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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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Isotope exchange at equilibrium between CO and 13CO2/NaH13CO3. The samples were prepared and the spectra recorded as indicated in . The two assays contained 90 (●) and 9 nM (◼) CODH-II monomers. The amplitudes and rate constants for these assays were 0.70 mM and 0.0139 min−1 (●) and 1.0 mM and 0.001 min−1 (◼), respectively. The data were fit according to eq 5 shown in the text. The concentrations of CO and 13CO2/H13CO3 were 0.7 atm and 50 mM, respectively.
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fig3: Isotope exchange at equilibrium between CO and 13CO2/NaH13CO3. The samples were prepared and the spectra recorded as indicated in . The two assays contained 90 (●) and 9 nM (◼) CODH-II monomers. The amplitudes and rate constants for these assays were 0.70 mM and 0.0139 min−1 (●) and 1.0 mM and 0.001 min−1 (◼), respectively. The data were fit according to eq 5 shown in the text. The concentrations of CO and 13CO2/H13CO3 were 0.7 atm and 50 mM, respectively.

Mentions: To improve our understanding of the origin of the CO line broadening, we measured the isotopic exchange between CO and CO2 in solution by following the incorporation of 13C from 13CO2 into 13CO in the presence of 12CO at lower concentrations of enzyme (<1 µM) than in the CO line broadening experiments. Under these conditions, broadening of the 13CO resonance was not noticeable. Since the concentrations of the substrates are much higher than that of enzyme, the assay is expected to follow the steady-state kinetics of isotope exchange at chemical equilibrium. The number of averaged transients was reduced to 768 scans per spectrum with 8192 data points (38 min collection time per spectrum) in the time domain for detection of the incorporation of 13C into CO. The amount of 13CO increased exponentially with time, as expected for a first-order exchange process, with an amplitude equivalent to the total CO concentration and with a kexc (vel/[CODH]) of ∼1.7 s−1, calculated according to eq 5 (Figure 3). The total concentrations of CO (∼0.7 atm), 13CO2, and H13CO3− (50 mM) did not change during the experiment, as determined from the T1-corrected peak integrations, indicating that an isotope exchange had occurred at equilibrium in which 13C from 13CO2 was incorporated into CO. The isotope exchange rate (vel) was found to be proportional to the CODH concentration (not shown). To compare this isotope exchange reaction with the steady-state initial velocity of CO2 reduction, the reduction of CO2 to CO was assessed at 20 °C by following the formation of Mb-CO (35) with 2 mM sodium dithionite as the reductant, yielding a KmCO2 of 0.5 mM and a kcat of 2 s−1 (Supplementary Figure 2). In contrast, the steady-state parameters for CO oxidation by CODH-II are as follows: KmCO = 18 µM, KmMV = 4 mM, and kcat = 15900 s−1 per monomer at 70 °C (2). At 20 °C, the kcat for CO oxidation decreases to 1500 s−1, while the Km values for the substrates are temperature-independent. Thus, for the isotope exchange between CO and CO2 in solution (kcat = 1.7 s−1), the reduction of free 13CO2 to 13CO is rate-limiting (kcat = 2 s−1), as this step is 750-fold slower than CO oxidation. Since the rate constant for 13CO line broadening (1080 s−1 at 20 °C) is comparable to that for CO oxidation (kcat = 1500 s−1 at 20 °C), the steps involved in 13CO exchange broadening could include CO binding and oxidation to a bound form of CO2, followed by conversion of bound CO2 back to bound CO and re-release of CO into solution. However, one can clearly rule out the possibility that the 13CO line broadening includes the release of CO2 from the enzyme and the reduction of solution CO2 back to CO, since these steps occur too slowly to be part of the 13CO exchange process and, as described above, only 13CO (and not 13CO2) line broadening is observed. These results also suggest that the equilibrium between the CO2/CO and CODH-IIox/CODH-IIred redox couples (eq 7), the “ping” phase of this ping-pong reaction, is significantly shifted to the right.


