Calorimetric and Spectropscopic Investigation

Calorimetric and Spectropscopic Investigation

Introduction

Nitrite reductase (NiR) from Alcaligenes faecalis strain S-6 is a copper containing protein that plays a key role in the denitrification processes , i. e., the conversion of nitrate and nitrite to nitric oxide, nitrous oxide and molecular nitrogen. Possible applications of the nitrite reductases in biosensors for the detection of nitrites and have raised the interest in these proteins.

Discussion

From the X-ray structure analysis of wild type NiR at 2.0 Å resolution and it appears that the macromolecule is a trimer of identical 37 kDa large subunits, each of which has 343 amino acids folded in two domains with an essentially ß-sandwich secondary structure. Each NiR monomer contains one type-1 Cu atom and one type-2 Cu atom. The type-1 Cu atom, buried within each subunit, accepts the electron needed for nitrite reduction, from pseudoazurin, its redox partner . It is coordinated by two Nd-atoms, from His95 and His145, and two S atoms, from Cys136 and Met150. The histidine and cysteine ligand atoms constitute the equatorial plane and coordinate the copper ion in a distorted trigonal planar configuration, with the Met residue interacting more weakly in the axial position. This geometry resembles that of the copper site in the monomeric blue copper proteins . The type-2 Cu atom, located at the interface of two adjacent subunits at the bottom of a 14-Å deep pocket, is the site of reduction of nitrite to NO and is bound by the Ne's of His100, His135, and His306, and a water molecule. The two copper centers are approximately 12.5 Å apart and are connected covalently through the Cys136 and His135 bridge, type-1 and type-2 ligands, respectively.

The native state of NiR is well characterized spectroscopically. The type-1 Cu displays two intense absorption bands at 460 nm and 590 nm (these optical features confer to the protein its characteristic bluish-green colour) and in the EPR spectrum shows a narrow parallel hyperfine splitting (A|| < 100 Gauss); the type-2 copper has a low optical absorption in the 700-810 nm range with a larger parallel hyperfine splitting (A|| > 120 Gauss) .

Nitrite reductases from different sources, such as Alcaligenes xylosoxidans and Pseudomonas aureofaciens, with a maximum optical absorption at ˜590 nm, are indicated as blue NiRs .

A complete characterization of a protein requires also information about the energetics of the system, which can be obtained by means of thermal denaturation studies. Differential scanning calorimetry (DSC) is one of the most efficient methods for assessing protein stability and the interaction between subunits and domains of a macromolecule and . According to the results so far reported in the literature, the thermal denaturation of multi-domain and, in particular, multi-copper proteins is irreversible revealing, in most cases, the presence of stable intermediates on the denaturation pathway. If the thermally induced denaturation is irreversible, it is not possible to apply the equilibrium thermodynamics. Therefore, theoretical models describing the thermal transition are of great importance for the analysis of the calorimetric traces. It has been found in many cases that the ...