Reversible inactivation of CO dehydrogenase with thiol compounds

• Published in: Biochemical and biophysical research communications Vol. 447; no. 3; pp. 413 - 418
• Main Authors: Kreß, Oliver, Gnida, Manuel, Pelzmann, Astrid M., Marx, Christian, Meyer-Klaucke, Wolfram, Meyer, Ortwin
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 09.05.2014
• Subjects: 60 APPLIED LIFE SCIENCES; ABSORPTION SPECTROSCOPY; acetylcysteine; Aldehyde Oxidoreductases - antagonists & inhibitors; Aldehyde Oxidoreductases - chemistry; Bacterial Proteins - antagonists & inhibitors; Bacterial Proteins - chemistry; beta-mercaptoethanol; Bradyrhizobiaceae - enzymology; CARBON DIOXIDE; CARBON MONOXIDE; Carbon monoxide dehydrogenase regulation; Catalytic Domain - drug effects; coenzyme A; COENZYMES; CONCENTRATION RATIO; Copper; Copper - chemistry; COPPER IONS; CYSTEINE; dithiothreitol; electron paramagnetic resonance spectroscopy; ELECTRON SPIN RESONANCE; Electron Spin Resonance Spectroscopy; enzyme inhibition; GLYCINE; HOMOCYSTEINE; HYDROXYLASES; IN VITRO; INACTIVATION; INHIBITION; LIGANDS; MOLYBDENUM; Molybdenum - chemistry; Multienzyme Complexes - antagonists & inhibitors; Multienzyme Complexes - chemistry; Oligotropha carboxidovorans; OXIDATION; Oxidation-Reduction; subcellular fractions; SUBSTRATES; Sulfhydryl Compounds - pharmacology; Thiol; thiols; X-ray absorption spectroscopy; X-RAY SPECTROSCOPY; Thiol; X-ray absorption spectroscopy; Oligotropha carboxidovorans; Copper; Molybdenum; Carbon monoxide dehydrogenase regulation
• ISSN: 0006-291X; 1090-2104; 1090-2104
• DOI: 10.1016/j.bbrc.2014.03.147

[Display omitted] •Rather large thiols (e.g. coenzyme A) can reach the active site of CO dehydrogenase.•CO- and H2-oxidizing activity of CO dehydrogenase is inhibited by thiols.•Inhibition by thiols was reversed by CO or upon lowering the thiol concentration.•Thiols coordinate the Cu ion in the [CuSMo(O)OH] active site as a third ligand. Carbon monoxide dehydrogenase (CO dehydrogenase) from Oligotropha carboxidovorans is a structurally characterized member of the molybdenum hydroxylase enzyme family. It catalyzes the oxidation of CO (CO+H2O→CO2+2e−+2H+) which proceeds at a unique [CuSMo(O)OH] metal cluster. Because of changing activities of CO dehydrogenase, particularly in subcellular fractions, we speculated whether the enzyme would be subject to regulation by thiols (RSH). Here we establish inhibition of CO dehydrogenase by thiols and report the corresponding Ki-values (mM): l-cysteine (5.2), d-cysteine (9.7), N-acetyl-l-cysteine (8.2), d,l-homocysteine (25.8), l-cysteine–glycine (2.0), dithiothreitol (4.1), coenzyme A (8.3), and 2-mercaptoethanol (9.3). Inhibition of the enzyme was reversed by CO or upon lowering the thiol concentration. Electron paramagnetic resonance spectroscopy (EPR) and X-ray absorption spectroscopy (XAS) of thiol-inhibited CO dehydrogenase revealed a bimetallic site in which the RSH coordinates to the Cu-ion as a third ligand {[MoVI(O)OH(2)SCuI(SR)S-Cys]} leaving the redox state of the Cu(I) and the Mo(VI) unchanged. Collectively, our findings establish a regulation of CO dehydrogenase activity by thiols in vitro. They also corroborate the hypothesis that CO interacts with the Cu-ion first. The result that thiol compounds much larger than CO can freely travel through the substrate channel leading to the bimetallic cluster challenges previous concepts involving chaperone function and is of importance for an understanding how the sulfuration step in the assembly of the bimetallic cluster might proceed.