiao, Yuming; Gu, Weiwei; Cramer, Stephen P.; Antholine, William E.; Krymov, Vladimir; Gerfen, Gary J.; Ragsdale, Stephen W.

In: Biochemistry, Vol. 43, No. 13, 06.04.2004, p. 3944-3955.

Research output: Contribution to journalArticle

Seravalli, J, Xiao, Y, Gu, W, Cramer, SP, Antholine, WE, Krymov, V, Gerfen, GJ & Ragsdale, SW 2004, 'Evidence that NiNi Acetyl-CoA Synthase Is Active and that the CuNi Enzyme Is Not', Biochemistry, vol. 43, no. 13, pp. 3944-3955. https://doi.org/10.1021/bi036194n
Seravalli, Javier ; Xiao, Yuming ; Gu, Weiwei ; Cramer, Stephen P. ; Antholine, William E. ; Krymov, Vladimir ; Gerfen, Gary J. ; Ragsdale, Stephen W. / Evidence that NiNi Acetyl-CoA Synthase Is Active and that the CuNi Enzyme Is Not. In: Biochemistry. 2004 ; Vol. 43, No. 13. pp. 3944-3955.
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