Crystallographic Snapshots of Cyanide- and Water-Bound C-Clusters from Bifunctional Carbon Monoxide Dehydrogenase/Acetyl-CoA Synthase^,

Abstract

Nickel-containing carbon monoxide dehydrogenases (CODHs) reversibly catalyze the oxidation of carbon monoxide to carbon dioxide and are of vital importance in the global carbon cycle. The unusual catalytic CODH C-cluster has been crystallographically characterized as either a NiFe 4 S 4 or a NiFe 4 S 5 metal center, the latter containing a fifth, additional sulfide that bridges Ni and a unique Fe site. To determine whether this bridging sulfide is catalytically relevant and to further explore the mechanism of the C-cluster, we obtained crystal structures of the 310 kDa bifunctional CODH/acetyl-CoA synthase complex from Moorella thermoacetica bound both with a substrate H 2 O/OH -molecule and with a cyanide inhibitor. X-ray diffraction data were collected from native crystals and from identical crystals soaked in a solution containing potassium cyanide. In both structures, the substrate H 2 O/OH -molecule exhibits binding to the unique Fe site of the C-cluster. We also observe cyanide binding in a bent conformation to Ni of the C-cluster, adjacent the substrate H 2 O/OH -molecule. Importantly, the bridging sulfide is not present in either structure. As these forms of the C-cluster represent the coordination environment immediately before the reaction takes place, our findings do not support a fifth, bridging sulfide playing a catalytic role in the enzyme mechanism. The crystal structures presented here, along with recent structures of CODHs from other organisms, have led us toward a unified mechanism for CO oxidation by the C-cluster, the catalytic center of an environmentally important enzyme.Carbon monoxide dehydrogenases (CODHs) are key enzymes in the global carbon cycle and catalyze the reversible conversion of CO to CO 2 . In some anaerobic bacteria, including the phototroph Rhodospirillum rubrum and the thermophile Carboxydothermus hydrogenoformans, monofunctional Ni-containing CODHs allow these organisms to use CO as their sole carbon and energy source (1, 2). CODH activity accounts for the removal of ∼10 8 tons of CO from the environment every year (3). Acetogenic bacteria, including well-characterized Moorella thermoacetica, couple CODH-catalyzed CO 2 reduction with acetyl-CoA synthesis in the bifunctional enzyme complex CODH/ acetyl-CoA synthase (ACS) as part of the Wood-Ljungdahl carbon fixation pathway (4-7). Briefly, in the "eastern" branch of the pathway, one molecule of CO 2 is reduced to a methyl group in a series of folate-dependent steps. The methyl group is then transferred from methyltetrahydrofolate to the corrinoid ironsulfur protein (CFeSP) by methyl-H 4 folate:CFeSP methyltransferase (MeTr). In the "western" branch (Scheme 1), where Nicontaining CODH/ACS is the principal player, a second molecule of CO 2 is reduced to a CO intermediate by the CODH active site C-cluster. CO then travels ∼70 Å through a remarkable tunnel within the enzyme to the ACS active site A-cluster (8-12), where it is combined with the CFeSP-derived methyl group and coenzyme A to form acetyl-CoA. Acetyl-CoA c...

Kinetics and mechanistic analysis of an extremely rapid carbon dioxide fixation reaction

“…nickel hydroxide | carbon dioxide-bicarbonate conversion | reaction mechanism R ecent research in this laboratory has been directed toward the attainment of synthetic analogues of the NiFe 3 S 4 active site of the enzyme carbon monoxide dehydrogenase (1,2), which catalyzes the interconversion reaction CO 2 þ 2H þ þ 2e − ⇌ CO þ H 2 O. In the course of this work, we have prepared binuclear Ni II ∕Fe II bridged species with the intention of simulating the Ni…Fe component of the enzyme site that is the locus of substrate binding, activation, and product release (3).…”

The extended reductive acetyl-CoA pathway: ATPases in metal cluster maturation and reductive activation

“…An additional sulfido ligand bridging Ni and Fe in a [Ni-Fe4-S5] cluster, previously assumed to be the active state of the cluster C Ha et al, 2007), is connected to a more oxidized inactive state of the enzyme (Wang et al, 2013a,b). The central Ni ion of cluster C is the place where the substrates CO and CO 2 ( Figure 2D) (Jeoung and Dobbek, 2007a), as well as inhibitors, such as cyanide, bind (Ha et al, 2007;Kung et al, 2009). The three Ni-ligands form a quasi-T-shaped arrangement leaving one coordination site at the Ni open to bind and activate the substrates.…”

“…The activity of CODHs is inhibited by several small molecules, many of which resemble CODH substrates to which they are isosteric and isoelectronic. Cyanide is a slow binding competitive inhibitor related to CO (Diekert and Thauer, 1978;Drake et al, 1980;Krzycki and Zeikus, 1984;Grahame and Stadtman, 1987) and binds to an open coordination site of the Ni ion (Ha et al, 2007;Seravalli and Ragsdale, 2008;Kung et al, 2009). Cyanate, acting as an analogue of CO 2 , binds selectively to the reduced state of CODH where it competes with CO 2 for binding to the active site (Wang et al, 2013a,b).…”

Investigations of the Efficient Electrocatalytic Interconversions of Carbon Dioxide and Carbon Monoxide by Nickel-Containing Carbon Monoxide Dehydrogenases

“…Several crystal structures of NiFe-containing CODH (Class IV) or CODH/ACS (Class III) from different organisms have been solved [4, 8, 15, 20–22]. All Class IV enzymes have a dimeric structure (shown in Figure 2) in which each monomer contains a unique active site (called the C-cluster) which is a [Ni4Fe-4S] (or [NiFe-5S]) cubane cluster linked to an extra-cuboidal pendant or ‘dangling’ Fe.…”