EPR Studies of the Active Sites of Carbon Monoxide Dehydrogenase from Clostridium thermoaceticum

The active sites of the nickel and iron-containing enzyme, carbon monoxide dehydrogenase (CODH) from clostridium thermoaceticum were investigated using Electron Paramagnetic Resonance (EPR) technique. CODH exhibits several spectral features called NiFeC, $g_{ave}=1.82$, $g_{ave}=1.86$. FCII signals which are originated from different clusters in this enzyme. CODH is know to catalyze two different kinds of reactions - acetyl-CoA synthesis and CO oxidation. The acetyl-CoA synthesis activity can be followed by monitoring CO/acetyl-CoA exchange. The addition of 1,10-phenanthroline (phen) to CODH selectively destroyed the CO/acetyl-CoA exchange activity and eliminated the NiFeC signal completely. CO oxidation activity and other EPR signals were unaffected. Such behavior demonstrates that CODH has two distinct active sites and that the NiFe complex is only responsible for the CO/acctyl-CoA exchange activity. Phen caused the removal of only 30% of Ni in the NiFe complex ($0.3Ni/{\alpha}{\beta}$) as shown by the quantitative metal analysis. The phen-treated CODH could be reactivated fully by incubation In $Ni^{2+}$ solution. Radioactive $^{63}Ni^{2+}$ was used to quantitate the amount of the $Ni^{2+}$ incorporated into phen-treated enzyme and showed that the amount was the same as the removed by the phen treatment. i.e. $0.3Ni/{\alpha}{\beta}$. This indicates that only 30% of NiFe complexes are labile and responsible for the CO/acctyl-CoA exchange activity, the other 70% are non-labile and have no exchange activity. This is the first clear evidence that the NiFe complex is heterogencous and labile and non-labile Ni sites arc interacting differently with substrates and chelating agents like phen.