Reversible inactivation of CO dehydrogenase with thiol compounds.
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 {[Mo(VI)(O)OH(2)SCu(I)(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.
Department of Microbiology, University of Bayreuth, 95440 Bayreuth, Germany.
Oliver
O
Kreß
Department of Chemistry, University of Paderborn, 33098 Paderborn, Germany.
Manuel
M
Gnida
Department of Microbiology, University of Bayreuth, 95440 Bayreuth, Germany.
Astrid M
AM
Pelzmann
Institute of Biochemistry and Biophysics, Friedrich-Schiller-University of Jena, 07745 Jena, Germany.
Christian
C
Marx
Department of Chemistry, University of Paderborn, 33098 Paderborn, Germany.
Wolfram
W
Meyer-Klaucke
Department of Microbiology, University of Bayreuth, 95440 Bayreuth, Germany. Electronic address: Ortwin.Meyer@uni-bayreuth.de.
Ortwin
O
Meyer
D000445:Aldehyde Oxidoreductases / Q000037:antagonists & inhibitors* / Q000737:chemistry; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry; D041841:Bradyrhizobiaceae / Q000201:enzymology*; D020134:Catalytic Domain / Q000187:drug effects; D003300:Copper / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D008982:Molybdenum / Q000737:chemistry; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors* / Q000737:chemistry; D010084:Oxidation-Reduction; D013438:Sulfhydryl Compounds / Q000494:pharmacology*
D001426:Bacterial Proteins; D009097:Multienzyme Complexes; D013438:Sulfhydryl Compounds; D003300:Copper; D008982:Molybdenum; D000445:Aldehyde Oxidoreductases; C019823:carbon monoxide dehydrogenase
Carbon monoxide dehydrogenase regulation; Copper; Molybdenum; Oligotropha carboxidovorans; Thiol; X-ray absorption spectroscopy