Low spin quantitation of NiFeC EPR signal from carbon monoxide dehydrogenase is not due to damage incurred during protein purification |
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| Affiliation: | 1. Surface and Nanoscience Division, Materials Science Group, Indira Gandhi Centre for Atomic Research, Kalpakkam, 603102, Tamil Nadu, India;2. Catalysis Division, National Chemical Laboratory, Pune, 411 008, India;1. School of Mechatronic Engineering, Xi’an Technological University, Xi’an 710021, China;2. Institute of Engineering and Technology, Department of Hydraulics and Hydraulic and Pneumatic Systems, South Ural State University, Lenin Prospect 76, Chelyabinsk 4540080, Russian Federation;3. Department of Chemistry, Gorgan Branch, Islamic Azad University, Gorgan, Iran;4. Department of Physics, Faculty of Sciences, Golestan University, Gorgan, Iran;5. Department of Chemistry, Azadshahr Branch, Islamic Azad University, Azadshahr, Golestan, Iran;6. Department of Chemistry and Biotechnology, Swinburne University of Technology, Hawthorn, VIC 3122, Australia;7. Department of Chemistry, Faculty of Science, Golestan University, Gorgan, Iran;8. Golestan Rheumatology Research Center, Golestan University of Medical Science, Gorgan, Iran;9. Department of Chemistry, Payan noor University, Behshahr, Iran;10. Department of Process Engineering, Iranian Central Oil Field Company, Tehran, Iran;11. Deprtment of Chemical Sciences, Bernal Institute, University of Limerick, Limerick, Ireland;1. National Institute for Lasers, Plasma & Radiation Physics, Lasers Department, P.O. Box MG-36, Bucharest-Magurele, Bucharest 769231, Romania;2. Politehnica University of Bucharest, Faculty of Applied Chemistry and Materials Science, Department of Science and Engineering of Oxide Materials and Nanomaterials, Polizu Street no 1-7, Bucharest 011061, Romania;3. Institute of Biochemistry, Romanian Academy, 296 Splaiul Independentei, 060031 Bucharest, Romania;4. Institute of Cellular Biology and Pathology “N. Simionescu”, Proteomics Department, B. P. Hasdeu Street, No. 8, 050568 Bucharest, Romania;5. University of Bucharest, Faculty of Biology, Microbiology Immunology Department, Aleea Portocalelor no 1-3, Bucharest 060101, Romania;6. National Institute of Materials Physics, Magurele, Ilfov, Romania;7. Research Institute of the University of Bucharest – ICUB, Bucharest, Romania;1. Department of Data Science and Knowledge Engineering (DKE), Maastricht University, Maastricht, the Netherlands;2. School of Computing Sciences, University of East Anglia, Norwich, United Kingdom;1. School of Physics and Astronomy, University of Exeter, Exeter, UK;2. School of Pharmacy and Biomolecular Science, Royal College of Surgeons in Ireland, Dublin, 2, Ireland;3. Department of Chemistry and Biochemistry and Science of Advanced Materials Program, Central Michigan University, Mt. Pleasant, MI, USA;4. Cranfield Forensic Institute, Cranfield University, Shrivenham, UK |
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| Abstract: | 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. |
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