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

Seravalli J, Ragsdale SW - Biochemistry (2008)

Isotope exchange at equilibrium between CO and 13CO2/NaH13CO3. The samples were prepared and the spectra recorded as indicated in . The two assays contained 90 (●) and 9 nM (◼) CODH-II monomers. The amplitudes and rate constants for these assays were 0.70 mM and 0.0139 min−1 (●) and 1.0 mM and 0.001 min−1 (◼), respectively. The data were fit according to eq 5 shown in the text. The concentrations of CO and 13CO2/H13CO3 were 0.7 atm and 50 mM, respectively.
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fig3: Isotope exchange at equilibrium between CO and 13CO2/NaH13CO3. The samples were prepared and the spectra recorded as indicated in . The two assays contained 90 (●) and 9 nM (◼) CODH-II monomers. The amplitudes and rate constants for these assays were 0.70 mM and 0.0139 min−1 (●) and 1.0 mM and 0.001 min−1 (◼), respectively. The data were fit according to eq 5 shown in the text. The concentrations of CO and 13CO2/H13CO3 were 0.7 atm and 50 mM, respectively.
Mentions: To improve our understanding of the origin of the CO line broadening, we measured the isotopic exchange between CO and CO2 in solution by following the incorporation of 13C from 13CO2 into 13CO in the presence of 12CO at lower concentrations of enzyme (<1 µM) than in the CO line broadening experiments. Under these conditions, broadening of the 13CO resonance was not noticeable. Since the concentrations of the substrates are much higher than that of enzyme, the assay is expected to follow the steady-state kinetics of isotope exchange at chemical equilibrium. The number of averaged transients was reduced to 768 scans per spectrum with 8192 data points (38 min collection time per spectrum) in the time domain for detection of the incorporation of 13C into CO. The amount of 13CO increased exponentially with time, as expected for a first-order exchange process, with an amplitude equivalent to the total CO concentration and with a kexc (vel/[CODH]) of ∼1.7 s−1, calculated according to eq 5 (Figure 3). The total concentrations of CO (∼0.7 atm), 13CO2, and H13CO3− (50 mM) did not change during the experiment, as determined from the T1-corrected peak integrations, indicating that an isotope exchange had occurred at equilibrium in which 13C from 13CO2 was incorporated into CO. The isotope exchange rate (vel) was found to be proportional to the CODH concentration (not shown). To compare this isotope exchange reaction with the steady-state initial velocity of CO2 reduction, the reduction of CO2 to CO was assessed at 20 °C by following the formation of Mb-CO (35) with 2 mM sodium dithionite as the reductant, yielding a KmCO2 of 0.5 mM and a kcat of 2 s−1 (Supplementary Figure 2). In contrast, the steady-state parameters for CO oxidation by CODH-II are as follows: KmCO = 18 µM, KmMV = 4 mM, and kcat = 15900 s−1 per monomer at 70 °C (2). At 20 °C, the kcat for CO oxidation decreases to 1500 s−1, while the Km values for the substrates are temperature-independent. Thus, for the isotope exchange between CO and CO2 in solution (kcat = 1.7 s−1), the reduction of free 13CO2 to 13CO is rate-limiting (kcat = 2 s−1), as this step is 750-fold slower than CO oxidation. Since the rate constant for 13CO line broadening (1080 s−1 at 20 °C) is comparable to that for CO oxidation (kcat = 1500 s−1 at 20 °C), the steps involved in 13CO exchange broadening could include CO binding and oxidation to a bound form of CO2, followed by conversion of bound CO2 back to bound CO and re-release of CO into solution. However, one can clearly rule out the possibility that the 13CO line broadening includes the release of CO2 from the enzyme and the reduction of solution CO2 back to CO, since these steps occur too slowly to be part of the 13CO exchange process and, as described above, only 13CO (and not 13CO2) line broadening is observed. These results also suggest that the equilibrium between the CO2/CO and CODH-IIox/CODH-IIred redox couples (eq 7), the “ping” phase of this ping-pong reaction, is significantly shifted to the right.

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