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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) 13C NMR of an exchange reaction mixture containing 15 µM CODH-II (blue) or no CODH-II (red), 0.7 atm of 13CO, and 5.0 mM NaH13CO3 in 0.1 M MES (pD 6.30) with >90% D2O. MES buffer peaks are not shown. Spectra were collected for 12 h with decoupling of the proton channel. The concentrations as estimated from the T1-corrected peak integrations are as follows: 0.7 mM 13CO, 4.41 mM 13CO2, and 1.31 mM H13CO3−. (B) Fits to a four-parameter Lorentzian function of the 13CO resonance peaks. Data without CODH-II show the fit in red and with CODH-II show the fit in blue. (C) Fits to a Lorentzian function of the 13CO2 resonance peaks for the sample without CODH-II (red fit) and with CODH-II (blue fit). (D) Fits to a Lorentzian function of the H13CO3− resonance peaks for the sample without CODH-II (red fit) and with CODH-II (blue fit). NMR peak intensities are normalized for clarity.
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fig1: (A) 13C NMR of an exchange reaction mixture containing 15 µM CODH-II (blue) or no CODH-II (red), 0.7 atm of 13CO, and 5.0 mM NaH13CO3 in 0.1 M MES (pD 6.30) with >90% D2O. MES buffer peaks are not shown. Spectra were collected for 12 h with decoupling of the proton channel. The concentrations as estimated from the T1-corrected peak integrations are as follows: 0.7 mM 13CO, 4.41 mM 13CO2, and 1.31 mM H13CO3−. (B) Fits to a four-parameter Lorentzian function of the 13CO resonance peaks. Data without CODH-II show the fit in red and with CODH-II show the fit in blue. (C) Fits to a Lorentzian function of the 13CO2 resonance peaks for the sample without CODH-II (red fit) and with CODH-II (blue fit). (D) Fits to a Lorentzian function of the H13CO3− resonance peaks for the sample without CODH-II (red fit) and with CODH-II (blue fit). NMR peak intensities are normalized for clarity.

Mentions: Upon addition of CODH-II to a solution containing 13CO and 13CO2, the 13CO resonance exhibits significant broadening (Figure 1A,B). The observed ΔυCO values were in the range between 1 and 30 Hz, depending on the conditions. To compare the results to normal steady-state conditions for CO oxidation, the concentrations of the substrates were at least 10-fold higher than that of CODH-II. As shown in Figure 1, when the solution contained 0.7 atm (0.69 mM) of 13CO and 5.0 mM NaH13CO3, the observed line widths for 13CO, 13CO2, and H13CO3− were 0.68, 0.63, and 1.73 Hz, respectively, without enzyme, as obtained from fits to eq 2. In the presence of 15 µM CODH-II, the resonances “broadened” to 4.32, 0.66, and 1.66 Hz, respectively, yielding line broadenings for 13CO, 13CO2, and H13CO3− of 3.64, 0.03, and −0.07 Hz, respectively. The broadening values for 13CO2 and H13CO3− are insignificant, given the error of the measurement, since line broadening values in the range of 0.5 Hz can originate from slight discrepancies in the tuning of the broadband probe of the instrument. Furthermore, there was no consistent dependence of the CO2 line width broadening on the CO2 or CODH-II concentration. The T1-corrected integrations for the resonances were used to calculate the concentrations of 13CO, 13CO2, and H13CO3− in the reaction mixture, which were 0.71, 4.41, and 1.31 mM, respectively.


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

Seravalli J, Ragsdale SW - Biochemistry (2008)

(A) 13C NMR of an exchange reaction mixture containing 15 µM CODH-II (blue) or no CODH-II (red), 0.7 atm of 13CO, and 5.0 mM NaH13CO3 in 0.1 M MES (pD 6.30) with >90% D2O. MES buffer peaks are not shown. Spectra were collected for 12 h with decoupling of the proton channel. The concentrations as estimated from the T1-corrected peak integrations are as follows: 0.7 mM 13CO, 4.41 mM 13CO2, and 1.31 mM H13CO3−. (B) Fits to a four-parameter Lorentzian function of the 13CO resonance peaks. Data without CODH-II show the fit in red and with CODH-II show the fit in blue. (C) Fits to a Lorentzian function of the 13CO2 resonance peaks for the sample without CODH-II (red fit) and with CODH-II (blue fit). (D) Fits to a Lorentzian function of the H13CO3− resonance peaks for the sample without CODH-II (red fit) and with CODH-II (blue fit). NMR peak intensities are normalized for clarity.
© Copyright Policy - open-access - ccc-price
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fig1: (A) 13C NMR of an exchange reaction mixture containing 15 µM CODH-II (blue) or no CODH-II (red), 0.7 atm of 13CO, and 5.0 mM NaH13CO3 in 0.1 M MES (pD 6.30) with >90% D2O. MES buffer peaks are not shown. Spectra were collected for 12 h with decoupling of the proton channel. The concentrations as estimated from the T1-corrected peak integrations are as follows: 0.7 mM 13CO, 4.41 mM 13CO2, and 1.31 mM H13CO3−. (B) Fits to a four-parameter Lorentzian function of the 13CO resonance peaks. Data without CODH-II show the fit in red and with CODH-II show the fit in blue. (C) Fits to a Lorentzian function of the 13CO2 resonance peaks for the sample without CODH-II (red fit) and with CODH-II (blue fit). (D) Fits to a Lorentzian function of the H13CO3− resonance peaks for the sample without CODH-II (red fit) and with CODH-II (blue fit). NMR peak intensities are normalized for clarity.
Mentions: Upon addition of CODH-II to a solution containing 13CO and 13CO2, the 13CO resonance exhibits significant broadening (Figure 1A,B). The observed ΔυCO values were in the range between 1 and 30 Hz, depending on the conditions. To compare the results to normal steady-state conditions for CO oxidation, the concentrations of the substrates were at least 10-fold higher than that of CODH-II. As shown in Figure 1, when the solution contained 0.7 atm (0.69 mM) of 13CO and 5.0 mM NaH13CO3, the observed line widths for 13CO, 13CO2, and H13CO3− were 0.68, 0.63, and 1.73 Hz, respectively, without enzyme, as obtained from fits to eq 2. In the presence of 15 µM CODH-II, the resonances “broadened” to 4.32, 0.66, and 1.66 Hz, respectively, yielding line broadenings for 13CO, 13CO2, and H13CO3− of 3.64, 0.03, and −0.07 Hz, respectively. The broadening values for 13CO2 and H13CO3− are insignificant, given the error of the measurement, since line broadening values in the range of 0.5 Hz can originate from slight discrepancies in the tuning of the broadband probe of the instrument. Furthermore, there was no consistent dependence of the CO2 line width broadening on the CO2 or CODH-II concentration. The T1-corrected integrations for the resonances were used to calculate the concentrations of 13CO, 13CO2, and H13CO3− in the reaction mixture, which were 0.71, 4.41, and 1.31 mM, respectively.

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