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.

Conformation of Pseudoazurin in the 152 kDa Electron Transfer Complex with Nitrite Reductase Determined by Paramagnetic NMR

“…It has been shown that this substitution does not affect the structure significantly 18 or influence the binding to Nir. 20 Attempts to determine a dissociation constant for the complex of NiR and Paz by NMR titration experiments have been hampered until now by the size of this complex. 20 A full titration of NiR into ZnPaz was obtained using perdeuterated Paz in combination with TROSY.…”

The X-ray crystal structure of a pseudoazurin from Sinorhizobium meliloti

“…4). This region of the protein structure has been shown to serve as the recognition site for a CuNIR for four of the five other pseudoazurins [14,[37][38][39][40]. A complete pathway for denitrification has been identified in the annotated S. meliloti genome, including a gene encoding a putative CuNIR [16] and a sequence alignment of the S. meliloti and A. faecalis CuNIRs using LALIGN [41] reveals that they share 82% sequence identity.…”

Non-covalent interactions in blue copper protein probed by Met16 mutation and electronic and resonance Raman spectroscopy of Achromobacter cycloclastes pseudoazurin

“…1b) [5]. In addition, the ligated to Cu(II) His81 interacts weakly with the side chain of Met16 residue, which is in the close vicinity of the PAz-NiR binding site [14][15][16].…”

“…Recent NMR spectroscopic studies revealed that Ala15 could be involved in the formation of the AcPAz-NiR complex [14,15]. Therefore, an investigation of the His81-Met16 interaction became necessary [36] in order to understand its role in modulation of the electronic structure and reactivity of the blue copper(II) site.…”