title: Redox-state-dependent complex formation between pseudoazurin and nitrite reductase.
abstract: Bacterial copper-containing nitrite reductase catalyzes the reduction of nitrite to nitric oxide as part of the denitrification process. Pseudoazurin interacts with nitrite reductase in a transient fashion to supply the necessary electrons. The redox-state dependence of complex formation between pseudoazurin and nitrite reductase was studied by nuclear magnetic resonance spectroscopy and isothermal titration calorimetry. Binding of pseudoazurin in the reduced state is characterized by the presence of two binding modes, a slow and a fast exchange mode, with a K(d)(app) of 100 microM. In the oxidized state of pseudoazurin, binding occurs in a single fast exchange mode with a similar affinity. Metal-substituted proteins have been used to show that the mode of binding of pseudoazurin is independent of the metal charge of nitrite reductase. Contrary to what was found for other cupredoxins, protonation of the exposed His ligand to the copper of pseudoazurin, His81, does not appear to be involved directly in the dual binding mode of the reduced form. A model assuming the presence of a minor form of pseudoazurin is proposed to explain the behavior of the complex in the reduced state.
authors.affiliation: Leiden Institute of Chemistry, Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands.
authors.forename: Antonietta
authors.initials: A
authors.lastname: Impagliazzo
authors.forename: Anneloes J
authors.initials: AJ
authors.lastname: Blok
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Cliff
authors.forename: John E
authors.initials: JE
authors.lastname: Ladbury
authors.forename: Marcellus
authors.initials: M
authors.lastname: Ubbink
mesh_terms: D042481:Alcaligenes faecalis / Q000201:enzymology; D000595:Amino Acid Sequence; D001400:Azurin / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry*; D002151:Calorimetry; D003300:Copper / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009572:Nitrite Reductases / Q000737:chemistry*; D010084:Oxidation-Reduction; D011487:Protein Conformation
chemical_list: D001426:Bacterial Proteins; C047163:pseudoazurin; D001400:Azurin; D003300:Copper; D009572:Nitrite Reductases; C052695:nitrite reductase, copper-containing