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13C NMR characterization of an exchange reaction between CO and CO2 catalyzed by carbon monoxide dehydrogenase.

Seravalli J, Ragsdale SW - Biochemistry (2008)

Bottom Line: 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 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Nebraska, Lincoln, Nebraska 68588, and Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109-0606, USA.

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 degrees 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.

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(A) Dependence of the broadening exchange rate (rateLB) on the CODH-II monomer concentration. Other conditions are 0.5−0.7 atm of 13CO, 50 mM NaH13CO3, and 0.1 M MES (pD 6.8). The T1-corrected integrations of the 13CO peak were used to calculate the 13CO concentrations. The slope of the linear regression is 2000 ± 200 s−1. (B) Dependence of rateLB after normalization for CODH-II concentration on the CO concentration. The line at 1700 s−1 represents the average of all the measurements shown, while the dashed lines indicate the standard error (300 s−1). (C) Effect of pD on the exchange broadening for the CO resonance (●) and pH dependence of kcat for CO oxidation (◼). The steady-state measurements were carried out at 20 °C, in 20 mM methyl viologen, 1 atm of CO, and 0.1 M MES buffer.
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fig2: (A) Dependence of the broadening exchange rate (rateLB) on the CODH-II monomer concentration. Other conditions are 0.5−0.7 atm of 13CO, 50 mM NaH13CO3, and 0.1 M MES (pD 6.8). The T1-corrected integrations of the 13CO peak were used to calculate the 13CO concentrations. The slope of the linear regression is 2000 ± 200 s−1. (B) Dependence of rateLB after normalization for CODH-II concentration on the CO concentration. The line at 1700 s−1 represents the average of all the measurements shown, while the dashed lines indicate the standard error (300 s−1). (C) Effect of pD on the exchange broadening for the CO resonance (●) and pH dependence of kcat for CO oxidation (◼). The steady-state measurements were carried out at 20 °C, in 20 mM methyl viologen, 1 atm of CO, and 0.1 M MES buffer.

Mentions: To ensure that the exchange process is in the slow exchange limit, we performed the experiment at varying concentrations of CODH-II. Although the amount of CO broadening is proportional to the CODH-II concentration (Figure 2A), the 13CO, as well as the HCO3− and CO2, chemical shifts were unchanged by addition of CODH-II (Figure 1B−D), and reproducible within 0.1 Hz for different experiments. Under the slow exchange regime of the NMR time scale, the exchange process can be expected to affect the line widths of the exchanging resonances and not the resonant frequencies or the longitudinal relaxation rate constants (1/T1 values). Therefore, the 13CO chemical exchange occurs at a much slower rate than the time scale of acquisition of the NMR spectra, i.e., 2πΔω, where Δω is the resonance frequency difference between the exchanging species. For a slow exchange process, 2πΔω*/Δυ ≫ 1 (25), where Δυ is the line broadening, as defined in . For a chemical exchange between CO and CO2, Δω (i.e., ωCO − ωCO2) = 60 ppm or 6000 Hz; thus, 2πΔω equals approximately 36000 s−1. Likewise, for the exchange between CO and HCO3−, Δ = 23 ppm or 2300 Hz. Thus, both of the resonance frequency differences are much larger than the maximum observed broadening for CO of ∼30 Hz. That the CO resonance exhibits line broadening but remains at the same chemical shift as free CO demonstrates conclusively that the 13CO chemical exchange process is within the slow exchange limit of the NMR experiment. Under this time regime, eq 3 is the appropriate treatment for the broadening (Δυ), and the 13CO line broadening is expected to be proportional to the exchange rate as shown by eq 4. According to eq 4, the rate of the 13CO chemical exchange is 16.8 mM s−1. This value equates to a rate constant of 1080 s−1 at 20 °C, based on a monomeric unit of CODH-II. The exchange rate (rateLB) in turn is proportional to the enzyme concentration, as shown in Figure 2A.


