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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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Revised mechanism of CO oxidation suggested by 13C NMR experiments. Steps 1 and 2, which are monitored by the 13CO line broadening experiments, are designated with blue arrows. Exchange of bound 13CO2 with solution CO2 is too slow to be involved in 13CO exchange broadening.
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fig4: Revised mechanism of CO oxidation suggested by 13C NMR experiments. Steps 1 and 2, which are monitored by the 13CO line broadening experiments, are designated with blue arrows. Exchange of bound 13CO2 with solution CO2 is too slow to be involved in 13CO exchange broadening.

Mentions: As shown in Figure 4, CO oxidation involves CO binding to the C-cluster, CO oxidation to bound CO2, release of protons and CO2 to solution, and electron transfer to the B- and D-clusters, which transfer electrons to external redox mediators. This scheme is similar to one proposed in a recent structural paper in which bound CO2 was trapped as a bridging ligand between Ni and an asymmetrically coordinated Fe, which has been called Fe1 (also called ferrous component II, FCII) (9). Another proposed step in the CO oxidation reaction based on the X-ray structure of dithionite-reduced CODH-II involves channeling of CO and water from the surface to the C-cluster (14). CO is proposed to migrate through a hydrophobic channel, and water molecules are proposed to move through a hydrophilic channel. Thus, NMR-detected chemical exchange reactions involving 13CO or competitive inhibitor 13CN− would include entry of the free species at the mouth of the CO channel and migration to the C-cluster.


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

Seravalli J, Ragsdale SW - Biochemistry (2008)

Revised mechanism of CO oxidation suggested by 13C NMR experiments. Steps 1 and 2, which are monitored by the 13CO line broadening experiments, are designated with blue arrows. Exchange of bound 13CO2 with solution CO2 is too slow to be involved in 13CO exchange broadening.
© Copyright Policy - open-access - ccc-price
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2664834&req=5

fig4: Revised mechanism of CO oxidation suggested by 13C NMR experiments. Steps 1 and 2, which are monitored by the 13CO line broadening experiments, are designated with blue arrows. Exchange of bound 13CO2 with solution CO2 is too slow to be involved in 13CO exchange broadening.
Mentions: As shown in Figure 4, CO oxidation involves CO binding to the C-cluster, CO oxidation to bound CO2, release of protons and CO2 to solution, and electron transfer to the B- and D-clusters, which transfer electrons to external redox mediators. This scheme is similar to one proposed in a recent structural paper in which bound CO2 was trapped as a bridging ligand between Ni and an asymmetrically coordinated Fe, which has been called Fe1 (also called ferrous component II, FCII) (9). Another proposed step in the CO oxidation reaction based on the X-ray structure of dithionite-reduced CODH-II involves channeling of CO and water from the surface to the C-cluster (14). CO is proposed to migrate through a hydrophobic channel, and water molecules are proposed to move through a hydrophilic channel. Thus, NMR-detected chemical exchange reactions involving 13CO or competitive inhibitor 13CN− would include entry of the free species at the mouth of the CO channel and migration to the C-cluster.

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