Mechanism of CO oxidation by carbon monoxide dehydrogenase from Clostridium thermoaceticum and its inhibition by anions

Seravalli, Javier; Kumar, Manoj; Lu, Wei; Ragsdale, Stephen W.

doi:10.1021/bi00024a012

Cited by 58 publications

(90 citation statements)

References 49 publications

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“…The effect of sulfide ion on CODH activity and spectral properties are qualitatively similar to those of other anions, including cyanide, cyanate, thiocyanate, and azide ions (15)(16)(17). Cyanide ion fully inhibits CODH's, apparently by binding to the C red1 state as evidenced by a shift in the C red1 EPR signal when CN -is added (15).…”

Section: Discussionmentioning

confidence: 64%

“…Similar to sulfide, inhibition by thiocyanate is partial (16). This reactivity pattern common to sulfide and other inhibitory anions suggests that these other anions might also bind to the enzyme in the same manner as sulfide -i.e.…”

Section: Discussionmentioning

confidence: 93%

“…Thiocyanate, cyanate and azide also appear to bind C red1 but not C red2 (16)(17). Similar to sulfide, inhibition by thiocyanate is partial (16).…”

Section: Discussionmentioning

confidence: 93%

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Effect of Sodium Sulfide on Ni-Containing Carbon Monoxide Dehydrogenases

Feng

Lindahl

2004

J. Am. Chem. Soc.

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Running Title: Effect of Sulfide on CODH 2 AbstractThe structure of the active-site C-cluster in CO dehydrogenase from Carboxythermus hydrogenoformans includes a µ 2 -sulfide ion bridged to the Ni and unique Fe, while the same cluster in enzymes from Rhodospirillum rubrum (CODH Rr ) and Moorella thermoacetica (CODH Mt ) lack this ion. This difference was investigated by exploring the effects of sodium sulfide on activity and spectral properties. Sulfide partially inhibited the CO oxidation activity of CODH Rr and generated a lag prior to steady-state. CODH Mt was inhibited similarly but without a lag. Adding sulfide to CODH Mt in the C red1 state caused the g av = 1.82 EPR signal to decline and new features to appear, including one with g = 1.95, 1.85 and (1.70 or 1.62). Removing sulfide caused the g av = 1.82 signal to reappear and activity to recover. Sulfide did not affect the g av = 1.86 signal from the C red2 state. A model was developed in which sulfide binds reversibly to C red1 , inhibiting catalysis. Reducing this adduct causes sulfide to dissociate, C red2 to develop, and activity to recover. Using this model, apparent K I values are 40 ± 10 nM for CODH Rr and 60 ± 30 µM for CODH Mt . Effects of sulfide are analogous to those of other anions, including the substrate hydroxyl group, suggesting that these ions also bridge the Ni and unique Fe. This proposed arrangement raises the possibility that CO binding labilizes the bridging hydroxyl and increases its nucleophilic tendency towards attacking Ni-bound carbonyl.3 Ni-containing carbon monoxide dehydrogenases are found in anaerobic bacteria and archaea that grow autotrophically on CO or CO 2 and reducing equivalents (1). There are other potentially important differences between these structures but these will not be discussed here.The C-cluster can be stabilized in four redox states, called C ox , C red1 , C int and C red2 (6-9).The fully-oxidized C ox state is S = 0 and appears not to be involved in CO/CO 2 redox catalysis. 4 Reduction by 1 electron to the S = ½ C red1 state activates the enzyme (10). During catalysis, CO probably coordinates to the Ni of the C-cluster in this state, followed by the attack of the carbonyl by a hydroxide ion, forming a Ni-bound carboxylate. The S = ½ C red2 state then forms as CO 2 dissociates. The C-cluster in the C red2 state transfers 2 electrons, in 1-electron steps, to the B-and D-clusters. In doing so, it passes through an EPR-silent C int state (9) and ultimately returns to the C red1 state (7).We were intrigued by the µ 2 -sulfide ion uniquely found in the CODH Ch C-cluster, and endeavored to understand the circumstances for its presence. One possibility was that this sulfide ion is required for catalysis and that the 3 structures lacking it might reflect enzyme crystallized in an inactive form. Alternatively, the sulfide ion might inhibit catalysis and CODH Ch might have been crystallized in an inactive form. Other possibilities are that the C-cluster in CODH Ch might differ intrinsically from those in the othe...

