Active Site Comparison of Co^II Blue and Green Nitrite Reductases

Abstract

Copper-containing nitrite reductases (NiRs) possess type 1 (T1) and type 2 (T2) copper sites and can be either green or blue in color owing to differences at their T1 centers. The active sites of a green and a blue NiR were studied by utilizing their T1CuI/T2CoII and T1CoII/T2CoII-substituted forms. The UV/Vis spectra of these derivatives highlight the similarity of the T2 centers in these enzymes and that T1 site differences are also present in the CoII forms. The paramagnetic NMR spectra of T1CuI/T2CoII enzymes allow hyperfine shifted resonances from the three T2 His ligands to be assigned: these exhibit remarkably similar positions in the spectra of both NiRs, emphasizing the homology of the T2 centers. The addition of nitrite results in subtle alterations in the paramagnetic NMR spectra of the T1CuI/T2CoII forms at pH<7, which indicate a geometry change upon the binding of substrate. Shifted resonances from all of the T1 site ligands have been assigned and the CoII--N(His) interactions are alike, whereas the CbetaH proton resonances of the Cys ligand exhibit subtle chemical shift differences in the blue and green NiRs. The strength of the axial CoII--S(Met) interaction is similar in the two NiRs studied, but the altered conformation of the side chain of this ligand results in a dramatically different chemical shift pattern for the CgammaH protons. This indicates an alteration in the bonding of the axial ligand in these derivatives, which could be influential in the CuII proteins.

Redox cycling and kinetic analysis of single molecules of solution-phase nitrite reductase

“…The Lys329Cys mutant of the bNiR from Alcaligenes xylosoxidans, which introduces a Cys residue 2 nm from the T1 Cu within the same monomer, was isolated and purified as described previously (25). The Cys was modified with Atto647N-maleimide yielding protein that was 9% labeled (26).…”

“…1. Single green NiR enzymes (gNiR) (25) from Alcaligenes faecalis were immobilized and fluorescence intensity fluctuations were shown to be indicative of enzyme turnovers (24), although individual turnovers To whom correspondence may be addressed. E-mail: wmoerner@stanford.edu or canters@chem.leidenuniv.nl.…”

Novel Denitrifying Bacterium Ochrobactrum anthropi YD50.2 Tolerates High Levels of Reactive Nitrogen Oxides

“…The reduced form of the protein generated by adding one grain of dithionite did not have these absorption peaks. These properties are characteristic of Nir (Cu-Nir) proteins containing copper, especially of the green-type Cu-Nir protein (32). Generally, Cu-Nir contains one each of the type 1 and 2 copper ions (32).…”

“…These properties are characteristic of Nir (Cu-Nir) proteins containing copper, especially of the green-type Cu-Nir protein (32). Generally, Cu-Nir contains one each of the type 1 and 2 copper ions (32). The deduced amino acid sequence of the nirK gene of O. anthropi YD50.2 contains residues ligating to the type 1 (His131, His181, Cys172, and Met186) and 2 (His136, His171, and His342) copper ions whose spectral properties closely resemble those of rNirK.…”

Refined Distances Between Paramagnetic Centers of a Multi‐Copper Nitrite Reductase Determined by Pulsed EPR (iDEER) Spectroscopy

“…[15] Conventional EPR and PELDOR experimental conditions are given in the Results section and in the figure legends. More details related to the sample preparation and EPR spectroscopy are provided in the text of the Supporting Information.…”