Functional copper at the acetyl-CoA synthase active site

doi:10.1073/pnas.0436720100

Functional copper at the acetyl-CoA synthase active site

^*Department of Biochemistry, University of Nebraska, Lincoln, NE 68588; ^‡Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853; and ^†Department of Applied Science, University of California, Davis, CA 95616

Edited by Jack Halpern, University of Chicago, Chicago, IL, and approved December 16, 2002 (received for review November 2, 2002)

Abstract

The bifunctional CO dehydrogenase/acetyl-CoA synthase (CODH/ACS) plays a central role in the Wood–Ljungdahl pathway of autotrophic CO₂ fixation. A recent structure of the Moorella thermoacetica enzyme revealed that the ACS active site contains a [4Fe-4S] cluster bridged to a binuclear Cu-Ni site. Here, biochemical and x-ray absorption spectroscopic (XAS) evidence is presented that the copper ion at the M. thermoacetica ACS active site is essential. Depletion of copper correlates with reduction in ACS activity and in intensity of the “NiFeC” EPR signal without affecting either the activity or the EPR spectroscopic properties associated with CODH. In contrast, Zn content is negatively correlated with ACS activity without any apparent relationship to CODH activity. Cu is also found in the methanogenic CODH/ACS from Methanosarcina thermophila. XAS studies are consistent with a distorted Cu(I)–S₃ site in the fully active enzyme in solution. Cu extended x-ray absorption fine structure analysis indicates an average Cu–S bond length of 2.25 Å and a metal neighbor at 2.65 Å, consistent with the Cu–Ni distance observed in the crystal structure. XAS experiments in the presence of seleno-CoA reveal a Cu–S₃Se environment with a 2.4-Å Se–Cu bond, strongly implicating a Cu–SCoA intermediate in the mechanism of acetyl-CoA synthesis. These results indicate an essential and functional role for copper in the CODH/ACS from acetogenic and methanogenic organisms.

Footnotes

↵ § To whom correspondence should be addressed at: Department of Biochemistry, Beadle Center, University of Nebraska, Lincoln, NE 68588-0664. E-mail: sragsdale1{at}unl.edu.
This paper was submitted directly (Track II) to the PNAS office.
↵ ¶ It is surprising that the Cu content was overlooked with such a well studied enzyme in which plasma emission analyses indicated that metals other than Ni and Fe were found at substoichiometric levels (19). Reexamining some early (circa 1983) plasma emission analyses corroborates that many preparations of CODH indeed lacked Cu; however, at that time, cells were cultured differently, the enzyme was isolated under different conditions, and the enzyme was not known to contain ACS activity. Therefore, a Cu-depleted enzyme with high CODH activity, but devoid of ACS activity, would not have been recognized. Once the M. thermoacetica CODH was recognized to be the ACS (22), preparations low in ACS were discarded without further analysis. Reexamining the results of metal analyses after 1987 and after the metal content had been “established” indeed reveals that Cu was present in variable amounts, similar to the amount observed in current enzyme preparations. Unfortunately, this Cu was overlooked because the focus of attention was on assessing integrity of each preparation by measuring the concentrations of the previously established metals. Another reason Cu was overlooked is that addition of Cu(II) to a solution containing CODH/ACS leads to inactivation (S.W.R., unpublished data).
Abbreviations:
1. XAS, x-ray absorption spectroscopy
2. XANES, x-ray absorption near-edge spectroscopy
3. EXAFS, extended x-ray absorption fine structure
4. CODH, CO dehydrogenase
5. ACS, acetyl-CoA synthase
6. U, unit(s)