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Section: Discussionmentioning

confidence: 64%

Section: Discussionmentioning

confidence: 93%

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Effect of Sodium Sulfide on Ni-Containing Carbon Monoxide Dehydrogenases

Feng

Lindahl

2004

J. Am. Chem. Soc.

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“…Finally, redox active sites must be present on the enzyme to accept the two electrons produced by CO oxidation. Identifying the electron acceptors has been a point of much discussion (2,5,7,8,(11)(12)(13)32). The discovery of a D-cluster provides insight into the issue of electron translocation within CODH.…”

Section: Resultsmentioning

confidence: 99%

Life on carbon monoxide: X-ray structure of Rhodospirillum rubrum Ni-Fe-S carbon monoxide dehydrogenase

Drennan

Heo

Sintchak

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

302

274

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A crystal structure of the anaerobic Ni-Fe-S carbon monoxide dehydrogenase (CODH) from Rhodospirillum rubrum has been determined to 2.8-Å resolution. The CODH family, for which the R. rubrum enzyme is the prototype, catalyzes the biological oxidation of CO at an unusual Ni-Fe-S cluster called the C-cluster. The Ni-Fe-S C-cluster contains a mononuclear site and a four-metal cubane. Surprisingly, anomalous dispersion data suggest that the mononuclear site contains Fe and not Ni, and the four-metal cubane has the form [NiFe3S4] and not [Fe4S4]. The mononuclear site and the four-metal cluster are bridged by means of Cys 531 and one of the sulfides of the cube. CODH is organized as a dimer with a previously unidentified [Fe4S4] cluster bridging the two subunits. Each monomer is comprised of three domains: a helical domain at the N terminus, an ␣͞␤ (Rossmann-like) domain in the middle, and an ␣͞␤ (Rossmann-like) domain at the C terminus. The helical domain contributes ligands to the bridging [Fe4S4] cluster and another [Fe4S4] cluster, the B-cluster, which is involved in electron transfer. The two Rossmann domains contribute ligands to the active site C-cluster. This x-ray structure provides insight into the mechanism of biological CO oxidation and has broader significance for the roles of Ni and Fe in biological systems. P hototrophic anaerobes such as Rhodospirillum rubrum have the ability to use CO as a sole carbon and energy source (1). This ability derives from the oxidation of CO to CO 2 catalyzed by carbon monoxide dehydrogenases (CODHs). Some CODH enzymes also participate in the synthesis or degradation of acetyl-CoA and are referred to as CODH͞acetyl CoA synthases (CODH͞ACS). The dual role of these CODH͞ACSs is found in CO 2 fixation to acetyl-CoA by acetogens via the well studied Wood͞Ljungdahl pathway (reviewed in ref.2). Methanogens also use CODH͞ACSs in the conversion of acetyl-CoA to methane. As a consequence of these activities, CODHs are essential for the proper regulation of environmental carbon monoxide. Each year, up to 10 8 tons of CO are oxidized to CO 2 by aerobic and anaerobic bacteria containing CODH enzymes (3). Prehistoric environments rich in CO and CO 2 and deficient in oxygen (4) prompt speculation that early life forms relied on CO͞CO 2 as prime sources of both carbon and energy. In an effort to understand the detailed biochemical workings of early and current life on CO, we present the three-dimensional structure of an anaerobic Ni-Fe-S CODH enzyme.CODHs catalyze the oxidation of carbon monoxide in the two-electron process.The mechanism of CO oxidation is thought to involve binding and deprotonation of an H 2 O molecule to form hydroxide at a unique Ni-Fe-S center called the C-cluster (5). CO is believed to bind to a site on the C-cluster adjacent to the hydroxide. Thus, a metal-bound hydroxide may attack the CO carbon. Then the resulting metal-COOH intermediate is deprotonated and CO 2 is lost to yield a two-electron-reduced C-cluster (reviewed in ref.2). In R. rubrum, electrons are pas...

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“…The most popular mechanism of CO oxidation (12,13,14,16,34) has CO binding to the Ni of the C-cluster and H 2 O binding to FCII (Figure 3, upper mechanism). This lowers the pK a of the H 2 O, yielding a nucleophilic OH -that attacks the Ni-bound CO. Ni binding to CO increases the electrophilicity of the carbonyl carbon and may induce dissociation of the OH - (16).…”

Section: Redox and Spectroscopic Properties Of B-and D-clustersmentioning

confidence: 99%