Function and CO binding properties of the NiFe complex in carbon monoxide dehydrogenase from Clostridium thermoaceticum.
Adding 1,10-phenanthroline to carbon monoxide dehydrogenase from Clostridium thermoaceticum results in the complete loss of the NiFeC EPR signal and the CO/acetyl-CoA exchange activity. Other EPR signals characteristic of the enzyme (the gav = 1.94 and gav = 1.86 signals) and the CO oxidation activity are completely unaffected by the 1,10-phenanthroline treatment. This indicates that there are two catalytic sites on the enzyme; the NiFe complex is required for catalyzing the exchange and acetyl-CoA synthase reactions, while some other site is responsible for CO oxidation. The strength of CO binding to the NiFe complex was examined by titrating dithionite-reduced enzyme with CO. During the titration, the NiFeC EPR signal developed to a final spin intensity of 0.23 spin/alpha beta. The resulting CO titration curve (NiFeC spins/alpha beta vs CO pha beta) was fitted using two reactions: binding of CO to the oxidized NiFe complex, and reduction of the CO-bound species to a form that exhibits the NiFeC signal. Best fits yielded apparent binding constants between 6000 and 14,000 M-1 (Kd = 70-165 microM). This sizable range is due to uncertainty whether CO binds to all or only a small fraction (approximately 23%) of the NiFe complexes. Reduction of the CO-bound NiFe complex is apparently required to activate it for catalysis. The electron used for this reduction originates from the CO oxidation site, suggesting that delivery of a low-potential electron to the CO-bound NiFe complex is the physiological function of the CO oxidation reaction catalyzed by this enzyme.
Shin
W
W
Department of Chemistry, Texas A&M University, College Station 77843.
Lindahl
P A
PA
D000445:Aldehyde Oxidoreductases / Q000378:metabolism*; D002248:Carbon Monoxide / Q000378:metabolism*; D003013:Clostridium / Q000201:enzymology*; D004227:Dithionite / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D007501:Iron / Q000378:metabolism*; D009097:Multienzyme Complexes; D009532:Nickel / Q000378:metabolism*; D010084:Oxidation-Reduction; D010618:Phenanthrolines / Q000494:pharmacology; D013816:Thermodynamics
D009097:Multienzyme Complexes; D010618:Phenanthrolines; D004227:Dithionite; D009532:Nickel; D002248:Carbon Monoxide; D007501:Iron; D000445:Aldehyde Oxidoreductases; C019823:carbon monoxide dehydrogenase; C025205:1,10-phenanthroline