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

Abstract

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.

A Unified Electrocatalytic Description of the Action of Inhibitors of Nickel Carbon Monoxide Dehydrogenase

“…43 These two (linear and bent) modes of cyanide binding are consistent with steady-state kinetic studies which demonstrate that CN − acts as a slow inhibitor, 20 thus suggesting that cyanide forms a rapid, reversible complex that undergoes a slow rearrangement to form a tight-binding complex. 28,43 …”

“…28 All chemicals were of analytical or equivalent grade. The gases, carbon monoxide, carbon dioxide, and argon, were purchased from BOC.…”

A Simple, Large-Scale Overexpression Method of Deriving Carbon Monoxide Dehydrogenase II from Thermophilic BacteriumCarboxydothermus hydrogenoformans

“…5,6) Recently, the expression system for cooS II gene encoding CODH II from C. hydrogenoformans in E. coli was reported. 7,8) Notably, recombinant CODH II (Rec-CODH II) shows CO oxidation activity nearly equal to native CODH II. 7,8) This culture process, however, poses a critical problem: the system inevitably produces hydrogen sulfide derived from Na 2 S, which is a sulfide source for [Fe-S] clusters and a reductant for the reduction of the medium.…”

“…7,8) Notably, recombinant CODH II (Rec-CODH II) shows CO oxidation activity nearly equal to native CODH II. 7,8) This culture process, however, poses a critical problem: the system inevitably produces hydrogen sulfide derived from Na 2 S, which is a sulfide source for [Fe-S] clusters and a reductant for the reduction of the medium. Hydrogen sulfide is known to be toxic and to have a corrosive effect on metallic equipment, and hence the culture method is not suitable for large-scale culture.…”

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

“…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).…”