ja0659830_si_001.pdf (73.41 kB)
Redox-State-Dependent Complex Formation between Pseudoazurin and Nitrite Reductase
journal contribution
posted on 2007-01-10, 00:00 authored by Antonietta Impagliazzo, Anneloes J. Blok, Matthew J. Cliff, John E. Ladbury, Marcellus UbbinkBacterial 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 Kdapp of 100 μM. 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.