13C NMR characterization of an exchange reaction between CO and CO2 catalyzed by carbon monoxide dehydrogenase.

Seravalli J, Ragsdale SW - Biochemistry (2008)

(A) Dependence of the broadening exchange rate (rateLB) on the CODH-II monomer concentration. Other conditions are 0.5−0.7 atm of 13CO, 50 mM NaH13CO3, and 0.1 M MES (pD 6.8). The T1-corrected integrations of the 13CO peak were used to calculate the 13CO concentrations. The slope of the linear regression is 2000 ± 200 s−1. (B) Dependence of rateLB after normalization for CODH-II concentration on the CO concentration. The line at 1700 s−1 represents the average of all the measurements shown, while the dashed lines indicate the standard error (300 s−1). (C) Effect of pD on the exchange broadening for the CO resonance (●) and pH dependence of kcat for CO oxidation (◼). The steady-state measurements were carried out at 20 °C, in 20 mM methyl viologen, 1 atm of CO, and 0.1 M MES buffer.
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fig2: (A) Dependence of the broadening exchange rate (rateLB) on the CODH-II monomer concentration. Other conditions are 0.5−0.7 atm of 13CO, 50 mM NaH13CO3, and 0.1 M MES (pD 6.8). The T1-corrected integrations of the 13CO peak were used to calculate the 13CO concentrations. The slope of the linear regression is 2000 ± 200 s−1. (B) Dependence of rateLB after normalization for CODH-II concentration on the CO concentration. The line at 1700 s−1 represents the average of all the measurements shown, while the dashed lines indicate the standard error (300 s−1). (C) Effect of pD on the exchange broadening for the CO resonance (●) and pH dependence of kcat for CO oxidation (◼). The steady-state measurements were carried out at 20 °C, in 20 mM methyl viologen, 1 atm of CO, and 0.1 M MES buffer.
Mentions: To ensure that the exchange process is in the slow exchange limit, we performed the experiment at varying concentrations of CODH-II. Although the amount of CO broadening is proportional to the CODH-II concentration (Figure 2A), the 13CO, as well as the HCO3− and CO2, chemical shifts were unchanged by addition of CODH-II (Figure 1B−D), and reproducible within 0.1 Hz for different experiments. Under the slow exchange regime of the NMR time scale, the exchange process can be expected to affect the line widths of the exchanging resonances and not the resonant frequencies or the longitudinal relaxation rate constants (1/T1 values). Therefore, the 13CO chemical exchange occurs at a much slower rate than the time scale of acquisition of the NMR spectra, i.e., 2πΔω, where Δω is the resonance frequency difference between the exchanging species. For a slow exchange process, 2πΔω*/Δυ ≫ 1 (25), where Δυ is the line broadening, as defined in . For a chemical exchange between CO and CO2, Δω (i.e., ωCO − ωCO2) = 60 ppm or 6000 Hz; thus, 2πΔω equals approximately 36000 s−1. Likewise, for the exchange between CO and HCO3−, Δ = 23 ppm or 2300 Hz. Thus, both of the resonance frequency differences are much larger than the maximum observed broadening for CO of ∼30 Hz. That the CO resonance exhibits line broadening but remains at the same chemical shift as free CO demonstrates conclusively that the 13CO chemical exchange process is within the slow exchange limit of the NMR experiment. Under this time regime, eq 3 is the appropriate treatment for the broadening (Δυ), and the 13CO line broadening is expected to be proportional to the exchange rate as shown by eq 4. According to eq 4, the rate of the 13CO chemical exchange is 16.8 mM s−1. This value equates to a rate constant of 1080 s−1 at 20 °C, based on a monomeric unit of CODH-II. The exchange rate (rateLB) in turn is proportional to the enzyme concentration, as shown in Figure 2A.

Bottom Line: 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 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Nebraska, Lincoln, Nebraska 68588, and Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109-0606, USA.

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 degrees 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.

Show MeSH
Related in: MedlinePlus