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Abstract: 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 (42.5kDa/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. [Copyright &y& Elsevier]

• Structural properties of Mo- and Cu-containing oxidoreductases. • Pyranopterin dithiolene role as electron transfer pathway in Mo-enzymes. • Influence of non-bonding interactions in catalytic and electron transfer rates. • Correlation between exchange interaction (J) and electronic coupling matrix (H DA). • Cys-His bridge role as electron transfer pathway in copper nitrite reductase. Oxidoreductases containing transition metal ions are widespread in nature and are essential for living organisms. In these enzymes, transition metal ions are present either as mononuclear centers or organized into clusters, accomplishing two main roles. One is to be the core of active sites where the substrate is converted into product, and the other is to serve as electron transfer centers. Oxidoreductases containing multiple redox cofactors bind both the substrate and an external electron donor/acceptor at distant protein sites for them to exchange the electrons involved in the redox reaction. Intra-protein electron transfer occurs through specific pathways that link distant metal cofactors, which may additionally be magnetically coupled. Here we review the current understanding on the molecular properties of these long chemical pathways involved in metal coordination, intra − protein electron transfer processes, and transmitting magnetic interactions in Mo- and Cu-containing oxidoreductases. [ABSTRACT FROM AUTHOR]

The Cys-His bridge as electron transfer conduit in the enzymatic catalysis of nitrite to nitric oxide by nitrite reductase from Sinorhizobium meliloti 2011 ( Sm Nir) was evaluated by site-directed mutagenesis, steady state kinetic studies, UV–vis and EPR spectroscopic measurements as well as computational calculations. The kinetic, structural and spectroscopic properties of the His171Asp (H171D) and Cys172Asp (C172D) Sm Nir variants were compared with the wild type enzyme. Molecular properties of H171D and C172D indicate that these point mutations have not visible effects on the quaternary structure of Sm Nir. Both variants are catalytically incompetent using the physiological electron donor pseudoazurin, though C172D presents catalytic activity with the artificial electron donor methyl viologen (k cat = 3.9(4) s − 1 ) lower than that of wt Sm Nir (k cat = 240(50) s − 1 ). QM/MM calculations indicate that the lack of activity of H171D may be ascribed to the N δ1 H…O C hydrogen bond that partially shortcuts the T1–T2 bridging Cys-His covalent pathway. The role of the N δ1 H…O C hydrogen bond in the pH-dependent catalytic activity of wt Sm Nir is also analyzed by monitoring the T1 and T2 oxidation states at the end of the catalytic reaction of wt Sm Nir at pH 6 and 10 by UV–vis and EPR spectroscopies. These data provide insight into how changes in Cys-His bridge interrupts the electron transfer between T1 and T2 and how the pH-dependent catalytic activity of the enzyme are related to pH-dependent structural modifications of the T1–T2 bridging chemical pathway. [ABSTRACT FROM AUTHOR]

A copper-containing nitrite reductase catalyzes the reduction of nitrite to nitric oxide in the denitrifier Sinorhizobium meliloti 2011 (Sm NirK), a microorganism used as bioinoculant in alfalfa seeds. Wild type Sm NirK is a homotrimer that contains two copper centers per monomer, one of type 1 (T1) and other of type 2 (T2). T2 is at the interface of two monomers in a distorted square pyramidal coordination bonded to a water molecule and three histidine side chains, H171 and H136 from one monomer and H342 from the other. We report the molecular, catalytic, and spectroscopic properties of the Sm NirK variant H342G, in which the interfacial H342 T2 ligand is substituted for glycine. The molecular properties of H342G are similar to those of wild type Sm NirK. Fluorescence-based thermal shift assays and FTIR studies showed that the structural effect of the mutation is only marginal. However, the kinetic reaction with the physiological electron donor was significantly affected, which showed a ∼ 100-fold lower turnover number compared to the wild type enzyme. UV–Vis, EPR and FTIR studies complemented with computational calculations indicated that the drop in enzyme activity are mainly due to the void generated in the protein substrate channel by the point mutation. The main structural changes involve the filling of the void with water molecules, the direct coordination to T2 copper ion of the second sphere aspartic acid ligand, a key residue in catalysis and nitrite sensing in NirK, and to the loss of the 3 N-O coordination of T2. The substitution of the interfacial histidine ligand for glycine in the copper active site of nitrite reductase showed a marginal effect on the protein trimeric structure, but the void produced by the mutation yielded a drastic decrease in catalytic activity due to structural changes in copper coordination. [Display omitted] • Rhizobia are denitrifiying bacteria massively used as bioinoculants in agriculture. • Nitrite reductases (NirK) of denitrifiers generate greenhouse gases. • The type 2 copper ion is not lost in the His342Gly variant of S. meliloti NirK. • The interfacial histidine copper ligand has a marginal effect on quaternary structure. • The lack of interfacial histidine drops significantly the catalytic efficiency of NirK. [ABSTRACT FROM AUTHOR]