¹³C NMR Characterization of an Exchange Reaction between CO and CO₂ Catalyzed by Carbon Monoxide Dehydrogenase

Carbon monoxide dehydrogenase (CODH) catalyzes the reversible oxidation of CO to CO₂ at a nickel−iron−sulfur cluster (the C-cluster). CO oxidation follows a ping-pong mechanism involving two-electron reduction of the C-cluster followed by electron transfer through an internal electron transfer chain to external electron acceptors. We describe ¹³C NMR studies demonstrating a CODH-catalyzed steady-state exchange reaction between CO and CO₂ in the absence of external electron acceptors. This reaction is characterized by a CODH-dependent broadening of the ¹³CO NMR resonance; however, the chemical shift of the ¹³CO resonance is unchanged, indicating that the broadening is in the slow exchange limit of the NMR experiment. The ¹³CO line broadening occurs with a rate constant (1080 s⁻¹ at 20 °C) that is approximately equal to that of CO oxidation. It is concluded that the observed exchange reaction is between ¹³CO and CODH-bound ¹³CO₂ because ¹³CO line broadening is pH-independent (unlike steady-state CO oxidation), because it requires a functional C-cluster (but not a functional B-cluster) and because the ¹³CO₂ line width does not broaden. Furthermore, a steady-state isotopic exchange reaction between ¹²CO and ¹³CO₂ in solution was shown to occur at the same rate as that of CO₂ reduction, which is approximately 750-fold slower than the rate of ¹³CO exchange broadening. The interaction between CODH and the inhibitor cyanide (CN⁻) was also probed by ¹³C NMR. A functional C-cluster is not required for ¹³CN⁻ broadening (unlike for ¹³CO), and its exchange rate constant is 30-fold faster than that for ¹³CO. The combined results indicate that the ¹³CO exchange includes migration of CO to the C-cluster, and CO oxidation to CO₂, but not release of CO₂ or protons into the solvent. They also provide strong evidence of a CO₂ binding site and of an internal proton transfer network in CODH. ¹³CN⁻ exchange appears to monitor only movement of CN⁻ between solution and its binding to and release from CODH

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

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 NiFe₄S₄ or a NiFe₄S₅ 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 <i>Moorella thermoacetica</i> bound both with a substrate H₂O/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 H₂O/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 H₂O/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

Biosynthesis and Reactivity of Cysteine Persulfides in Signaling

Hydrogen sulfide (H₂S) elicits pleiotropic physiological effects ranging from modulation of cardiovascular to CNS functions. A dominant method for transmission of sulfide-based signals is via posttranslational modification of reactive cysteine thiols to persulfides. However, the source of the persulfide donor and whether its relationship to H₂S is as a product or precursor is controversial. The transsulfuration pathway enzymes can synthesize cysteine persulfide (Cys–SSH) from cystine and H₂S from cysteine and/or homocysteine. Recently, Cys–SSH was proposed as the primary product of the transsulfuration pathway with H₂S representing a decomposition product of Cys–SSH. Our detailed kinetic analyses demonstrate a robust capacity for Cys–SSH production by the human transsulfuration pathway enzymes, cystathionine beta-synthase and γ-cystathionase (CSE) and for homocysteine persulfide synthesis from homocystine by CSE only. However, in the reducing cytoplasmic milieu where the concentration of reduced thiols is significantly higher than of disulfides, substrate level regulation favors the synthesis of H₂S over persulfides. Mathematical modeling at physiologically relevant hepatic substrate concentrations predicts that H₂S rather than Cys–SSH is the primary product of the transsulfuration enzymes with CSE being the dominant producer. The half-life of the metastable Cys–SSH product is short and decomposition leads to a mixture of polysulfides (Cys–S–(S)_<i>n</i>–S–Cys). These in vitro data, together with the intrinsic reactivity of Cys–SSH for cysteinyl versus sulfur transfer, are consistent with the absence of an observable increase in protein persulfidation in cells in response to exogenous cystine and evidence for the formation of polysulfides under these conditions