Functional copper at the acetyl-CoA synthase active site

Abstract

The bifunctional CO dehydrogenase͞acetyl-CoA synthase (CODH͞ACS) plays a central role in the Wood-Ljungdahl pathway of autotrophic CO 2 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 CODH͞ACS from Methanosarcina thermophila. XAS studies are consistent with a distorted Cu ( T he bifunctional enzyme CO dehydrogenase͞acetyl-CoA synthase (CODH͞ACS; EC 1.2.99.2) plays a central role in the Wood-Ljungdahl pathway of autotrophic CO 2 fixation (1, 2). CODH catalyzes the two-electron reduction of CO 2 to CO (Eq. 1). The CO is directed through a 70-Å channel to the ACS active site, where it is condensed with a methyl group (donated by the methylated corrinoid iron-sulfur protein, CH 3 -CFeSP), and CoA to generate acetyl-CoA (Eq. 2) (3).A recent crystal structure reveals that CODH͞ACS is a 300-kDa ␣ 2 ␤ 2 protein with two core CODH ␤ subunits tethered on each side to two ACS ␣ subunits (4). The A cluster in each ␣ subunit contains 1 Ni, 1 Cu, and 4 Fe ions, whereas the ␤ subunit contains 1 Ni and 10 Fe ions arranged into three clusters, known as B, C, and D. Various studies indicate that Ni is a required component of CODH in acetogenic bacteria (5, 6) and Rhodospirillum rubrum (7). Ni also is essential for ACS activity, and a subpopulation of the Ni ions in the ACS active site, called the ''labile Ni,'' is required for ACS activity and for generation of an EPR signal called the ''NiFeC signal'' (8). When CODH͞ACS is reacted with CO, this EPR signal forms and, upon reaction with the CH 3 -CFeSP, it decays, both reactions occurring at catalytically relevant rates, indicating intermediacy of the NiFeC species in the ACS catalytic cycle (9). Until recently (4), Cu was not known to be a component of CODH͞ACS. The discovery of Cu at the ACS active site was surprising, given that this enzyme has been studied for many years and this metal had never been previously reported in a CODH͞ACS. ¶ Cu was found in the Methanosarcina barkeri CODH (10); however, ACS was not present in this preparation of methanogenic enzyme, and Cu has not been reported in the three CODH crystal structures (4, 11, 12) or in other CODHs that have been characterized. A relationship between Cu content and ACS activity was indicated in an earlier report (4); here we provide convincing biochemical and spectroscopic evidence for the importance of Cu in the ACS mechanism. Materials and MethodsMaterials. CO (99.99%) and N 2 (99.998%) were purchase...

A functional Ni-Ni-[4Fe-4S] cluster in the monomeric acetyl-CoA synthase from Carboxydothermus hydrogenoformans

“…A promiscuous proximal metal site in cluster A leaves the question of the true catalytic metal open. An essential and functional role of Cu in the ACS͞CODH from acetogenic and methanogenic microorganisms was suggested from the correlation of Cu contents and acetyl-CoA͞CO exchange activities in combination with spectroscopic measurements (15). On the other hand, data have been obtained consistent with a catalytically active Ni 2 -cubane site.…”

“…The Ni-K edge x-ray absorption near edge structure (XANES) of ACS Ch (Fig. 5, which is published as supporting information on the PNAS web site), a finger print for the metal binding motif, differs from the XANES patterns published for the Ni-d site in ACS Mt ͞ CODH Mt (15,44). We attribute these differences to the Ni-p contribution.…”

“…Free CODHIII Ch was not detectable under any of these conditions. Table 1) at rates that exceeded those reported before of the ACS͞CODH from other sources (15,19). The exchange activity of ACS Ch required the presence of reducing agents such as Ti(III) citrate or dithionite.…”

The compositional and evolutionary logic of metabolism

“…Several enzyme iron-sulfur centers have been recognized [149,150] [151,152], but it was subsequently shown that the Cu-containing cluster represents an inactivated form of ACS [101]. Similarly, it has also been shown that the open form of the Ni-Ni ACS may switch to a closed, inactivated, form by exchanging one of the nickel atoms for a zinc atom [100].…”

Application of Stopped-Flow and Time-Resolved X-Ray Absorption Spectroscopy to the Study of Metalloproteins Molecular Mechanisms

“…Since XAS accurately reports the structure of metal-protein centers, early workers focused on providing additional high resolution structural and electronic information on crystallographically characterized samples. For instance, XAS enabled detailed structural investigation of metal active sites in imidazolonepropionase 25 , cytochrome P450 26,27 , CO dehydrogenase/acetyl-CoA synthase [28][29][30] , manganese catalases 31,32 , and lipoxygenase 33 by providing key insights into their electronic states and atomic structures. Moreover, insights into the enzymatic reaction mechanisms could be derived from XAS analysis, as was shown for tyrosine hydroxylase 34 , molybdenum(Mo)-nitogenase [35][36][37] , and farnesyltransferase 38 .…”