Overexpression, purification, and biochemical and spectroscopic characterization of copper-containing nitrite reductase from Sinorhizobium meliloti 2011. Study of the interaction of the catalytic copper center with nitrite and NO.
The entire nirK gene coding for a putative copper-nitrite reductase (Nir) from Sinorhizobium meliloti 2011 (Sm) was cloned and overexpressed heterologously in Escherichia coli for the first time. The spectroscopic and molecular properties of the enzyme indicate that SmNir is a green Nir with homotrimeric structure (42.5 kDa/subunit) containing two copper atoms per monomer, one of type 1 and the other of type 2. SmNir follows a Michaelis-Menten mechanism and is inhibited by cyanide. EPR spectra of the as-purified enzyme exhibit two magnetically different components associated with type 1 and type 2 copper centers in a 1:1 ratio. EPR characterization of the copper species obtained upon interaction of SmNir with nitrite, and catalytically-generated and exogenous NO reveals the formation of a Cu-NO EPR active species not detected before in closely related Nirs.
Departamento de Física, Facultad de Bioquímica y Ciencias Biológicas, Universidad Nacional del Litoral, Ciudad Universitaria, Paraje El Pozo, S3000ZAA Santa Fe, Argentina.
Félix M
FM
Ferroni
Sergio A
SA
Guerrero
Alberto C
AC
Rizzi
Carlos D
CD
Brondino
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D003001:Cloning, Molecular; D003300:Copper / Q000737:chemistry*; D003486:Cyanides / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli; D015870:Gene Expression; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009569:Nitric Oxide / Q000096:biosynthesis / Q000737:chemistry*; D009572:Nitrite Reductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009573:Nitrites / Q000737:chemistry; D055503:Protein Multimerization; D021122:Protein Subunits / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016962:Sinorhizobium meliloti / Q000737:chemistry / Q000201:enzymology*
D001426:Bacterial Proteins; D003486:Cyanides; D009573:Nitrites; D021122:Protein Subunits; D011994:Recombinant Proteins; D009569:Nitric Oxide; D003300:Copper; D009572:Nitrite Reductases; C052695:nitrite reductase, copper-containing