Low spin quantitation of NiFeC EPR signal from carbon monoxide dehydrogenase is not due to damage incurred during protein purification.
Evidence is presented that the O2-sensitive, nickel- and iron-containing enzyme carbon monoxide dehydrogenase from Clostridium thermoaceticum was purified without significantly inactivating either its CO oxidation or CO/acetyl-CoA exchange activities. All CO oxidation activity from the crude extract was recovered in the purified enzyme (and side fractions). The exchange activity could not be quantified similarly, because the crude extract and early purification step fractions exhibited little or no exchange activity. Later purification fractions exhibited much more exchange activity, suggesting that an inhibitor was present in the impure fractions. The NiFeC EPR signal intensity was used as an indicator of the enzyme's capacity to catalyze exchange. This signal was extremely sensitive to oxygen; exposure to as little as 0.5 equiv/mol enzyme dimer resulted in substantial loss of intensity. The NiFeC intensities at each step in the purification were virtually invariant, indicating that the enzyme had not been exposed to oxygen and had not been inactivated towards catalyzing exchange. The ability to purify carbon monoxide dehydrogenase (CODH) without inactivating nearly any of the molecules suggests that it is quite stable under anaerobic conditions. The purified enzyme, which could not have lost functional metal ions during purification, contained 1.9 Ni and 11.3 Fe, similar to previous reports. The NiFeC EPR signal intensity from each purification fraction (0.2 spins/mol enzyme dimer) was as low as from previous preparations, indicating that its low spin quantitation is not the result of damage incurred during purification. If the low intensity arises from heterogeneity as proposed earlier, the heterogeneity must originate prior to purification.
Department of Chemistry, Texas A&M University, College Station 77843.
W
W
Shin
P A
PA
Lindahl
D000445:Aldehyde Oxidoreductases / Q000737:chemistry* / Q000302:isolation & purification; D002850:Chromatography, Gel; D003013:Clostridium / Q000201:enzymology; D004578:Electron Spin Resonance Spectroscopy; D004789:Enzyme Activation; D009097:Multienzyme Complexes; D009532:Nickel / Q000737:chemistry; D010100:Oxygen / Q000737:chemistry
D009097:Multienzyme Complexes; D009532:Nickel; D000445:Aldehyde Oxidoreductases; C019823:carbon monoxide dehydrogenase; D010100:Oxygen