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Tyr25 is a ligand to the active site d1 heme in as isolated, oxidized cytochrome cd1 nitrite reductase from Paracoccus pantotrophus. This form of the enzyme requires reductive activation, a process that involves not only displacement of Tyr25 from the d1 heme but also switching of the ligands at the c heme from bis-histidinyl to His/Met. A Y25S variant retains this bis-histidinyl coordination in the crystal of the oxidized state that has sulfate bound to the d1 heme iron. This Y25S form of the enzyme does not require reductive activation, an observation previously interpreted as meaning that the presence of the phenolate oxygen of Tyr25 is the critical determinant of the requirement for activation. This interpretation now needs re-evaluation because, unexpectedly, the oxidized as prepared Y25S protein, unlike the wild type, has different heme iron ligands in solution at room temperature, as judged by magnetic circular dichroism and electron spin resonance spectroscopies, than in the crystal. In addition, the binding of nitrite and cyanide to oxidized Y25S cytochrome cd1 is markedly different from the wild type enzyme, thus providing insight into the affinity of the oxidized d1 heme ring for anions in the absence of the steric barrier presented by Tyr25.

Original publication

DOI

10.1074/jbc.M501890200

Type

Journal article

Journal

J Biol Chem

Publication Date

15/07/2005

Volume

280

Pages

26073 - 26079

Keywords

Anions, Binding Sites, Crystallography, X-Ray, Cytochromes, Electron Spin Resonance Spectroscopy, Escherichia coli, Heme, Histidine, Hydrogen-Ion Concentration, Kinetics, Ligands, Mutation, Nitric Oxide, Nitrite Reductases, Nitrites, Oxygen, Paracoccus pantotrophus, Potassium Cyanide, Protein Binding, Spectrophotometry, Temperature, Tyrosine, Ultraviolet Rays